An early key event in the activation of neutrophil granulocytes is Ca2+ influx. Members of the transient receptor potential (TRP) channel family may be held responsible for this. The aim of the present study is to analyse the expression pattern of TRP mRNA and identify characteristic currents unambiguously attributable to particular TRP channels. mRNA was extracted from human neutrophils, isolated by gradient centrifugation and also by magnetically labelled CD15 antibodies. The presence of mRNA was demonstrated using reverse transcriptase–PCR in neutrophils (controlled to be CD5-negative) as well as in human leukaemic cell line 60 (HL-60) cells, for the following TRP species: the long TRPC2 (LTRPC2), the vanilloid receptor 1, the vanilloid receptor-like protein 1 and epithelial Ca2+ channels 1 and 2. TRPC6 was specific for neutrophils, whereas only in HL-60 cells were TRPC1, TRPC2, TRPC3, melastatin 1 and melastatin-related 1 found. Patch-clamp measurements in neutrophils revealed non-selective cation currents evoked by intracellular ADP-ribose and by NAD+. Both these modes of activation have been found to be characteristic of LTRPC2. Furthermore, single-channel activity was resolved in neutrophils and it was indistinguishable from that in LTRPC2-transfected HEK-293 cells. The results provide evidence that LTRPC2 in neutrophil granulocytes forms an entry pathway for Na+ and Ca2+, which is regulated by ADP-ribose and the redox state.
Abbreviations used: ADPR, ADP-ribose; BVF, bivalent-ion-free; [Ca2+]i, intracellular cytosolic Ca2+ concentration; cADPR, cyclic ADPR; ECaC, epithelial Ca2+ channel; fMLP, N-formylmethionyl-leucylphenylalanine; HL-60, human leukaemic cell line 60; IL-8R, interleukin-8 receptor; MACS, magnetic antibody cell separation; MLSN1, melastatin 1; MTR1, melastatin-related 1; NMDG, N-methyl-d-glucamine; OAG, 1-oleoyl-2-acetyl-sn-glycerol; RT, reverse transcriptase; SOCE, store-operated Ca2+ entry; TRP, transient receptor potential; TRPC, TRPC channel; LTRPC2, long TRP channel 2: VR1, vanilloid receptor 1.