Efflux of intracellular K+ and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K+] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K+ ion surrogate 86Rb+, we have demonstrated an efflux of intracellular K+ during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K+ ions in apoptosis, cytoprotection by elevated extracellular [K+] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K+] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing ≈ 700kDa complex and an inactive ≈ 1.4 MDa complex. Increasing [K+] inhibited caspase activation by preventing formation of the ≈ 700kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K+] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress.
Abbreviations used: Apaf-1, apoptotic protease-activating factor 1; DISC, death-inducing signalling complex; [K+]ext, extracellular K+ concentration; [K+]i, intracellular K+ concentration; MG132, carbobenzoxy-Leu-Leu-leucinal; [Na+]ext, extracellular Na+ concentration; NMDG, N-methyl-d-glucamine; Ψm, mitochondrial membrane potential; PtdSer, phosphatidylserine; TMRE, tetramethylrhodamine ethyl ester; TNF, tumour-necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; DEVDase, Asp-Glu-Val-Asp-ase; Z-DEVD.AFC, benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; Z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp O-methyl ester fluoromethylketone; FCS, fetal-calf serum.
Present address: Amersham-Pharmacia Biotech, Forest Farm Laboratories, Whitchurch, Cardiff CF14 7YT, Wales, U.K.