Nociception — the ability to detect painful stimuli — is an invaluable sense that warns against present or imminent damage. In patients with chronic pain, however, this warning signal persists in the absence of any genuine threat and affects all aspects of everyday life. Neuropathic pain, a form of chronic pain caused by damage to sensory nerves themselves, is dishearteningly refractory to drugs that may work in other types of pain and is a major unmet medical need begging for novel analgesics. Hyperpolarisation-activated cyclic nucleotide (HCN)-modulated ion channels are best known for their fundamental pacemaker role in the heart; here, we review data demonstrating that the HCN2 isoform acts in an analogous way as a ‘pacemaker for pain’, in that its activity in nociceptive neurons is critical for the maintenance of electrical activity and for the sensation of chronic pain in pathological pain states. Pharmacological block or genetic deletion of HCN2 in sensory neurons provides robust pain relief in a variety of animal models of inflammatory and neuropathic pain, without any effect on normal sensation of acute pain. We discuss the implications of these findings for our understanding of neuropathic pain pathogenesis, and we outline possible future opportunities for the development of efficacious and safe pharmacotherapies in a range of chronic pain syndromes.

Introduction

Pain is as old as animals — a low-level sensory system that has warned us of harm from time immemorial. For many years, pain was considered to be in the realm of emotions, beyond the reach of reductionist science. The great early neurobiologist, Charles Sherrington, first brought pain into physical science by postulating the existence of nociceptor-specific sensory nerve terminals, which would only detect stimuli strong enough to pose a danger to the organism [1]. Early work by Ed Perl confirmed that nociceptors did indeed exist and, surprisingly, responded to a wide variety of harmful stimuli, unlike other sensory receptors that specialise in responses to particular stimuli [2]. Patrick Wall took matters further by demonstrating that single neurons in the spinal cord responded to painful stimuli, and that axonal pathways ascended from these neurons to the brain [3]. In the 1990s, cell and molecular biologists began to enter the field of pain. Many of the ion channels and receptors important for initiating and modulating pain were cloned in this decade, and the study of pain moved into the realm of molecular biology. Neuropathic pain — caused by damage to the nervous system — remains the most medically intractable form of pain but is also at last beginning to yield to a molecular approach. We review here recent research suggesting that activity of the hyperpolarisation-activated cyclic nucleotide-gated (HCN) ion channel family, and HCN2 in particular, may provide the key to this distressing and poorly understood syndrome.

Definitions

Nociception: The detection of harmful or potentially harmful stimuli by specific peripheral nerve terminals or nociceptors.

Hypersensitivity: An exaggerated pain response of the nociceptive nervous system.

Hyperalgesia: Increased pain response to noxious (painful) stimuli (overlaps with hypersensitivity).

Allodynia: Augmented pain response to innocuous (harmless) stimuli, such as light touch.

Acute pain: Short-term pain initiated by a suprathreshold stimulus. Rapidly subsides once stimulus is removed.

Chronic pain: Pain that lasts for an extended period (usually defined as >12 weeks). Can persist in the absence of stimulation or of visible underlying damage.

Neuropathic pain: Pain arising as a direct consequence of a lesion or disease affecting somatosensory nervous system.

Inflammatory pain: A state of increased pain sensitivity due to local inflammation. Typically short-lived but the inflammation and associated pain can become chronic in conditions such as arthritis.

Painful diabetic neuropathy (PDN): Neuropathic pain caused by damage to peripheral nerves in diabetes.

Post-herpetic neuralgia (PHN): Neuropathic pain caused by damage to peripheral nerves following Herpes Zoster infection (shingles).

Pain — what is it?

Acute pain

Pain is initiated in nociceptive nerve terminals (i.e. those sensitive to noxious stimuli) innervating almost all parts of the body apart from the brain. Unlike many other types of peripheral nerve fibres, such as thermoreceptors and sympathetic fibres, in which tonic electrical activity is typical, nociceptive neurons are usually silent and can only be engaged by noxious (pain-causing) stimuli. Noxious stimuli (chemical, mechanical or thermal in nature) activate ion channels in the nerve terminal membrane. These ion channels carry an inward current that depolarises the nerve terminal and, if threshold is reached, action potentials (APs) are elicited which propagate along the sensory fibre to alert the central nervous system (CNS) to the painful stimulus. Nociceptors encode the noxious stimulus intensity into a frequency of firing; thus, an innocuous (non-painful) stimulus will not depolarise the nerve terminal sufficiently for generation of an AP, whereas a noxious stimulus will trigger a train of impulses at a frequency that increases with stimulus intensity [4]. The cell bodies of nociceptive neurons lie in the dorsal root or trigeminal ganglia (DRG and TG, respectively), which reside adjacent to the spinal cord. The pattern of impulse firing is relayed to the CNS via synapses that incoming DRG neuronal afferents make with second-order neurons in the spinal cord, and from there to the brain via ascending spinothalamic projections. In the brain, the coded signal is evaluated, the perception of pain is born and an appropriate escape response is generated through motor pathways.

Under normal conditions, the nociceptive system is only engaged by noxious stimuli that pose a danger to the organism, that is, suprathreshold stimuli that can induce APs in nociceptive nerve fibres. The type of pain initiated by this kind of stimulus (e.g. noxious heat or intense mechanical pressure) is termed acute pain and serves a clear survival function by facilitating protective responses such as withdrawal from the pain-inducing source. The value of this protective system is emphasised by case studies of rare individuals with congenital insensitivity to pain [5]. In these people, mutations render their nociceptive system inoperative, despite other sensory functions being normal. The pain-free phenotype, far from being a source of happiness, is typically linked to a short lifespan as a consequence of a failure to protect the organism from damage. This illustrates the first difficulty in developing new pain treatments — an ideal analgesic must not silence acute pain.

A distinct form of pain which is present within a few minutes of injury is inflammatory pain. Unlike other sensations, which adapt with prolonged presentation of a stimulus, pain tends to increase as a result of sensitisation of nociceptors — a phenomenon also known as hyperalgesia (enhanced pain). One important mechanism underlying hyperalgesia is that tissue damage causes release of inflammatory mediators such as prostaglandin E2 (PGE2), bradykinin and nerve growth factor, among many others. The resulting inflammatory pain has a significant protective value because (1) it prevents further damage by increasing pain sensitivity to both noxious and innocuous stimulation (termed hyperalgesia and allodynia, respectively) and (2) it facilitates healing by engaging the immune system to remove damaged cells or irritants and to fight pathogens. Although the inflammatory state normally subsides in synchrony with resolution of the underlying damage, in certain cases, such as an arthritic knee, the inflammation does not resolve and therefore the pain becomes chronic (i.e. long-lived).

Chronic pain

Chronic pain sufferers are affected by hypersensitivity to stimulation as well as ongoing spontaneous pain in the absence of any overt stimulation. In chronic pain, the protective value of pain is outweighed by the imposed restraints on everyday life (e.g. interference with mobility), forcing patients to seek treatment. Currently available analgesics for chronic inflammatory pain include non-steroid anti-inflammatory drugs (NSAIDs) and opiates and although these show good efficacy, they also feature major side effects (e.g. gastric damage caused by NSAIDs and addiction and problems with gastric motility caused by opiates) that can limit their clinical application. The risk of death from respiratory depression caused by opiates is also significant and it's incidence has risen in recent years [68].

Chronic pain caused by direct damage to the nervous system, known as neuropathic pain, is usually regarded as being distinct from inflammatory pain. Common causes of neuropathic pain are mechanical injury to peripheral nerves (e.g. car accidents and lower back injury); metabolic disorders [e.g. the pain in type 2 diabetes, called painful diabetic neuropathy (PDN)]; infection [e.g. post-herpetic neuralgia (PHN) and HIV-associated neuropathy] and cancer (e.g. bone cancer, or the pain caused by cancer chemotherapy, for instance vincristine-induced neuropathy). PDN, in particular, affects approximately 1 in 4 diabetics and is the predominant cause of neuropathic pain in the clinic and, given the epidemic growth of type 2 diabetes, constitutes a large and increasing medical need [9]. Injury to the CNS (e.g. stroke and spinal cord injury) can also cause neuropathic pain, but this is relatively uncommon and the pathophysiology involved is even more poorly understood; therefore, this area will not be discussed further here.

Current pharmacotherapies for neuropathic pain

Treatments that work for inflammatory pain (NSAIDs and opiates) are typically ineffective for neuropathic pain. Even front-line treatments for neuropathic pain are bedevilled by poor efficacy (as well as numerous side effects), as evidenced by their poor ‘number needed to treat’ (NNT) values (where NNT is the number of patients needing to be treated to obtain 50% pain relief in just one single patient) [10]. These include antidepressants [tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors and serotonin and norepinephrine reuptake inhibitors (SNRIs)], anticonvulsants (gabapentinoids and carbamazepine), opioids and local anaesthetics. For instance, a recent meta-analysis of 229 independent studies reports that first-line treatments such as gabapentin and pregabalin are ineffective for the majority of patients with neuropathic pain (NNT of 7.2 and 7.7, respectively) [11]. A summary of drugs currently used in the treatment of neuropathic pain is given in Table 1.

Table 1
A summary of drug classes commonly used in the treatment of neuropathic pain, their mechanisms of action, clinical indications, advantages and disadvantages of use
Class of drug Target Proposed mechanism of action Clinical uses Disadvantages Efficacy in neuropathic pain 
Non-steroidal anti-inflammatory drugs (NSAIDs) COX enzymes Inhibition of COX reduces production of the inflammatory mediator PGE2 The most commonly used class of analgesic across all pain categories [12]
Indicated for mild–moderate pain and inflammatory pain [13
  • Gastrointestinal ulceration and bleeding [14]

  • Increased risk of cardiovascular events [15]

  • Risk of acute kidney injury

  • Hypersensitivity reactions are common [11]

 
Poor evidence, no randomised clinical trials in existence [16], substantial placebo component suggested [17
Opioids μ-opioid receptor Activation of descending modulatory systems [18,19Moderate–severe pain [20Rapid-onset, short-term side effects include:
  • Constipation

  • Nausea

  • Dizziness

  • Sedation

More problematic complications occurring with long-term use include [21]:
  • Immunological compromise

  • Hypogonadism

  • Hyperalgesia

  • Misuse and addiction

Overdose may cause death [68]
NNH 11.7 [11
Some beneficial effects, but restricted to second-/third-line treatment due to adverse side effects [11,21,22]
NNT 4.3 [11
Antidepressants Monoamine reuptake transporters Inhibition of reuptake transporters increases monoamine levels. This increases activity in descending antinociceptive pathways
Possible role for neuroimmune influences [23
Neuropathic pain including PDN [24Side effects of tricyclic antidepressants [21]
  • Dry mouth

  • Hypotension

  • Cardiac toxicity

Low safety/tolerability (NNH 13.4) [11]
Use of TCAs for neuropathic pain is ‘off-label’, potentially causing legal issues
Noradrenaline reuptake inhibitors [20]
  • Problems with blood pressure

  • Cardiac toxicity

NNH 11.8 [11
Moderately effective
NNT for TCAs 3.6, NNT for SNRIs 11.8 [11
Anti-epileptics Various ion channels including voltage-gated sodium and calcium channels Decreased neuronal activity Neuropathic pain including trigeminal neuralgia [24Side effects include:
  • Dizziness and somnolence

  • Cognitive impairment

  • Psychiatric disturbance

  • Liver damage

  • Anti-epileptic hypersensitivity syndrome

Tolerability and safety moderate (NNH 13.9 for gabapentin, but 2.0 for other anti-epileptics) [11
Moderately effective (NNT 7.7) [11
Topical treatments TRPV1 channels (capsaicin)
Voltage-gated sodium channels (lidocaine) 
Decreased firing of peripheral nerves via channel inhibition (lidocaine) or ‘desensitisation’ of fibres (capsaicin) [25Localised neuropathic pain in patients wishing to avoid oral treatments Capsaicin may cause local burning, irritation or pruritus [26Low efficacy, but good safety and tolerability hence recommended as second-line treatment [11
Class of drug Target Proposed mechanism of action Clinical uses Disadvantages Efficacy in neuropathic pain 
Non-steroidal anti-inflammatory drugs (NSAIDs) COX enzymes Inhibition of COX reduces production of the inflammatory mediator PGE2 The most commonly used class of analgesic across all pain categories [12]
Indicated for mild–moderate pain and inflammatory pain [13
  • Gastrointestinal ulceration and bleeding [14]

  • Increased risk of cardiovascular events [15]

  • Risk of acute kidney injury

  • Hypersensitivity reactions are common [11]

 
Poor evidence, no randomised clinical trials in existence [16], substantial placebo component suggested [17
Opioids μ-opioid receptor Activation of descending modulatory systems [18,19Moderate–severe pain [20Rapid-onset, short-term side effects include:
  • Constipation

  • Nausea

  • Dizziness

  • Sedation

More problematic complications occurring with long-term use include [21]:
  • Immunological compromise

  • Hypogonadism

  • Hyperalgesia

  • Misuse and addiction

Overdose may cause death [68]
NNH 11.7 [11
Some beneficial effects, but restricted to second-/third-line treatment due to adverse side effects [11,21,22]
NNT 4.3 [11
Antidepressants Monoamine reuptake transporters Inhibition of reuptake transporters increases monoamine levels. This increases activity in descending antinociceptive pathways
Possible role for neuroimmune influences [23
Neuropathic pain including PDN [24Side effects of tricyclic antidepressants [21]
  • Dry mouth

  • Hypotension

  • Cardiac toxicity

Low safety/tolerability (NNH 13.4) [11]
Use of TCAs for neuropathic pain is ‘off-label’, potentially causing legal issues
Noradrenaline reuptake inhibitors [20]
  • Problems with blood pressure

  • Cardiac toxicity

NNH 11.8 [11
Moderately effective
NNT for TCAs 3.6, NNT for SNRIs 11.8 [11
Anti-epileptics Various ion channels including voltage-gated sodium and calcium channels Decreased neuronal activity Neuropathic pain including trigeminal neuralgia [24Side effects include:
  • Dizziness and somnolence

  • Cognitive impairment

  • Psychiatric disturbance

  • Liver damage

  • Anti-epileptic hypersensitivity syndrome

Tolerability and safety moderate (NNH 13.9 for gabapentin, but 2.0 for other anti-epileptics) [11
Moderately effective (NNT 7.7) [11
Topical treatments TRPV1 channels (capsaicin)
Voltage-gated sodium channels (lidocaine) 
Decreased firing of peripheral nerves via channel inhibition (lidocaine) or ‘desensitisation’ of fibres (capsaicin) [25Localised neuropathic pain in patients wishing to avoid oral treatments Capsaicin may cause local burning, irritation or pruritus [26Low efficacy, but good safety and tolerability hence recommended as second-line treatment [11

NNT: number needed to treat, defined as the number of patients needing to be treated to obtain 50% pain relief in just one single patient.

NNH: number needed to harm, defined as the number of patients who needed to be treated for one patient to drop out due to adverse effects — note that patients may of course suffer serious side effects yet persist in taking treatments and thus not be included in this measure.

Theories of neuropathic pain

The question of whether neuropathic pain ‘resides’ in the peripheral nervous system (PNS) or the CNS is critical both for an understanding of this distressing phenomenon and for its treatment. There is general agreement that peripheral nerve injury, triggering activity in primary nociceptive afferent nerve fibres, is the precipitating event in neuropathic pain. Nerve activity has been documented in a variety of traumatic and non-traumatic injury models in animals, where it develops concurrently with pain symptoms, as well as in human patients [2733]. Importantly, preventing this peripheral input from entering the spinal cord by pharmacological block or surgical sectioning of the dorsal root of the affected DRG hinders the development of pain hypersensitivity [3437]. It therefore appears that activity in nociceptive afferents is both sufficient and necessary for at least the initiation of neuropathic pain.

The peripherally driven barrage of impulses in nociceptive nerve fibres is important in at least two respects: (1) it can explain the spontaneous pain that is characteristic of neuropathic pain and (2) it can elicit a process of central sensitisation that is thought to further amplify nociceptive input [38,39]. Central sensitisation causes the threshold for pain detection to fall dramatically, so that pain can be triggered by innocuous stimuli such as light touch (e.g. the brush of a feather), applied even some distance away from an injury [40]. The argument that central sensitisation resides in the CNS (probably at early synapses in the spinal cord) is well founded, based on the observations that brush-evoked hypersensitivity spreads far from the initial site of injury, and is rapidly reversed when the pain of an injury is cut off by cooling or by injection of local anaesthetic at the site of the original injury — too rapidly to be explained by the diffusion of some peripheral signal from injured to uninjured areas [41,42].

Mechanistically, central sensitisation shares features with long-term potentiation (LTP) of glutamatergic transmission, which underlies physiological processes such as learning and memory in other parts of the CNS [43]. Strictly speaking, the term central sensitisation describes the potentiation of spinal nociceptive and wide dynamic range neuron responsiveness (‘wind-up’) to post-synaptic excitation [44]. Similar to LTP, wind-up is usually coupled with feed-forward recruitment of N-methyl-d-aspartate (NMDA) receptor currents, by a mechanism that involves relief of Mg2+ pore blockade and suppression of kinase/phosphatase modulation of post-synaptic NMDA receptors [45]. In practice, however, the term central sensitisation has come to encompass all injury-induced changes that increase spinal gain, such as potentiation of NMDA-independent glutamate receptor responses, gene expression changes in both primary afferents and spinal neurons, receptive field changes and de novo synapse formation between dorsal horn neurons, suppression of intraspinal inhibition, activation of protein kinase cascades and loss of descending modulation [4650]. More recently, the role of glial–neuronal interactions in central sensitisation has also been recognised. Microglia, in particular, become rapidly activated following injury and secrete a number of pro-nociceptive mediators, such as interleukin (IL)-1β, IL-6, IL-10, tumour necrosis factor-α and fractalkine, which can increase responsiveness of spinal cord neurons [51,52].

A key area of disagreement, however, is whether central sensitisation is constantly maintained by peripheral input, that is, depends moment-by-moment on continued activity in nociceptive fibres, or whether an initial blast of activity in nociceptors is sufficient to trigger a long-lasting state of central sensitisation, analogous to a ‘pain memory’, which can continue (perhaps indefinitely) in the absence of further input. Clarification of the precise mechanism involved has major implications for treatment:

  • (i)

    If continued peripheral input is a requirement, then this ongoing nerve activity could perhaps be suppressed by a peripherally acting compound with potentially fewer side effects than a drug acting in the CNS. Examples of such agents are local anaesthetics, such as lidocaine, that inhibit AP propagation by blocking sodium ion channels. These blockers are analgesic but require continual application and cause complete anaesthesia (i.e. loss of all sensation, including normal touch). Skin patch formulations such as lidocaine and capsaicin patches can provide longer lasting relief (up to 12 weeks with a single application) but only work in some patients (NNT = 10.6) [11,53]. Other known sodium channel blockers such as carbamazepine reduce peripheral drive and pain, but are CNS penetrant and so have unpleasant side effects attributable to actions within the CNS (see below).

  • (ii)

    If a central action is required to suppress a long-lasting ‘pain memory’, then a CNS-acting drug will be needed, with the attendant side effects. Among the first-line treatments for neuropathic pain are the gabapentinoids that inhibit synaptic transmission by inhibiting expression of calcium channels in the synaptic membrane [54], but may also interact with γ-aminobutyric acid biosynthesis enzymes, NMDA receptors, protein kinase C and inflammatory cytokines. Other commonly used CNS-acting compounds are TCAs such as amitriptyline, SNRIs, such as duloxetine, and anticonvulsants like carbamazepine. All these interventions are associated with unwanted side effects, such as drowsiness, dizziness, headaches, nausea, diarrhoea, constipation and impairment of motor coordination, which may become so severe that they preclude continuation of the drug [10,55].

To decipher which critical elements are primarily responsible for maintaining neuropathic pain it is informative to consider the identity of the nerve fibres that generate ongoing activity, and the molecular constituents underlying this process. Ongoing activity was first documented in A-fibres, arising within 16–24 h after nerve injury [27,28]. These include large myelinated Aβ fibres, which are predominantly low-threshold mechanoreceptors activated by light touch and therefore not considered to play a major role in pain under physiological conditions, as well as thinly myelinated Aδ fibres, of which many are nociceptive in nature [3]. Spontaneous activity has, for technical reasons, been more challenging to detect in small unmyelinated C-fibres, the archetypal pain signalling neurons; however, advanced techniques such as microneurography have made it possible to demonstrate ongoing C-fibre discharge in a wide array of animal neuropathic pain models, including those arising from peripheral nerve injury, diabetic and HIV-associated neuropathy [29,30]. Importantly, such activity can be detected even years after nerve injury in animals and patients with neuropathic pain and can also be used as a pain biomarker [30,32,33,56]; thus, C-fibre hyperexcitability is significantly greater in polyneuropathy patients with pain, compared with those without pain [57,58].

Since nociceptive fibres are equipped with the molecular machinery for the detection and transmission of noxious signals, ongoing activity in these neurons provides a framework to explain spontaneous pain and central sensitisation in chronic pain syndromes. Impulse firing in these neurons is reliant on the function of ion channels that facilitate membrane depolarisation, AP generation and spike propagation. These ion channels have divergent actions such as influencing firing threshold, sculpting the AP waveform and determining spike frequency, all of which influence pain signalling [5963]. A group of ion channels that have recently emerged as important regulators of AP firing in nociceptors are the HCN channels [64,65].

What are HCN ion channels?

HCN channels are Na+- and K+-permeable pore-forming membrane proteins expressed in a range of excitable cells, with major functions in both peripheral and central neurons and in cardiac cells [66]. They are structurally homologous to potassium channels, but are less selective for K+ ions (Erev ≈ −30 mV), which means that when active at the resting neuronal membrane potential, they produce a depolarising inward current and so tend to enhance neuronal excitability (Figure 1). In marked contrast with other ‘classical’ ion channels (sodium, potassium and calcium channels), HCN family members are activated by hyperpolarisation, usually in the range between −60 and −100 mV. The current carried by HCN channels in neurons is usually referred to as Ih (activated by hyperpolarisation) or in cardiac cells as If (for ‘funny’, reflecting the surprise of its discoverers that a current could be activated in this way). During the AP cycle of a repetitively firing neuron, hyperpolarisation into the range of activation of HCN channels occurs after AP firing, due to the after-hyperpolarisation brought about by potassium channels [67]. Activation of HCN channels causes the membrane to rebound, increasing the probability of subsequent AP firing. In this manner, Ih can modulate resting potential, interspike interval and the frequency of firing in electrically excitable cells.

Structure and topology of HCN channels.

Figure 1.
Structure and topology of HCN channels.

HCN1–4 subunits form homo- or heterotetramers in the cell membrane (top) and when opened allow the passage of Na+, the main inward current carrier at the normal neuronal resting potential of −60 mV, as well as K+ and to a lesser extent Ca2+ (not shown). Each subunit comprises six transmembrane α helices (S1–S6, bottom), with intracellular amino and carboxyl termini. The positively charged S4 helix constitutes the voltage sensor, whereas the pore is formed by a pore loop between the S5 and S6 domains. The carboxyl terminus contains a CNBD (green) which can interact with cAMP (red) to induce a conformational change that causes a positive shift in the range of membrane potentials over which the channel is activated, and so facilitates channel opening.

Figure 1.
Structure and topology of HCN channels.

HCN1–4 subunits form homo- or heterotetramers in the cell membrane (top) and when opened allow the passage of Na+, the main inward current carrier at the normal neuronal resting potential of −60 mV, as well as K+ and to a lesser extent Ca2+ (not shown). Each subunit comprises six transmembrane α helices (S1–S6, bottom), with intracellular amino and carboxyl termini. The positively charged S4 helix constitutes the voltage sensor, whereas the pore is formed by a pore loop between the S5 and S6 domains. The carboxyl terminus contains a CNBD (green) which can interact with cAMP (red) to induce a conformational change that causes a positive shift in the range of membrane potentials over which the channel is activated, and so facilitates channel opening.

A notable characteristic of Ih is that the range of activation can be shifted towards more positive potentials upon cAMP binding, allowing a greater inward current which in turn can depolarise the resting membrane potential (RMP) and promote higher spike firing frequencies (Figure 2). Modulation by cAMP led to the recognition of the first physiological roles of HCN channels in cardiac muscle, where HCN function in specialised pacemaking cells of the sinoatrial (SA) node underlies rhythmic activity [68]. Adrenergic agonists such as epinephrine and norepinephrine bind to G-protein-coupled β1-adrenoceptors that activate adenylyl cyclase to form cAMP. Binding of cAMP to HCN channels in the SA node potentiates HCN activity, leading to an increased cardiac AP firing frequency [69]. Conversely, a reduction in cAMP caused by the parasympathetic transmitter acetylcholine mediates slowing of the heart rate.

HCN2 activity in physiological vs. pathological pain states and the effect of channel block.

Figure 2.
HCN2 activity in physiological vs. pathological pain states and the effect of channel block.

Under normal physiological conditions, the CNBD (pink) of HCN2 channels is not liganded by cAMP (top) and so the channels pass little inward current at the neuronal RMP (RMP ≈ −60 mV, black trace on IV graph at left). Injected current, supplied when a painful stimulus (heat, cold and strong mechanical stimulus) activates nociceptor-specific ion channels, causes modest nociceptor firing and a mild sensation of pain (top right). Increased cAMP caused by inflammation or nerve injury gives enhanced occupancy of the CNBD by cAMP (middle), and a rightward shift in the voltage-dependence of HCN2 activation (red trace on IV graph). The increased inward current at the RMP adds to the current injected by a noxious stimulus, augmenting the nociceptor firing frequency and therefore enhancing the sensation of pain (middle right). The HCN inhibitor ivabradine (bottom) is a state-dependent HCN blocker that can gain access to its intracellular channel-blocking site only when the channel is in its open state. Binding of ivabradine results in a reduced influx of Na+, which attenuates the firing frequency and so reduces the sensation of pain (bottom right). Figure adapted from ref. [132].

Figure 2.
HCN2 activity in physiological vs. pathological pain states and the effect of channel block.

Under normal physiological conditions, the CNBD (pink) of HCN2 channels is not liganded by cAMP (top) and so the channels pass little inward current at the neuronal RMP (RMP ≈ −60 mV, black trace on IV graph at left). Injected current, supplied when a painful stimulus (heat, cold and strong mechanical stimulus) activates nociceptor-specific ion channels, causes modest nociceptor firing and a mild sensation of pain (top right). Increased cAMP caused by inflammation or nerve injury gives enhanced occupancy of the CNBD by cAMP (middle), and a rightward shift in the voltage-dependence of HCN2 activation (red trace on IV graph). The increased inward current at the RMP adds to the current injected by a noxious stimulus, augmenting the nociceptor firing frequency and therefore enhancing the sensation of pain (middle right). The HCN inhibitor ivabradine (bottom) is a state-dependent HCN blocker that can gain access to its intracellular channel-blocking site only when the channel is in its open state. Binding of ivabradine results in a reduced influx of Na+, which attenuates the firing frequency and so reduces the sensation of pain (bottom right). Figure adapted from ref. [132].

Direct binding of intracellular cAMP to HCN channels in the SA node potentiates HCN activity, leading to an increased cardiac AP firing frequency [69]. Conversely, a reduction in cAMP caused by the parasympathetic transmitter acetylcholine mediates slowing of the heart rate. Note that in this case cAMP has a direct action on the HCN ion channel and does not act via its traditional route of phosphorylation by protein kinase A.

The four members of the HCN family (named HCN1 through HCN4) were identified in the late 1990s and share approximately 60% sequence identity [70,71]. In common with the K+ channel family, all have six transmembrane α-helices with intracellular N- and C-terminals and can assemble in either homomeric or heteromeric tetrameric compositions (Figure 1). One distinguishing feature among the HCN channels is their activation/deactivation rate; HCN1 is activated/deactivated rapidly (time constant approximately 60 ms at the half-activation membrane voltage), whereas HCN2 and HCN3 have an intermediate time-course, and HCN4 exhibits slow kinetics (time constant approximately 400 ms) [7174]. A second important distinction is the cAMP sensitivity; whereas HCN2 and HCN4 are strongly modulated by cAMP, with a positive shift in the voltage of half-activation of around 15 mV when fully liganded by cAMP, HCN1 and HCN3 appear relatively insensitive to cAMP [75]. The shift in voltage activation is mediated via a direct interaction of cAMP with the cyclic nucleotide-binding domain (CNBD), a cAMP-binding motif in the C-terminal domain of HCN channels [75]. Note also that native channels assembling as heterotetramers exhibit a mixture of biophysical properties, contributing to the diversity of native Ih in distinct neurons [76]. Although the factors governing homomeric or heteromeric assembly in vivo are currently unknown, some data suggest that this process can be dynamically driven by neuronal activity [77,78].

Modulation of cardiac activity by cAMP is attributed mainly to HCN4, the principal isoform expressed in the SA node [79]. Eliminating the HCN4-driven component causes an 80% reduction of cardiac If [80,81], while deletion of the other cAMP-sensitive subunit, HCN2, diminishes current by only 20% [82]. In accordance with this, cardiac deletion of the gene encoding HCN4, but not HCN2, leads to marked bradycardia in mice [81]. Interestingly, recent data suggest that HCN1 co-localises with HCN4 in the SA node and may also play a role in determination of the heart rate, though in view of the cAMP insensitivity of HCN1, this isoform is unlikely to contribute to adrenergic modulation of the heart rate [83].

HCN channels in the nervous system

HCN ion channels have important roles in the CNS, where they influence processes such as setting the neuronal baseline excitability, modulating dendritic integration and fine-tuning synaptic strength [8486]. In the brain, HCN1 and HCN2 are found in cerebral, hippocampal and cerebellar cortices, with HCN2 showing a near-ubiquitous expression [87,88]. HCN1 and HCN2 dysfunction in the CNS is associated with aberrant excitability and epilepsy in humans, underscoring the vital importance of these subunits in regulating network activity [89]. In contrast, the expression of HCN3 and HCN4 in the CNS appears less abundant. HCN3 is detected at low levels in the olfactory bulb and some areas of the hypothalamus, whereas HCN4 is found in restricted brain areas such as the nucleus of the lateral olfactory tract and thalamus [87,88]. Interestingly, Ih currents have also been reported in reactive brain astrocytes as well as pain-processing neurons in the spinal cord [9092]. This review will focus on the role of HCN channels in the PNS; for more information on CNS functions, including supraspinal pain pathways, readers are referred to other recent studies [85,86,9396].

Several studies have investigated HCN expression in sensory neurons of the PNS, often reporting divergent results owing to differences among species and developmental stages, as well as to suboptimal specificity of antibodies raised against the distinct isoforms [97]. However, combining these data with functional in vitro assays has allowed a more confident assessment of HCN channel distribution in sensory neuron subpopulations. Thus, HCN1 is reported to be predominantly expressed in large myelinated A-fibre neurons [98100], consistent with their fast and relatively cAMP-insensitive Ih which is absent in HCN1 knockout (KO) mice [101]. The most abundant isoform in small nociceptive neurons is HCN2, where it underlies a slow cAMP-sensitive Ih [64]. HCN2 has also been detected in myelinated fibres, including nociceptive Aδ fibres [102105]. Following HCN2 genetic deletion, a cAMP-insensitive, slowly-relaxing Ih remains in small neurons, and this can be attributed to HCN3, whose expression is widespread in DRG neurons [98,105,106, Lainez, in preparation]. Finally, the HCN4 isoform appears to be absent or expressed at low levels in sensory neurons [98,105,108].

Recent data indicate that HCN channels in the periphery play a vital role in regulating nociceptive excitability. The cAMP-sensitive HCN2 isoform in particular appears to exert a ‘pacemaking’ function in nociceptive neurons, in an analogous way to that of HCN4 in the heart.

The HCN2 ion channel — a novel peripheral target for neuropathic pain?

Preclinical evaluation of candidate drugs for neuropathic pain is conducted using animal models, in which a peripheral nerve (usually the sciatic nerve, which innervates the hind paw) is partially injured, in an attempt to reproduce the partial nerve injury that is a common cause of neuropathic pain in humans. Widely used examples include the Bennett and Xie chronic constriction injury model, in which a loose constriction of the sciatic nerve causes a partial interruption of afferent nerve fibres [109]; the Chung model, in which one or two of the three nerve roots making up the sciatic nerve are tightly tied with ligatures [110]; or the Seltzer model, in which a ligature interrupts around half of the nerve fibres in the sciatic nerve [111]. In each of these models, changes in the hind paw withdrawal thresholds — hypersensitivity — in response to heat, cold or mechanical stimuli can be readily measured. There are some fine-print differences between these models, but there is general agreement that they broadly reproduce human neuropathic pain caused by traumatic nerve injury. What is less clear is how well they reproduce other common causes of human neuropathic pain, such as PDN or PHN. Defects in how well these models reproduce human neuropathic pain conditions may explain why novel analgesics which work well in preclinical (i.e. animal) models often fail to translate their efficacy to humans.

The first indications of HCN channel involvement in neuropathic pain modulation came from experiments showing that the pan-HCN blocker ZD-7288 was able to reverse mechanical and thermal hypersensitivity, as well as spontaneous pain, in a variety of peripheral nerve injury models [98,112114]. Although off-target effects of ZD-7288 on other ion channels have been reported, including sodium and calcium channels [115117], the analgesic effect of Ih inhibition was reproduced using the more specific HCN blocker ivabradine, which was found to reverse neuropathic hypersensitivity as efficiently as the ‘gold standard’ analgesic gabapentin [65,118].

As discussed earlier, the majority of human pain syndromes are not caused by direct nerve trauma; in fact, clinical trials for analgesics are almost exclusively conducted on patients with non-traumatic neuropathies. It is therefore imperative to test candidate drugs in animal pain models that reproduce as much as possible other aetiologies of human chronic pain. Intriguingly, the evidence so far suggests that HCN inhibition may also be efficacious in such clinically relevant neuropathies. For instance, patients with cancer who are undergoing chemotherapy often develop a persisting peripheral neuropathy manifesting as hypersensitivity to mechanical and cold stimuli, and this phenotype can be recapitulated in animals via systematic treatment with the antineoplastic agent oxaliplatin [119]. A single administration of ivabradine can completely reverse both mechanical and cold allodynia induced by oxaliplatin at least as effectively as gabapentin [65,120]. Pharmacological HCN inhibition also attenuates chronic visceral pain [121] and mechanical hypersensitivity associated with PDN, the most common human pain syndrome [Tsantoulas, in preparation]. Put together, the evidence points towards a fundamental role of HCN function in a plethora of neuropathic pain states in humans. Furthermore, it provides a rationale for investigating the effectiveness of HCN block in other prevalent neuropathic pain syndromes, such as PHN and HIV-associated neuropathy.

HCN2 is the culprit

Although the analgesia produced by existing HCN blockers is robust and wide-ranging across several animal pain models, their clinical use is hampered by the induced bradycardia due to inhibition of HCN4-dependent pacemaking activity in the heart [65,118]. To circumvent this, it is imperative to clarify which HCN isoform(s) confer the analgesic effect and to selectively target those responsible. Existing pharmacological Ih blockers do not allow dissection of the relative involvement of distinct HCN subunits, because all four isoforms are blocked with approximately equal potency by available inhibitors [65,123,124]. However, an insight into the responsible isoform(s) can be acquired via genetic deletion of specific isoforms and examination of the ensuing pain phenotypes. Global deletion of HCN1 has no effect on mechanical and thermal pain modalities following nerve injury, but it does cause some reduction of pain caused by mild cold stimuli [101]. This suggests some involvement of HCN1 in cold allodynia and ties in with detection of this subunit in large neurons and in a restricted population of cold-sensitive small neurons [98101,125]. Characterisation of global HCN3 KO mice proved less exciting, as these mice develop both inflammatory and neuropathic pain normally, implying that HCN3 does not play a significant role in chronic pain states [107]. The relevance of HCN4 in pain has been more challenging to pin down due its vital role in heart physiology, illustrated by severe bradycardia, cardiac arrest and death following inducible global or cardiac-specific gene deletion [80,81]. A neuron-specific HCN4 KO mice may evade these difficulties, but has not been generated so far. Nevertheless, given its limited expression in the PNS (see above), HCN4 seems unlikely to play a significant role in pain signalling.

When HCN2 is deleted globally, mice display signs of epilepsy and ataxia, an effect attributable to HCN2 deficiency in the CNS, leading to premature death at around 4 weeks, before pain phenotypes can be properly evaluated [82]. Further investigation of the role of HCN2 in nociception was made feasible by deleting the gene in peripheral neurons only. The sodium channel gene NaV1.8 is expressed only in small somatosensory neurons [126], and using a Nav1.8-specific promoter to drive recombination via a Cre-lox system [127], HCN2 was selectively ablated in nociceptive neurons. Strikingly, assessment of pain behaviours revealed that these mice exhibited attenuated mechanical, heat or cold pain following nerve injury [64]. Moreover, HCN2 deletion from nociceptors reduced inflammatory pain [64,128] and completely precludes the development of mechanical allodynia in diabetic neuropathy [122]. Put together, these data suggest that the potent analgesia produced by pharmacological HCN inhibition is principally mediated by the HCN2 isoform, thus appointing this isoform with a central role in pain hypersensitivity across divergent neuropathic states.

What is the trail linking HCN2 activity to abnormal pain sensations? A first clue is provided by the observation that HCN2 inhibition does not affect pain processing under normal conditions. Thus, Nav1.8-HCN2-targeted KO mice exhibit acute pain thresholds indistinguishable from their control littermates when challenged with noxious mechanical, heat, cold or chemical stimuli [64]. Identical conclusions are reached when Ih is blocked with ZD-7288 or ivabradine, which also leave acute pain unaltered [65,112]. Thus, HCN2 ion channels do not contribute to the sensation of acute pain under normal (non-pathological) conditions, an advantage which is likely to be useful in developing HCN2-selective blockers as novel analgesics. As discussed above, one of the hallmarks of neuropathic pain is the development of aberrant spontaneous activity in peripheral nerves [30,129131]. Blocking this ongoing activity prevents the onset of neuropathic pain, and it therefore seems likely that nerve damage causing neuropathic pain can sensitise HCN2 activity in peripheral nerves and consequently promote spontaneous firing. Direct evidence for this hypothesis was provided by Sun et al. [114], who used in vivo teased fibre recordings to illustrate that inhibiting Ih with local application of ZD-7288 supresses the spontaneous firing that develops in DRG neurons following injury.

How does augmented HCN2 activity enhance nociceptive firing?

An elevation in intracellular cAMP causes a depolarising shift in the voltage-dependence of HCN2 activation and so allows nociceptor-expressed HCN2 channels to be more easily activated at rest. The increased inward Ih leads to a small membrane depolarisation and therefore to a lower threshold for the initiation of spontaneous spikes, and can also facilitate higher firing rates in the presence of neuronal activity [132] (Figure 2). In agreement with this, ivabradine reduces cAMP-induced neuronal firing in small nociceptors, and genetic deletion of HCN2 abolishes both the enhancement of firing caused by an elevation of cAMP and the inhibitory effect of HCN2 block on firing frequency [64,65]. A cAMP elevation is well documented in pain associated with inflammation; thus, PGE2 and other inflammatory mediators activate G-protein-coupled receptors coupled with Gs, leading to engagement of adenylate cyclase and cAMP production [133]. PGE2 injection into the mouse paw induces pain that can be reversed by the HCN2 blocker ZD-7288 [128], whereas Nav1.8-HCN2 KO mice do not exhibit pain hypersensitivity following intraplantar administration of PGE2 [64] or a cAMP analogue [128]. These experiments suggest that HCN2 is the major downstream mediator of PGE2 and therefore is the final target of the NSAID analgesic family, which includes many common analgesics such as aspirin and ibuprofen.

Given the substantial evidence for an involvement of HCN2 in neuropathic pain, at least in animal models (see above), which extracellular mediators might be responsible for causing an elevation in cAMP and so an enhancement in HCN2 activation? A body of evidence indicates that PGE2 signalling may contribute to neuropathic pain as well as to inflammatory pain. After nerve injury, PGE2 and its corresponding receptors EP1 and EP4 are up-regulated in the nerve and DRG, respectively, and blocking this signalling pathway relieves pain symptoms [134]. Accordingly, mice that lack membrane-associated PGE synthase, an enzyme required for PGE2 synthesis, show normal acute pain but diminished neuropathic pain [135]. Genetic deletion of the enzyme catalysing cAMP synthesis downstream of PGE2 also lessens neuropathic pain following nerve injury [136].

Some data suggest that non-traumatic neuropathic pain syndromes such as PDN may also be associated with a pro-inflammatory phenotype involving the PGE2/cAMP/HCN2 pathway. For instance, cAMP signalling is potentiated in peripheral terminals of diabetic rodents [137], whereas levels of cyclooxygenase-2 (COX-2), which is upstream of PGE2, are increased in diabetic DRG [138] and sciatic nerve [139]. Accordingly, reducing cAMP via genetic or pharmacological COX-2 inhibition lowers pain following diabetes induction [140142]. Further confidence in the translational value of these results stems from the finding that a similar induction of inflammatory mediators is present in diabetic patients [143]. Consistent with a role of cAMP-to-HCN2 signalling, conditional HCN2 deletion in nociceptive neurons precludes the development of PDN [122]. Put together, the data suggest that a pro-inflammatory phenotype may be a causal event driving neuropathic pain of diverse origin via potentiation of HCN2 activity in nociceptive neurons. Despite these data from animal studies suggesting a role for PGE2 in driving HCN2 activity in neuropathic pain, however, the evidence for an important role of PGE2 in human neuropathic pain is not strong and NSAIDs are seldom recommended for neuropathic pain in clinical practice [144]. A possible way of reconciling the compelling evidence for a role of HCN2 in neuropathic pain with the relative lack of efficacy of NSAIDs is to postulate that other extracellular mediators may drive an elevation of cAMP and so an enhancement of HCN2 function in neuropathic pain, but the identity of these mediators currently remains mysterious.

Another possible cause of enhanced HCN2 activity, especially at later time points, is via a transcriptional up-regulation of the channel. The result of a nociceptive membrane more densely packed with HCN2 channels would be an increased net inward current upon HCN2 activation during the AP cycle, favouring higher rates of firing. In inflammatory pain models, HCN2 up-regulation in cell bodies and terminals of nociceptive neurons has been shown to occur as early as 24 h following induction of inflammation [103,104,106,128,145,146], in tandem with an increase in Ih and C-fibre hyperexcitability [106,147]. Most studies in neuropathic pain models, however, report either no change or a reduction of HCN2 expression in the DRG soma, where the majority of spontaneous discharge appears to originate [27,98,99]. Interestingly, HCN2 channels appear to accumulate at the injury site, a known locus of ectopic impulse generation, in a similar fashion to sodium channels [99,148]. This raises the possibility that at least in traumatic neuropathies, augmented HCN2 expression at a neuroma could contribute to local hyperexcitability and spontaneous firing, and thus promote neuropathic pain.

Clinical development of HCN2-based analgesics

Ivabradine

Ivabradine is a broad-spectrum HCN inhibitor, clinically approved for the reduction of angina, which it achieves by inhibiting cardiac pacemaker HCN channels (notably HCN4), thus lowering the heart rate and reducing the cardiac metabolic demand. Ivabradine shows a lack of off-target effects and consistent antinociceptive efficacy in several chronic pain models [65,118]. Ivabradine is a substrate for P-glycoprotein (PgP), an ATP-dependent drug transporter found in the luminal membrane of the brain capillary endothelial cells that make up the blood–brain barrier [149]. Active efflux of ivabradine via PgP means that ivabradine levels are low in the CNS and therefore the observed analgesia must arise via the block of peripheral HCN channels. This is a significant finding, because it maps the pain-relevant HCN function to sensory neurons and dissociates it from other HCN loci along the neuraxis. Given the involvement of HCN2 in neuropathic pain (see above), the mechanism of action of ivabradine in peripheral neurons most likely involves inhibition of HCN2 channels in small nociceptive C-fibres [64,65] (Figure 2).

An important point is that ivabradine is effective as an analgesic even long after induction of pain in several models of neuropathic pain. Together with the fact that ivabradine is peripherally restricted, this observation provides a strong argument against the idea of a central ‘pain memory’ that can maintain pain hypersensitivity in the absence of peripheral mechanisms [65,118]. Central sensitisation must therefore be a process that is dynamically dependent on ongoing peripheral activity in nociceptive C-fibre afferents. Removing this peripheral nociceptive drive by inhibiting HCN2 is able to rapidly alleviate pain symptoms, even long after establishment of a neuropathic pain state. These observations give hope that a peripherally restricted HCN2 blocker will have efficacy as an analgesic even in long-established neuropathic pain, and moreover that the unpleasant psychotropic effects of currently prescribed analgesics for neuropathic pain will be absent, because analgesia will be possible without CNS penetration.

Another interesting property of ivabradine is that it can only gain access to the intracellular HCN-binding site when the channel pore is opened by hyperpolarisation [150]. This use-dependency may amplify the efficacy of ivabradine when the membrane is hyperpolarised following an AP and HCN channels are opened, and may explain the significant analgesic effect in neuropathic pain where ongoing firing is present. Consistent with this, potent suppression of cAMP-induced spontaneous activity by ivabradine has been demonstrated in vitro [65]. Intriguingly, ivabradine also appears to have a cumulative analgesic effect on repeated application, although the underlying mechanism is not clear at present [118].

Despite the consistent antinociceptive effect of ivabradine in preclinical models of chronic pain, its use for human pain pharmacotherapy is hindered by the lack of selectivity among the different HCN isoforms. As noted above, blocking the HCN4-dependent pacemaking activity in the heart induces significant bradycardia (up to 40% reduction in heart rate) in rodents [65,80,81,118]. This effect is present in humans, as evidenced by the bradycardic action of ivabradine and HCN4 mutations leading to idiosyncratic bradycardia [151154]. Ivabradine-induced bradycardia is a safety concern in patients with a normal heart rate, especially since there appears to be no therapeutic window between the analgesic and bradycardic effects of the drug (Figure 3) [65]. This probably precludes the use of ivabradine as an analgesic in the clinic, necessitating the development of alternative compounds with greater selectivity for HCN2 over HCN4.

The effect of ivabradine on heart rate and pain behaviour in mice.

Figure 3.
The effect of ivabradine on heart rate and pain behaviour in mice.

There is no clinically significant difference between the doses of ivabradine shown to reduce the heart rate and pain behaviours by 50% of maximum (EC50) in mice. The effect of ivabradine on the heart rate was recorded 30 min after administration, and this was compared to the effect of ivabradine on pain behaviours displayed 15–20 min after injection of the inflammatory agent formalin into the hind paw. Plotting dose–response curves for both effects on the same axis demonstrates the EC50 values to be similar, with a dose of 1.8 mg/kg required to suppress pain behaviours by 50% and a dose of 2.7 mg/kg causing a decrease in heart rate of 50%.

Figure 3.
The effect of ivabradine on heart rate and pain behaviour in mice.

There is no clinically significant difference between the doses of ivabradine shown to reduce the heart rate and pain behaviours by 50% of maximum (EC50) in mice. The effect of ivabradine on the heart rate was recorded 30 min after administration, and this was compared to the effect of ivabradine on pain behaviours displayed 15–20 min after injection of the inflammatory agent formalin into the hind paw. Plotting dose–response curves for both effects on the same axis demonstrates the EC50 values to be similar, with a dose of 1.8 mg/kg required to suppress pain behaviours by 50% and a dose of 2.7 mg/kg causing a decrease in heart rate of 50%.

Small-molecule inhibitors

Ivabradine could serve as a lead structure for the development of superior small-molecule analgesics with a more favourable safety profile. As discussed above, HCN2 is the crucial isoform for analgesia; therefore, a compound that is selective for HCN2 over HCN4 is predicted to produce analgesia of the same magnitude as ivabradine, but crucially, should be devoid of bradycardic side effects, because HCN2 is not highly expressed in pacemaker regions of the heart. Selectivity over HCN1 may also be a desirable goal, because inhibition of this channel in retinal cells is responsible for the alterations in visual sensation in a minority of patients following ivabradine treatment [155]. In addition, recent data suggest that HCN1 may also play an important stabilising role in cardiac pacemaking, as evidenced by a severely reduced cardiac output in HCN1-deficient mice [83]. Derivatives based on the ivabradine backbone could perhaps achieve the required subtype specificity [156]. Additional agents that have been found to inhibit Ih and could be strategically used in drug design are loperamide, clonidine, nicotine and even some general anaesthetics [157162].

A blocker targeting HCN2 needs to be peripherally restricted, because HCN2 function in the brain is fundamental for neuronal homeostasis. The detrimental effects of HCN2 dysfunction in the CNS are illustrated by the occurrence of epileptic seizures in HCN2-deficient mice [82,163], as well as in humans with HCN2 loss-of-function mutations [164,165]. Therefore, a HCN2 inhibitor that is a PgP substrate, or can be chemically altered to acquire such characteristics, is essential.

RNA interference

Gene silencing holds promise as an alternative avenue for reducing HCN2 activity in neurons. RNA interference (RNAi) therapeutics comprise small-interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) and microRNA precursors, with siRNA-based methods already under clinical trial assessment for other targets [166]. The chief advantage of RNAi is that it allows any gene to be selectively targeted; indeed, so far there has not been any account of failing to inhibit a molecular target of interest with siRNA in vitro. It may therefore be relatively straightforward to design siRNAs that exclusively target HCN2, but are devoid of any effect on other isoforms.

One consideration for siRNA application in whole organisms is that the knockdown is often modest. Although this may be a favourable quality in cases where preserving a baseline of activity is essential, repeated and prolonged administration may be required to observe a therapeutic outcome in cases where a large knockdown is needed to produce a therapeutic effect. Systemic delivery of naked siRNAs, sometimes in combination with a transfection reagent, has already been successfully used in animals to target ion channels and receptors involved in nociception. Daily intrathecal infusion of siRNAs against P2X3, a pain-related receptor found in a subset of nociceptive neurons, relieved neuropathic pain induced by partial nerve ligation [167]. Conversely, a pain phenotype was produced by experimental down-regulation of the potassium channel subunit Kv9.1 using intrathecal siRNAs [168], while Luo et al. [169] used a similar regime to knockdown the δ-opioid receptor and thus block opioid agonist-induced antinociception. The RNAi effect in these studies was sustained during siRNA administration, but subsided rapidly after cessation of treatment. The need for continuous delivery through an invasive method (intrathecal or intravenous) makes siRNA-based RNAi suboptimal for clinical use at present.

Longer lasting RNAi-based down-regulation of HCN2 in the nervous system can be achieved through viral delivery of shRNA, a method that has been used for a number of other neurological disorders such as Huntington's chorea and Alzheimer's disease using adeno-associated virus (AAV) and lentivirus [170172]. Viral delivery also has the potential for more targeted treatments; intrathecal administration of siRNAs targeting the DRG may lead to widespread siRNA dispersal, triggering unwanted side effects due to HCN2 inhibition in the CNS. This could be circumvented by using serotype-specific viruses with natural tropism towards DRG neurons, namely AA5, AAV9 and AAV2/3. Sensory neuron knockdown utilising AAVs has been achieved for transient receptor potential cation channel subfamily V member 1 (TRPV1) [173] and Nav1.3 [174,175], and this was coupled to analgesia in chronic pain models. Despite the recent success of virally mediated RNAi in animals, consistency and predictability of expression levels, as well as ability to temporally regulate expression, are still major concerns for translation to the clinic.

Monoclonal antibodies

The use of humanised monoclonal antibodies (mAbs) to fight disease has a strong precedent, including therapy for inflammatory [176] and neuropathic pain [177,178]. All mAbs are high-molecular-weight immunoglobulins (typically of the IgG class) with two heavy and two light chains with variable domains that bind to antigens and Fc constant domains with effector functions. They can be engineered to interact with particular antigens with very high affinity, and thus have the potential to target ion channels such as HCN2 with exquisite selectivity. This virtue of mAbs provides a significant advantage in drug discovery, because it is theoretically possible to achieve specificity even among highly homologous isoforms, such as those comprising the HCN family. Another important feature of mAbs is that their distribution is peripherally restricted, which as discussed is essential in avoiding HCN2-associated side effects due to penetration in the CNS. Moreover, mAbs exhibit minimal cytotoxicity and a long plasma half-life (typically several weeks), which allows infrequent administration [179]. Finally, mAb effector functions can be fine-tuned by protein engineering to tailor functional attributes, including reducing cytotoxicity and modulating clearance rate.

A main hurdle in developing mAbs for ion channels is associated with technical difficulties in purifying these large proteins in an appropriate format for antibody drug discovery. Another complication is that mAbs are too big to cross the membrane, which necessitates an extracellular interaction. Although extracellular regions in some ligand-gated ion channels such as P2X7 and TRPA1 have been successfully utilised for generation of functional mAbs [180,181], voltage-gated ion channels like HCN2 are more challenging, because their small extracellular loops are less amenable to this approach. Nevertheless, functional mAbs have already been produced for some voltage-gated ion channels such as Nav1.7 [182] and Eag-1 [183]. The Nav1.7 mAb, in particular, is promising as it was shown to be analgesic in preclinical studies [182]. In summary, it is likely that developments in biochemical methodology and structural understanding will render HCN2 mAb therapies a realistic goal in the foreseeable future.

Conclusion

Despite the recognition of the instrumental role of HCN channels in cardiac pacemaking more than a decade ago, it was only recently recognised that these channels carry out analogous modulatory tasks in the nervous system. As a result, we now have a significant conceptual and experimental grasp on the impact of HCN channels in neuronal physiology and disease pathology. Several lines of evidence point to peripherally expressed HCN2 as a protagonist in the regulation of nociceptive excitability, offering a fresh perspective in neuropathic pain research. Developing second generation agents to selectively block HCN2 activity in the PNS holds great potential for robust and clinically safe analgesia in chronic pain of diverse aetiologies, including those arising from nerve injury, cancer treatment and diabetic complications.

Abbreviations

AAV, adeno-associated virus; AP, action potential; CNBD, cyclic nucleotide-binding domain; CNS, central nervous system; COX, cyclooxygenase; DRG, dorsal root ganglion; HCN, hyperpolarisation-activated cyclic nucleotide-gated channel; IL, interleukin; KO, knockout; LTP, long-term potentiation; mAb, monoclonal antibody; NMDA, N-methyl-d-aspartate; NNT, number needed to treat; NSAIDs, non-steroid anti-inflammatory drugs; PDN, painful diabetic neuropathy; PGE2, prostaglandin E2; PgP, P-glycoprotein; PHN, post-herpetic neuralgia; PNS, peripheral nervous system; RNAi, RNA interference; RMP, resting membrane potential; SA, sinoatrial; shRNA, short hairpin RNA; siRNA, small-interfering RNA; SNRIs, serotonin and noradrenaline reuptake inhibitors; TCAs, tricyclic antidepressants; TG, trigeminal ganglion.

Author contribution

C.T., E.R.M. and P.A.M. wrote the manuscript.

Funding

This work was supported by the Medical Research Council [MR/J013129/1].

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Sherrington
,
C.S
. (
1906
)
The Integrative Action of the Nervous System
,
Scribner
,
New York
2
Bessou
,
P.
and
Perl
,
E.R.
(
1969
)
Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli
.
J. Neurophysiol.
32
,
1025
1043
PMID:
[PubMed]
3
Wall
,
P.D.
and
Dubner
,
R.
(
1972
)
Somatosensory pathways
.
Annu. Rev. Physiol.
34
,
315
336
doi:
4
Zotterman
,
Y.
(
1939
)
Touch, pain and tickling: an electro-physiological investigation on cutaneous sensory nerves
.
J. Physiol.
95
,
1
28
doi:
5
Cox
,
J.J.
,
Reimann
,
F.
,
Nicholas
,
A.K.
,
Thornton
,
G.
,
Roberts
,
E.
,
Springell
,
K.
et al. 
(
2006
)
An SCN9A channelopathy causes congenital inability to experience pain
.
Nature
444
,
894
898
doi:
6
Fischer
,
B.
,
Gooch
,
J.
,
Goldman
,
B.
,
Kurdyak
,
P.
and
Rehm
,
J.
(
2014
)
Non-medical prescription opioid use, prescription opioid-related harms and public health in Canada: an update 5 years later
.
Can. J. Public Health
105
,
e146
e149
PMID:
[PubMed]
7
Gwira Baumblatt
,
J.A.
,
Wiedeman
,
C.
,
Dunn
,
J.R.
,
Schaffner
,
W.
,
Paulozzi
,
L.J.
and
Jones
,
T.F.
(
2014
)
High-risk use by patients prescribed opioids for pain and its role in overdose deaths
.
JAMA Intern. Med.
174
,
796
doi:
8
Zedler
,
B.
,
Xie
,
L.
,
Wang
,
L.
,
Joyce
,
A.
,
Vick
,
C.
,
Kariburyo
,
F.
et al. 
(
2014
)
Risk factors for serious prescription opioid-related toxicity or overdose among Veterans Health Administration patients
.
Pain Med.
15
,
1911
1929
doi:
9
Schmader
,
K.E.
(
2002
)
Epidemiology and impact on quality of life of postherpetic neuralgia and painful diabetic neuropathy
.
Clin. J. Pain
18
,
350
354
doi:
10
Dworkin
,
R.H.
,
O'Connor
,
A.B.
,
Backonja
,
M.
,
Farrar
,
J.T.
,
Finnerup
,
N.B.
,
Jensen
,
T.S.
et al. 
(
2007
)
Pharmacologic management of neuropathic pain: evidence-based recommendations
.
Pain
132
,
237
251
doi:
11
Finnerup
,
N.B.
,
Attal
,
N.
,
Haroutounian
,
S.
,
McNicol
,
E.
,
Baron
,
R.
,
Dworkin
,
R.H.
et al. 
(
2015
)
Pharmacotherapy for neuropathic pain in adults: a systematic review and meta-analysis
.
Lancet Neurol.
14
,
162
173
doi:
13
Bjarnason
,
I.
(
2013
)
Gastrointestinal safety of NSAIDs and over-the-counter analgesics
.
Int. J. Clin. Pract.
,
67
,
37
42
doi:
14
Salvo
,
F.
,
Antoniazzi
,
S.
,
Duong
,
M.
,
Molimard
,
M.
,
Bazin
,
F.
,
Fourrier-Réglat
,
A.
et al. 
(
2014
)
Cardiovascular events associated with the long-term use of NSAIDs: a review of randomized controlled trials and observational studies
.
Expert Opin. Drug Saf.
13
,
573
585
doi:
15
NICE
.
Non-steroidal anti-inflammatory drugs
.
16
Vo
,
T.
,
Rice
,
A.S.
and
Dworkin
,
R.H.
(
2009
)
Non-steroidal anti-inflammatory drugs for neuropathic pain: how do we explain continued widespread use?
Pain
143
,
169
171
doi:
17
Basbaum
,
A.J.T.
(
2000
) The perception of pain. In
Principles of Neural Science
,
4th edn
(
Eric
R. Kandel
,
James
H. Schwartz
,
Thomas
M. Jessell
, eds), pp.
472
92
,
McGraw-Hill
,
USA
18
Pasternak
,
G.W.
(
2014
)
Opiate pharmacology and relief of pain
.
J. Clin. Oncol.
32
,
1655
1661
doi:
19
World Health Organisation
(
1996
)
20
Attal
,
N.
and
Finnerup
,
N.B.
(
2010
)
Pharmacological management of neuropathic pain
.
Pain Clin. Updat.
18
,
1
8
. http://iasp.files.cms-plus.com/Content/ContentFolders/Publications2/PainClinicalUpdates/Archives/PCU_18-9_final_1390260608342_7.pdf
21
Dworkin
,
R.H.
,
O'Connor
,
A.B.
,
Audette
,
J.
,
Baron
,
R.
,
Gourlay
,
G.K.
,
Haanpää
,
M.L.
et al. 
(
2010
)
Recommendations for the pharmacological management of neuropathic pain: an overview and literature update
.
Mayo Clin. Proc.
85
(
3 Suppl
),
S3
S14
doi:
22
Attal
,
N.
,
Bouhassira
,
D.
,
Gautron
,
M.
,
Vaillant
,
J.N.
,
Mitry
,
E.
,
Lepère
,
C.
et al. 
(
2009
)
Thermal hyperalgesia as a marker of oxaliplatin neurotoxicity: a prospective quantified sensory assessment study
.
Pain
144
,
245
252
doi:
23
Mika
,
J.
,
Zychowska
,
M.
,
Makuch
,
W.
,
Rojewska
,
E.
and
Przewlocka
,
B.
(
2013
)
Neuronal and immunological basis of action of antidepressants in chronic pain — clinical and experimental studies
.
Pharmacol. Rep.
65
,
1611
1621
doi:
24
NICE
.
Neuropathic pain in adults: pharmacological management in non-specialist settings
.
25
Anand
,
P.
and
Bley
,
K.
(
2011
)
Topical capsaicin for pain management: therapeutic potential and mechanisms of action of the new high-concentration capsaicin 8% patch
.
Br. J. Anaesth.
107
,
490
502
doi:
27
Kajander
,
K.C.
,
Wakisaka
,
S.
and
Bennett
,
G.J.
(
1992
)
Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat
.
Neurosci. Lett.
138
,
225
228
doi:
28
Liu
,
C.N.
,
Wall
,
P.D.
,
Ben-Dor
,
E.
,
Michaelis
,
M.
,
Amir
,
R.
and
Devor
,
M.
(
2000
)
Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury
.
Pain
85
,
503
521
doi:
29
Serra
,
J.
,
Bostock
,
H.
and
Navarro
,
X.
(
2010
)
Microneurography in rats: a minimally invasive method to record single C-fiber action potentials from peripheral nerves in vivo
.
Neurosci. Lett.
470
,
168
174
doi:
30
Serra
,
J.
,
Bostock
,
H.
,
Solà
,
R.
,
Aleu
,
J.
,
García
,
E.
,
Cokic
,
B.
et al. 
(
2012
)
Microneurographic identification of spontaneous activity in C-nociceptors in neuropathic pain states in humans and rats
.
Pain
153
,
42
55
doi:
31
Sun
,
Q.
,
Tu
,
H.
,
Xing
,
G.G.
,
Han
,
J.S.
and
Wan
,
Y.
(
2005
)
Ectopic discharges from injured nerve fibers are highly correlated with tactile allodynia only in early, but not late, stage in rats with spinal nerve ligation
.
Exp. Neurol.
191
,
128
136
doi:
32
Orstavik
,
K.
,
Namer
,
B.
,
Schmidt
,
R.
,
Schmelz
,
M.
,
Hilliges
,
M.
,
Weidner
,
C.
et al. 
(
2006
)
Abnormal function of C-fibers in patients with diabetic neuropathy
.
J. Neurosci.
26
,
11287
11294
doi:
33
Orstavik
,
K.
(
2003
)
Pathological C-fibres in patients with a chronic painful condition
.
Brain
126 (Pt 3)
,
567
–578| doi:
34
Sheen
,
K.
and
Chung
,
J.M.
(
1993
)
Signs of neuropathic pain depend on signals from injured nerve fibers in a rat model
.
Brain Res.
610
,
62
68
doi:
35
Yoon
,
Y.W.
,
Na
,
H.S.
and
Chung
,
J.M.
(
1996
)
Contributions of injured and intact afferents to neuropathic pain in an experimental rat model
.
Pain
64
,
27
36
doi:
36
Sukhotinsky
,
I.
,
Ben-Dor
,
E.
,
Raber
,
P.
and
Devor
,
M.
(
2004
)
Key role of the dorsal root ganglion in neuropathic tactile hypersensibility
.
Eur. J. Pain
8
,
135
143
doi:
37
Gracely
,
R.H.
,
Lynch
,
S.A.
and
Bennett
,
G.J.
(
1992
)
Painful neuropathy: altered central processing maintained dynamically by peripheral input
.
Pain
51
,
175
194
doi:
38
Woolf
,
C.J.
(
1983
)
Evidence for a central component of post-injury pain hypersensitivity
.
Nature
306
,
686
688
doi:
39
Baron
,
R.
,
Hans
,
G.
and
Dickenson
,
A.H.
(
2013
)
Peripheral input and its importance for central sensitization
.
Ann. Neurol.
74
,
630
636
doi:
40
Woolf
,
C.J.
and
Mannion
,
R.J.
(
1999
)
Neuropathic pain: aetiology, symptoms, mechanisms, and management
.
Lancet
353
,
1959
1964
doi:
41
Raja
,
S.N.
,
Campbell
,
J.N.
and
Meyer
,
R.A.
(
1984
)
Evidence for different mechanisms of primary and secondary hyperalgesia following heat injury to the glabrous skin
.
Brain
107
1179
1188
doi:
42
Campbell
,
J.N.
and
Meyer
,
R.A.
(
2006
)
Mechanisms of neuropathic pain
.
Neuron
52
,
77
92
doi:
43
Ji
,
R.R.
,
Kohno
,
T.
,
Moore
,
K.A.
and
Woolf
,
C.J.
(
2003
)
Central sensitization and LTP: do pain and memory share similar mechanisms?
Trends Neurosci.
26
,
696
705
doi:
44
Mendell
,
L.M.
(
1984
)
Modifiability of spinal synapses
.
Physiol. Rev.
64
,
260
324
PMID:
[PubMed]
45
Woolf
,
C.J.
and
Thompson
,
S.W.
(
1991
)
The induction and maintenance of central sensitization is dependent on N-methyl-d-aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states
.
Pain
44
,
293
299
doi:
46
Latremoliere
,
A.
and
Woolf
,
C.J.
(
2009
)
Central sensitization: a generator of pain hypersensitivity by central neural plasticity
.
J. Pain
10
,
895
926
doi:
47
Cook
,
A.J.
,
Woolf
,
C.J.
,
Wall
,
P.D.
and
McMahon
,
S.B.
(
1987
)
Dynamic receptive field plasticity in rat spinal cord dorsal horn following C-primary afferent input
.
Nature
325
,
151
153
doi:
48
Ji
,
R.R.
,
Baba
,
H.
,
Brenner
,
G.J.
and
Woolf
,
C.J.
(
1999
)
Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity
.
Nat. Neurosci.
2
,
1114
1119
doi:
49
Suzuki
,
R.
,
Morcuende
,
S.
,
Webber
,
M.
,
Hunt
,
S.P.
and
Dickenson
,
A.H.
(
2002
)
Superficial NK1-expressing neurons control spinal excitability through activation of descending pathways
.
Nat. Neurosci.
5
,
1319
1326
doi:
50
D'Mello
,
R.
and
Dickenson
,
A.H.
(
2008
)
Spinal cord mechanisms of pain
.
Br. J. Anaesth.
101
,
8
16
doi:
51
Old
,
E.A.
,
Clark
,
A.K.
and
Malcangio
,
M.
(
2015
)
The role of glia in the spinal cord in neuropathic and inflammatory pain
.
Handb. Exp. Pharmacol.
227
,
145
170
doi:
52
Scholz
,
J.
and
Woolf
,
C.J.
(
2007
)
The neuropathic pain triad: neurons, immune cells and glia
.
Nat. Neurosci.
10
,
1361
1368
doi:
53
Derry
,
S.
,
Sven-Rice
,
A.
,
Cole
,
P.
,
Tan
,
T.
and
Moore
,
R.A.
(
2013
)
Topical capsaicin (high concentration) for chronic neuropathic pain in adults
.
Cochrane Database Syst. Rev.
2
,
Cd007393
doi:
54
Dolphin
,
A.C.
(
2012
)
Calcium channel auxiliary α2δ and β subunits: trafficking and one step beyond
.
Nat. Rev. Neurosci.
13
,
664
doi:
55
Wiffen
,
P.J.
,
Derry
,
S.
,
Moore
,
R.A.
and
Kalso
,
E.A.
(
2014
)
Carbamazepine for chronic neuropathic pain and fibromyalgia in adults
.
Cochrane Database Syst. Rev.
4
,
Cd005451
doi:
56
Ochoa
,
J.L.
,
Campero
,
M.
,
Serra
,
J.
and
Bostock
,
H.
(
2005
)
Hyperexcitable polymodal and insensitive nociceptors in painful human neuropathy
.
Muscle Nerve
32
,
459
472
doi:
57
Kleggetveit
,
I.P.
and
Jørum
,
E.
(
2010
)
Large and small fiber dysfunction in peripheral nerve injuries with or without spontaneous pain
.
J. Pain
11
,
1305
1310
doi:
58
Kleggetveit
,
I.P.
,
Namer
,
B.
,
Schmidt
,
R.
,
Helås
,
T.
,
Rückel
,
M.
,
Ørstavik
,
K.
et al. 
(
2012
)
High spontaneous activity of C-nociceptors in painful polyneuropathy
.
Pain
153
,
2040
2047
doi:
59
Dib-Hajj
,
S.D.
,
Cummins
,
T.R.
,
Black
,
J.A.
and
Waxman
,
S.G.
(
2010
)
Sodium channels in normal and pathological pain
.
Annu. Rev. Neurosci.
33
,
325
347
doi:
60
Waxman
,
S.G.
and
Zamponi
,
G.W.
(
2014
)
Regulating excitability of peripheral afferents: emerging ion channel targets
.
Nat. Neurosci.
17
,
153
163
doi:
61
Busserolles
,
J.
,
Tsantoulas
,
C.
,
Eschalier
,
A.
and
López García
,
J.A.
(
2016
)
Potassium channels in neuropathic pain: advances, challenges, and emerging ideas
.
Pain
157
(
Suppl 1
),
S7
S14
doi:
62
Tsantoulas
,
C.
and
McMahon
,
S.B.
(
2014
)
Opening paths to novel analgesics: the role of potassium channels in chronic pain
.
Trends Neurosci.
37
,
146
158
doi:
63
Tsantoulas
,
C.
,
Zhu
,
L.
,
Yip
,
P.
,
Grist
,
J.
,
Michael
,
G.J.
and
McMahon
,
S.B.
(
2014
)
Kv2 dysfunction after peripheral axotomy enhances sensory neuron responsiveness to sustained input
.
Exp. Neurol.
251
,
115
126
doi:
64
Emery
,
E.C.
,
Young
,
G.T.
,
Berrocoso
,
E.M.
,
Chen
,
L.
and
McNaughton
,
P.A.
(
2011
)
HCN2 ion channels play a central role in inflammatory and neuropathic pain
.
Science
333
,
1462
1466
doi:
65
Young
,
G.T.
,
Emery
,
E.C.
,
Mooney
,
E.R.
,
Tsantoulas
,
C.
and
McNaughton
,
P.A.
(
2014
)
Inflammatory and neuropathic pain are rapidly suppressed by peripheral block of hyperpolarisation-activated cyclic nucleotide-gated ion channels
.
Pain
155
,
1708
1719
doi:
66
Biel
,
M.
,
Wahl-Schott
,
C.
,
Michalakis
,
S.
and
Zong
,
X.
(
2009
)
Hyperpolarization-activated cation channels: from genes to function
.
Physiol. Rev.
89
,
847
885
doi:
67
Tsantoulas
,
C.
(
2015
)
Emerging potassium channel targets for the treatment of pain
.
Curr. Opin. Support. Palliat. Care
9
,
147
154
doi:
68
Baruscotti
,
M.
,
Bucchi
,
A.
and
Difrancesco
,
D.
(
2005
)
Physiology and pharmacology of the cardiac pacemaker (‘funny’) current
.
Pharmacol. Ther.
107
,
59
79
doi:
69
DiFrancesco
,
D.
and
Tortora
,
P.
(
1991
)
Direct activation of cardiac pacemaker channels by intracellular cyclic AMP
.
Nature
351
,
145
147
doi:
70
Gauss
,
R.
,
Seifert
,
R.
and
Kaupp
,
U.B.
(
1998
)
Molecular identification of a hyperpolarization-activated channel in sea urchin sperm
.
Nature
393
,
583
587
doi:
71
Ludwig
,
A.
,
Zong
,
X.
,
Stieber
,
J.
,
Hullin
,
R.
,
Hofmann
,
F.
and
Biel
,
M.
(
1999
)
Two pacemaker channels from human heart with profoundly different activation kinetics
.
EMBO J.
18
,
2323
2329
doi:
72
Ishii
,
T.M.
,
Takano
,
M.
,
Xie
,
L.H.
,
Noma
,
A.
and
Ohmori
,
H.
(
1999
)
Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node
.
J. Biol. Chem.
274
,
12835
9
doi:
73
Seifert
,
R.
,
Scholten
,
A.
,
Gauss
,
R.
,
Mincheva
,
A.
,
Lichter
,
P.
and
Kaupp
,
U.B.
(
1999
)
Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis
.
Proc. Natl Acad. Sci. USA
96
,
9391
9396
doi:
74
Mistrik
,
P.
,
Mader
,
R.
,
Michalakis
,
S.
,
Weidinger
,
M.
,
Pfeifer
,
A.
and
Biel
,
M.
(
2005
)
The murine HCN3 gene encodes a hyperpolarization-activated cation channel with slow kinetics and unique response to cyclic nucleotides
.
J. Biol. Chem.
280
,
27056
27061
doi:
75
Wainger
,
B.J.
,
DeGennaro
,
M.
,
Santoro
,
B.
,
Siegelbaum
,
S.A.
and
Tibbs
,
G.R.
(
2001
)
Molecular mechanism of cAMP modulation of HCN pacemaker channels
.
Nature
411
,
805
810
doi:
76
Ulens
,
C.
and
Tytgat
,
J.
(
2001
)
Functional heteromerization of HCN1 and HCN2 pacemaker channels
.
J. Biol. Chem.
276
,
6069
6072
doi:
77
Brewster
,
A.L.
,
Bernard
,
J.A.
,
Gall
,
C.M.
and
Baram
,
T.Z.
(
2005
)
Formation of heteromeric hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the hippocampus is regulated by developmental seizures
.
Neurobiol. Dis.
19
,
200
207
doi:
78
Zha
,
Q.
,
Brewster
,
A.L.
,
Richichi
,
C.
,
Bender
,
R.A.
and
Baram
,
T.Z.
(
2008
)
Activity-dependent heteromerization of the hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels: role of N-linked glycosylation
.
J. Neurochem.
105
,
68
77
doi:
79
Shi
,
W.
,
Wymore
,
R.
,
Yu
,
H.
,
Wu
,
J.
,
Wymore
,
R.T.
,
Pan
,
Z.
et al. 
(
1999
)
Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues
.
Circ. Res.
85
,
e1
e6
doi:
80
Stieber
,
J.
,
Herrmann
,
S.
,
Feil
,
S.
,
Loster
,
J.
,
Feil
,
R.
,
Biel
,
M.
et al. 
(
2003
)
The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart
.
Proc. Natl Acad. Sci. USA
100
,
15235
15240
doi:
81
Baruscotti
,
M.
,
Bucchi
,
A.
,
Viscomi
,
C.
,
Mandelli
,
G.
,
Consalez
,
G.
,
Gnecchi-Rusconi
,
T.
et al. 
(
2011
)
Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4
.
Proc. Natl Acad. Sci. USA
108
,
1705
1710
doi:
82
Ludwig
,
A.
(
2003
)
Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2
.
EMBO J.
22
,
216
224
doi:
83
Fenske
,
S.
,
Krause
,
S.C.
,
Hassan
,
S.I.H.
,
Becirovic
,
E.
,
Auer
,
F.
,
Bernard
,
R.
et al. 
(
2013
)
Sick sinus syndrome in HCN1-deficient mice
.
Circulation
128
,
2585
2594
doi:
84
Huang
,
H.
and
Trussell
,
L.O.
(
2014
)
Presynaptic HCN channels regulate vesicular glutamate transport
.
Neuron
84
,
340
346
doi:
85
Benarroch
,
E.E.
(
2013
)
HCN channels: function and clinical implications
.
Neurology
80
,
304
310
doi:
86
Maroso
,
M.
,
Szabo
,
G.G.
,
Kim
,
H.K.
,
Alexander
,
A.
,
Bui
,
A.D.
,
Lee
,
S.H.
et al. 
(
2016
)
Cannabinoid control of learning and memory through HCN channels
.
Neuron
89
,
1059
1073
doi:
87
Santoro
,
B.
,
Chen
,
S.
,
Luthi
,
A.
,
Pavlidis
,
P.
,
Shumyatsky
,
G.P.
,
Tibbs
,
G.R.
et al. 
(
2000
)
Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS
.
J. Neurosci.
20
,
5264
5275
PMID:
[PubMed]
88
Notomi
,
T.
and
Shigemoto
,
R.
(
2004
)
Immunohistochemical localization of Ih channel subunits, HCN1-4, in the rat brain
.
J. Comp. Neurol.
471
,
241
276
doi:
89
Bender
,
R.A.
and
Baram
,
T.Z.
(
2008
)
Hyperpolarization activated cyclic-nucleotide gated (HCN) channels in developing neuronal networks
.
Prog. Neurobiol.
86
,
129
140
doi:
90
Hu
,
T.
,
Liu
,
N.
,
Lv
,
M.
,
Ma
,
L.
,
Peng
,
H.
,
Peng
,
S.
et al. 
(
2016
)
Lidocaine inhibits HCN currents in rat spinal substantia gelatinosa neurons
.
Anesth. Analg.
122
,
1048
1059
doi:
91
Honsa
,
P.
,
Pivonkova
,
H.
,
Harantova
,
L.
,
Butenko
,
O.
,
Kriska
,
J.
,
Dzamba
,
D.
et al. 
(
2014
)
Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia
.
Glia
62
,
2004
2021
doi:
92
Rivera-Arconada
,
I.
,
Roza
,
C.
and
Lopez-Garcia
,
J.A.
(
2013
)
Characterization of hyperpolarization-activated currents in deep dorsal horn neurons of neonate mouse spinal cord in vitro
.
Neuropharmacology
70
,
148
155
doi:
93
Reid
,
C.A.
,
Phillips
,
A.M.
and
Petrou
,
S.
(
2012
)
HCN channelopathies: pathophysiology in genetic epilepsy and therapeutic implications
.
Br. J. Pharmacol.
165
,
49
56
doi:
94
Du
,
L.
,
Wang
,
S.J.
,
Cui
,
J.
,
He
,
W.J.
and
Ruan
,
H.Z.
(
2013
)
Inhibition of HCN channels within the periaqueductal gray attenuates neuropathic pain in rats
.
Behav. Neurosci.
127
,
325
329
doi:
95
Santello
,
M.
and
Nevian
,
T.
(
2015
)
Dysfunction of cortical dendritic integration in neuropathic pain reversed by serotoninergic neuromodulation
.
Neuron
86
,
233
246
doi:
96
Cordeiro Matos
,
S.
,
Zhang
,
Z.
and
Seguela
,
P.
(
2015
)
Peripheral neuropathy induces HCN channel dysfunction in pyramidal neurons of the medial prefrontal cortex
.
J. Neurosci.
35
,
13244
13256
doi:
97
Hou
,
B.
,
Chen
,
H.
,
Qu
,
X.
,
Lin
,
X.
,
Luo
,
F.
and
Li
,
C.
(
2015
)
Characteristics of hyperpolarization-activated cyclic nucleotide-gated channels in dorsal root ganglion neurons at different ages and sizes
.
NeuroReport
26
,
981
987
doi:
98
Chaplan
,
S.R.
,
Guo
,
H.Q.
,
Lee
,
D.H.
,
Luo
,
L.
,
Liu
,
C.
,
Kuei
,
C.
et al. 
(
2003
)
Neuronal hyperpolarization-activated pacemaker channels drive neuropathic pain
.
J. Neurosci.
23
,
1169
1178
PMID:
[PubMed]
99
Jiang
,
Y.Q.
,
Xing
,
G.G.
,
Wang
,
S.L.
,
Tu
,
H.Y.
,
Chi
,
Y.N.
,
Li
,
J.
et al. 
(
2008
)
Axonal accumulation of hyperpolarization-activated cyclic nucleotide-gated cation channels contributes to mechanical allodynia after peripheral nerve injury in rat
.
Pain
137
,
495
506
doi:
100
Moosmang
,
S.
,
Stieber
,
J.
,
Zong
,
X.
,
Biel
,
M.
,
Hofmann
,
F.
and
Ludwig
,
A.
(
2001
)
Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues
.
Eur. J. Biochem.
268
,
1646
1652
doi:
101
Momin
,
A.
,
Cadiou
,
H.
,
Mason
,
A.
and
McNaughton
,
P.A.
(
2008
)
Role of the hyperpolarization-activated current Ih in somatosensory neurons
.
J. Physiol.
586
,
5911
5929
doi:
102
Tu
,
H.
,
Deng
,
L.
,
Sun
,
Q.
,
Yao
,
L.
,
Han
,
J.S.
and
Wan
,
Y.
(
2004
)
Hyperpolarization-activated, cyclic nucleotide-gated cation channels: roles in the differential electrophysiological properties of rat primary afferent neurons
.
J. Neurosci. Res.
76
,
713
722
doi:
103
Acosta
,
C.
,
McMullan
,
S.
,
Djouhri
,
L.
,
Gao
,
L.
,
Watkins
,
R.
,
Berry
,
C.
et al. 
(
2012
)
HCN1 and HCN2 in rat DRG neurons: levels in nociceptors and non-nociceptors, NT3-dependence and influence of CFA-induced skin inflammation on HCN2 and NT3 expression
.
PLoS ONE
7
,
e50442
doi:
104
Cho
,
H.J.
,
Staikopoulos
,
V.
,
Furness
,
J.B.
and
Jennings
,
E.A.
(
2009
)
Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine
.
Neuroscience
162
,
453
461
doi:
105
Kouranova
,
E.V.
,
Strassle
,
B.W.
,
Ring
,
R.H.
,
Bowlby
,
M.R.
and
Vasilyev
,
D.V.
(
2008
)
Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein expression in large versus small diameter dorsal root ganglion neurons: correlation with hyperpolarization-activated current gating
.
Neuroscience
153
,
1008
1019
doi:
106
Weng
,
X.
,
Smith
,
T.
,
Sathish
,
J.
and
Djouhri
,
L.
(
2012
)
Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Adelta-nociceptors
.
Pain
153
,
900
914
doi:
107
Reference deleted
108
Cho
,
H.J.
,
Staikopoulos
,
V.
,
Ivanusic
,
J.J.
and
Jennings
,
E.A.
(
2009
)
Hyperpolarization-activated cyclic-nucleotide gated 4 (HCN4) protein is expressed in a subset of rat dorsal root and trigeminal ganglion neurons
.
Cell Tissue Res.
338
,
171
177
doi:
109
Bennett
,
G.J.
and
Xie
,
Y.-K.
(
1988
)
A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man
.
Pain
33
,
87
107
doi:
110
Kim
,
S.H.
and
Chung
,
J.M.
(
1992
)
An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat
.
Pain
50
,
355
363
doi:
111
Seltzer
,
Z.
,
Dubner
,
R.
and
Shir
,
Y.
(
1990
)
A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury
.
Pain
43
,
205
218
doi:
112
Takasu
,
K.
,
Ono
,
H.
and
Tanabe
,
M.
(
2010
)
Spinal hyperpolarization-activated cyclic nucleotide-gated cation channels at primary afferent terminals contribute to chronic pain
.
Pain
151
,
87
96
doi:
113
Luo
,
L.
,
Chang
,
L.
,
Brown
,
S.M.
,
Ao
,
H.
,
Lee
,
D.H.
,
Higuera
,
E.S.
et al. 
(
2007
)
Role of peripheral hyperpolarization-activated cyclic nucleotide-modulated channel pacemaker channels in acute and chronic pain models in the rat
.
Neuroscience
144
,
1477
1485
doi:
114
Sun
,
Q.
,
Xing
,
G.G.
,
Tu
,
H.Y.
,
Han
,
J.S.
and
Wan
,
Y.
(
2005
)
Inhibition of hyperpolarization-activated current by ZD7288 suppresses ectopic discharges of injured dorsal root ganglion neurons in a rat model of neuropathic pain
.
Brain Res.
1032
,
63
69
doi:
115
Sánchez-Alonso
,
J.L.
,
Halliwell
,
J.V.
and
Colino
,
A.
(
2008
)
ZD 7288 inhibits T-type calcium current in rat hippocampal pyramidal cells
.
Neurosci. Lett.
439
,
275
280
doi:
116
Felix
,
R.
,
Sandoval
,
A.
,
Sánchez
,
D.
,
Gómora
,
J.C.
,
De la Vega-Beltrán
,
J.L.
,
Treviño
,
C.L.
et al. 
(
2003
)
ZD7288 inhibits low-threshold Ca(2+) channel activity and regulates sperm function
.
Biochem. Biophys. Res. Commun.
311
,
187
192
doi:
117
Wu
,
X.
,
Liao
,
L.
,
Liu
,
X.
,
Luo
,
F.
,
Yang
,
T.
and
Li
,
C.
(
2012
)
Is ZD7288 a selective blocker of hyperpolarization-activated cyclic nucleotide-gated channel currents?
Channels
6
,
438
442
doi:
118
Noh
,
S.
,
Kumar
,
N.
,
Bukhanova
,
N.
,
Chen
,
Y.
,
Stemkowsi
,
P.L.
and
Smith
,
P.A.
(
2014
)
The heart-rate-reducing agent, ivabradine, reduces mechanical allodynia in a rodent model of neuropathic pain
.
Eur. J. Pain
18
,
1139
1147
doi:
119
Carozzi
,
V.A.
,
Canta
,
A.
and
Chiorazzi
,
A.
(
2015
)
Chemotherapy-induced peripheral neuropathy: what do we know about mechanisms?
Neurosci. Lett.
596
,
90
107
doi:
120
Descoeur
,
J.
,
Pereira
,
V.
,
Pizzoccaro
,
A.
,
Francois
,
A.
,
Ling
,
B.
,
Maffre
,
V.
et al. 
(
2011
)
Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors
.
EMBO Mol. Med.
3
,
266
278
doi:
121
Chen
,
Y.
(
2014
)
ZD 7288, an HCN channel blocker, attenuates chronic visceral pain in irritable bowel syndrome-like rats
.
World J. Gastroenterol.
20
,
2091
2097
doi:
122
Reference deleted
123
Wickenden
,
A.D.
,
Maher
,
M.P.
and
Chaplan
,
S.R.
(
2009
)
HCN pacemaker channels and pain: a drug discovery perspective
.
Curr. Pharm. Des.
15
,
2149
2168
doi:
124
Stieber
,
J.
(
2006
)
Bradycardic and proarrhythmic properties of sinus node inhibitors
.
Mol. Pharmacol.
69
,
1328
1337
doi:
125
Orio
,
P.
,
Madrid
,
R.
, D
e la Peña
,
E.
,
Parra
,
A.
,
Meseguer
,
V.
,
Bayliss
,
D.A.
et al. 
(
2009
)
Characteristics and physiological role of hyperpolarization activated currents in mouse cold thermoreceptors
.
J. Physiol.
587
(
Pt 9
),
1961
1976
doi:
126
Akopian
,
A.N.
,
Sivilotti
,
L.
and
Wood
,
J.N.
(
1996
)
A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons
.
Nature
379
,
257
262
doi:
127
Stirling
,
L.C.
,
Forlani
,
G.
,
Baker
,
M.D.
,
Wood
,
J.N.
,
Matthews
,
E.A.
,
Dickenson
,
A.H.
et al. 
(
2005
)
Nociceptor-specific gene deletion using heterozygous NaV1.8-Cre recombinase mice
.
Pain
113
,
27
36
doi:
128
Schnorr
,
S.
,
Eberhardt
,
M.
,
Kistner
,
K.
,
Rajab
,
H.
,
Kasser
,
J.
,
Hess
,
A.
et al. 
(
2014
)
HCN2 channels account for mechanical (but not heat) hyperalgesia during long-standing inflammation
.
Pain
155
,
1079
1090
doi:
129
Djouhri
,
L.
(
2006
)
Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors
.
J. Neurosci.
26
,
1281
1292
doi:
130
Xie
,
W.
,
Strong
,
J.A.
,
Meij
,
J.T.
,
Zhang
,
J.M.
and
Yu
,
L.
(
2005
)
Neuropathic pain: early spontaneous afferent activity is the trigger
.
Pain
116
,
243
256
doi:
131
Song
,
Y.
,
Li
,
H.M.
,
Xie
,
R.G.
,
Yue
,
Z.F.
,
Song
,
X.J.
,
Hu
,
S.J.
et al. 
(
2012
)
Evoked bursting in injured Abeta dorsal root ganglion neurons: a mechanism underlying tactile allodynia
.
Pain
153
,
657
665
doi:
132
Emery
,
E.C.
,
Young
,
G.T.
and
McNaughton
,
P.A.
(
2012
)
HCN2 ion channels: an emerging role as the pacemakers of pain
.
Trends Pharmacol. Sci.
33
,
456
463
doi:
133
Momin
,
A.
and
McNaughton
,
P.A.
(
2009
)
Regulation of firing frequency in nociceptive neurons by pro-inflammatory mediators
.
Exp. Brain Res.
196
,
45
52
doi:
134
Ma
,
W.
,
Hatzis
,
C.
and
Eisenach
,
J.C.
(
2003
)
Intrathecal injection of cAMP response element binding protein (CREB) antisense oligonucleotide attenuates tactile allodynia caused by partial sciatic nerve ligation
.
Brain Res.
988
,
97
104
doi:
135
Mabuchi
,
T.
,
Kojima
,
H.
,
Abe
,
T.
,
Takagi
,
K.
,
Sakurai
,
M.
,
Ohmiya
,
Y.
et al. 
(
2004
)
Membrane-associated prostaglandin E synthase-1 is required for neuropathic pain
.
Neuroreport
15
,
1395
1398
doi:
136
Kim
,
K.-S.
,
Kim
,
J.
,
Back
,
S.K.
,
Im
,
J.Y.
,
Na
,
H.S.
and
Han
,
P.L.
(
2007
)
Markedly attenuated acute and chronic pain responses in mice lacking adenylyl cyclase-5
.
Genes Brain Behav.
6
,
120
127
doi:
137
Ahlgren
,
S.C.
and
Levine
,
J.D.
(
1993
)
Mechanical hyperalgesia in streptozotocin-diabetic rats
.
Neuroscience
52
,
1049
1055
doi:
138
Cheng
,
H.T.
,
Dauch
,
J.R.
,
Oh
,
S.S.
,
Hayes
,
J.M.
,
Hong
,
Y.
and
Feldman
,
E.L.
(
2010
)
P38 mediates mechanical allodynia in a mouse model of type 2 diabetes
.
Mol. Pain
6
,
28
doi:
139
Pop-Busui
,
R.
,
Marinescu
,
V.
,
Van Huysen
,
C.
,
Li
,
F.
,
Sullivan
,
K.
,
Greene
,
D.A.
et al. 
(
2002
)
Dissection of metabolic, vascular, and nerve conduction interrelationships in experimental diabetic neuropathy by cyclooxygenase inhibition and acetyl-l-carnitine administration
.
Diabetes
51
,
2619
2628
doi:
140
Kimura
,
S.
and
Kontani
,
H.
(
2009
)
Demonstration of antiallodynic effects of the cyclooxygenase-2 inhibitor meloxicam on established diabetic neuropathic pain in mice
.
J. Pharmacol. Sci.
110
,
213
217
doi:
141
Juárez-Rojop
,
I.E.
,
Morales-Hernández
,
P.E.
,
Tovilla-Zárate
,
C.A.
,
Bermúdez-Ocaña
,
D.Y.
,
Torres-Lopez
,
J.E.
,
Ble-Castillo
,
J.L.
et al. 
(
2015
)
Celecoxib reduces hyperalgesia and tactile allodynia in diabetic rats
.
Pharmacol. Rep.
67
,
545
552
doi:
142
Kellogg
,
A.P.
and
Pop-Busui
,
R.
(
2005
)
Peripheral nerve dysfunction in experimental diabetes is mediated by cyclooxygenase-2 and oxidative stress
.
Antioxid. Redox Signal.
7
,
1521
1529
doi:
143
Herder
,
C.
,
Baumert
,
J.
,
Zierer
,
A.
,
Roden
,
M.
,
Meisinger
,
C.
,
Karakas
,
M.
et al. 
(
2011
)
Immunological and cardiometabolic risk factors in the prediction of type 2 diabetes and coronary events: MONICA/KORA Augsburg case-cohort study
.
PLoS ONE
6
,
e19852
doi:
144
Moore
,
R.A.
,
Chi
,
C.C.
,
Wiffen
,
P.J.
,
Derry
,
S.
and
Rice
,
A.S.
(
2015
)
Oral nonsteroidal anti-inflammatory drugs for neuropathic pain
.
Cochrane Database Syst. Rev.
10
,
CD010902
doi:
145
Papp
,
I.
,
Holló
,
K.
and
Antal
,
M.
(
2010
)
Plasticity of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 expression in the spinal dorsal horn in inflammatory pain
.
Eur. J. Neurosci.
32
,
1193
1201
doi:
146
Richards
,
N.
and
Dilley
,
A.
(
2015
)
Contribution of hyperpolarization-activated channels to heat hypersensitivity and ongoing activity in the neuritis model
.
Neuroscience
284
,
87
98
doi:
147
Djouhri
,
L.
,
Al Otaibi
,
M.
,
Kahlat
,
K.
,
Smith
,
T.
,
Sathish
,
J.
and
Weng
,
X.
(
2015
)
Persistent hindlimb inflammation induces changes in activation properties of hyperpolarization-activated current (Ih) in rat C-fiber nociceptors in vivo
.
Neuroscience
301
,
121
133
doi:
148
Black
,
J.A.
,
Nikolajsen
,
L.
,
Kroner
,
K.
,
Jensen
,
T.S.
and
Waxman
,
S.G.
(
2008
)
Multiple sodium channel isoforms and mitogen-activated protein kinases are present in painful human neuromas
.
Ann. Neurol.
64
,
644
653
doi:
149
Schinkel
,
A.H.
(
1999
)
P-Glycoprotein, a gatekeeper in the blood-brain barrier
.
Adv. Drug Deliv. Rev.
36
,
179
194
doi:
150
Bucchi
,
A.
,
Baruscotti
,
M.
and
DiFrancesco
,
D.
(
2002
)
Current-dependent block of rabbit sino-atrial node I(f) channels by ivabradine
.
J. Gen. Physiol.
120
,
1
13
doi:
151
Postea
,
O.
and
Biel
,
M.
(
2011
)
Exploring HCN channels as novel drug targets
.
Nat. Rev. Drug Discov.
10
,
903
914
doi:
152
Schulze-Bahr
,
E.
,
Neu
,
A.
,
Friederich
,
P.
,
Kaupp
,
U.B.
,
Breithardt
,
G.
,
Pongs
,
O.
et al. 
(
2003
)
Pacemaker channel dysfunction in a patient with sinus node disease
.
J. Clin. Invest.
111
,
1537
1545
doi:
153
Nof
,
E.
,
Luria
,
D.
,
Brass
,
D.
,
Marek
,
D.
,
Lahat
,
H.
,
Reznik-Wolf
,
H.
et al. 
(
2007
)
Point mutation in the HCN4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia
.
Circulation
116
,
463
470
doi:
154
Milanesi
,
R.
,
Baruscotti
,
M.
,
Gnecchi-Ruscone
,
T.
and
DiFrancesco
,
D.
(
2006
)
Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel
.
N. Engl. J. Med.
354
,
151
157
doi:
155
Cervetto
,
L.
,
Demontis
,
G.C.
and
Gargini
,
C.
(
2007
)
Cellular mechanisms underlying the pharmacological induction of phosphenes
.
Br. J Pharmacol.
150
,
383
390
doi:
156
Melchiorre
,
M.
,
Del Lungo
,
M.
,
Guandalini
,
L.
,
Martini
,
E.
,
Dei
,
S.
,
Manetti
,
D.
et al. 
(
2010
)
Design, synthesis, and preliminary biological evaluation of new isoform-selective f-current blockers
.
J. Med. Chem.
53
,
6773
6777
doi:
157
Vasilyev
,
D.V.
,
Shan
,
Q.
,
Lee
,
Y.
,
Mayer
,
S.C.
,
Bowlby
,
M.R.
,
Strassle
,
B.W.
et al. 
(
2007
)
Direct inhibition of Ih by analgesic loperamide in rat DRG neurons
.
J. Neurophysiol.
97
,
3713
3721
doi:
158
Yagi
,
J.
and
Sumino
,
R.
(
1998
)
Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons
.
J. Neurophysiol.
80
,
1094
1104
159
Griguoli
,
M.
,
Maul
,
A.
,
Nguyen
,
C.
,
Giorgetti
,
A.
,
Carloni
,
P.
and
Cherubini
,
E.
(
2010
)
Nicotine blocks the hyperpolarization-activated current Ih and severely impairs the oscillatory behavior of oriens-lacunosum moleculare interneurons
.
J. Neurosci.
.
30
,
10773
10783
doi:
160
Sirois
,
J.E.
,
Lynch
, III,
C.
and
Bayliss
,
D.A.
(
2002
)
Convergent and reciprocal modulation of a leak K+ current and I(h) by an inhalational anaesthetic and neurotransmitters in rat brainstem motoneurones
.
J. Physiol.
541
(
Pt 3
)
717
729
doi:
161
Budde
,
T.
,
Coulon
,
P.
,
Pawlowski
,
M.
,
Meuth
,
P.
,
Kanyshkova
,
T.
,
Japes
,
A.
et al. 
(
2008
)
Reciprocal modulation of I (h) and I (TASK) in thalamocortical relay neurons by halothane
.
Pflugers Arch.
456
,
1061
1073
doi:
162
Osaki
,
Y.
,
Nodera
,
H.
,
Banzrai
,
C.
,
Endo
,
S.
,
Takayasu
,
H.
,
Mori
,
A.
et al. 
(
2015
)
Effects of anesthetic agents on in vivo axonal HCN current in normal mice
.
Clin. Neurophysiol.
126
,
2033
2039
doi:
163
Chung
,
W.K.
,
Shin
,
M.
,
Jaramillo
,
T.C.
,
Leibel
,
R.L.
,
LeDuc
,
C.A.
,
Fischer
,
S.G.
et al. 
(
2009
)
Absence epilepsy in apathetic, a spontaneous mutant mouse lacking the h channel subunit, HCN2
.
Neurobiol. Dis.
33
,
499
508
doi:
164
Baruscotti
,
M.
,
Bottelli
,
G.
,
Milanesi
,
R.
,
DiFrancesco
,
J.C.
and
DiFrancesco
,
D.
(
2010
)
HCN-related channelopathies
.
Pflugers Arch.
460
,
405
415
doi:
165
DiFrancesco
,
J.C.
,
Barbuti
,
A.
,
Milanesi
,
R.
,
Coco
,
S.
,
Bucchi
,
A.
,
Bottelli
,
G.
et al. 
(
2011
)
Recessive loss-of-function mutation in the pacemaker HCN2 channel causing increased neuronal excitability in a patient with idiopathic generalized epilepsy
.
J. Neurosci.
31
,
17327
17337
doi:
166
Tabernero
,
J.
,
Shapiro
,
G.I.
,
LoRusso
,
P.M.
,
Cervantes
,
A.
,
Schwartz
,
G.K.
,
Weiss
,
G.J.
et al. 
(
2013
)
First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement
.
Cancer Discov.
3
,
406
417
doi:
167
Dorn
,
G.
(
2004
)
siRNA relieves chronic neuropathic pain
.
Nucleic Acids Res.
32
,
e49
doi:
168
Tsantoulas
,
C.
,
Zhu
,
L.
,
Shaifta
,
Y.
,
Grist
,
J.
,
Ward
,
J.P.T.
,
Raouf
,
R.
et al. 
(
2012
)
Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury
.
J. Neurosci.
32
,
17502
17513
doi:
169
Luo
,
M.C.
,
Zhang
,
D.Q.
,
Ma
,
S.W.
,
Huang
,
Y.Y.
,
Shuster
,
S.J.
,
Porreca
,
F.
et al. 
(
2005
)
An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons
.
Mol. Pain
1
,
29
doi:
170
Harper
,
S.Q.
,
Staber
,
P.D.
,
He
,
X.
,
Eliason
,
S.L.
,
Martins
,
I.H.
,
Mao
,
Q.
et al. 
(
2005
)
RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model
.
Proc. Natl Acad. Sci. USA
102
,
5820
5825
doi:
171
Rodriguez-Lebron
,
E.
,
Denovan-Wright
,
E.M.
,
Nash
,
K.
,
Lewin
,
A.S.
and
Mandel
,
R.J.
(
2005
)
Intrastriatal rAAV-mediated delivery of anti-Huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington's disease transgenic mice
.
Mol. Ther.
12
,
618
633
doi:
172
Singer
,
O.
,
Marr
,
R.A.
,
Rockenstein
,
E.
,
Crews
,
L.
,
Coufal
,
N.G.
,
Gage
,
F.H.
et al. 
(
2005
)
Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model
.
Nat. Neurosci.
8
,
1343
1349
doi:
173
Hirai
,
T.
,
Enomoto
,
M.
,
Kaburagi
,
H.
,
Sotome
,
S.
,
Yoshida-Tanaka
,
K.
,
Ukegawa
,
M.
et al. 
(
2014
)
Intrathecal AAV serotype 9-mediated delivery of shRNA against TRPV1 attenuates thermal hyperalgesia in a mouse model of peripheral nerve injury
.
Mol. Ther.
22
,
409
419
doi:
174
Samad
,
O.A.
,
Tan
,
A.M.
,
Cheng
,
X.
,
Foster
,
E.
,
Dib-Hajj
,
S.D.
and
Waxman
,
S.G.
(
2013
)
Virus-mediated shRNA knockdown of Na(v)1.3 in rat dorsal root ganglion attenuates nerve injury-induced neuropathic pain
.
Mol. Ther.
21
,
49
56
doi:
175
Tan
,
A.M.
,
Samad
,
O.A.
,
Dib-Hajj
,
S.D.
and
Waxman
,
S.G.
(
2015
)
Virus-mediated knockdown of Nav1.3 in dorsal root ganglia of STZ-Induced diabetic rats alleviates tactile allodynia
.
Mol. Med.
21
,
544
552
doi:
176
Djouhri
,
L.
(
2016
)
PG110, a humanized anti-NGF antibody, reverses established pain hypersensitivity in persistent inflammatory pain, but not peripheral neuropathic pain, rat models
.
Pain Med.
doi:
177
Yao
,
C.Y.
,
Weng
,
Z.L.
,
Zhang
,
J.C.
,
Feng
,
T.
,
Lin
,
Y.
and
Yao
,
S.
(
2015
)
Interleukin-17A acts to maintain neuropathic pain through activation of CaMKII/CREB signaling in spinal neurons
.
Mol. Neurobiol.
PMID:
[PubMed]
178
Kersten
,
C.
,
Cameron
,
M.G.
,
Laird
,
B.
and
Mjåland
,
S.
(
2015
)
Epidermal growth factor receptor-inhibition (EGFR-I) in the treatment of neuropathic pain
.
Br. J. Anaesth.
115
,
761
767
doi:
179
Hogarth
,
P.M.
and
Pietersz
,
G.A.
(
2012
)
Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond
.
Nat. Rev. Drug Discov.
11
,
311
331
doi:
180
Buell
,
G.
,
Chessell
,
I.P.
,
Michel
,
A.D.
,
Collo
,
G.
,
Salazzo
,
M.
,
Herren
,
S.
et al. 
(
1998
)
Blockade of human P2X7 receptor function with a monoclonal antibody
.
Blood
92
,
3521
3528
PMID:
[PubMed]
181
Lee
,
K.J.
,
Wang
,
W.
,
Padaki
,
R.
,
Bi
,
V.
,
Plewa
,
C.A.
and
Gavva
,
N.R.
(
2014
)
Mouse monoclonal antibodies to transient receptor potential ankyrin 1 act as antagonists of multiple modes of channel activation
.
J. Pharmacol. Exp. Ther.
350
,
223
231
doi:
182
Lee
,
J.H.
,
Park
,
C.K.
,
Chen
,
G.
,
Han
,
Q.
,
Xie
,
R.G.
,
Liu
,
T.
et al. 
(
2014
)
A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief
.
Cell
157
,
1393
1404
doi:
183
Gomez-Varela
,
D.
,
Zwick-Wallasch
,
E.
,
Knotgen
,
H.
,
Sanchez
,
A.
,
Hettmann
,
T.
,
Ossipov
,
D.
et al. 
(
2007
)
Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity
.
Cancer Res.
67
,
7343
7349
doi: