title: Pore collapse underlies irreversible inactivation of TRPM2 cation channel currents.
abstract: The Ca(2+)-permeable cation channel transient receptor potential melastatin 2 (TRPM2) plays a key role in pathogen-evoked phagocyte activation, postischemic neuronal apoptosis, and glucose-evoked insulin secretion, by linking these cellular responses to oxidative stress. TRPM2 channels are coactivated by binding of intracellular ADP ribose and Ca(2+) to distinct cytosolically accessible sites on the channels. These ligands likely regulate the activation gate, conserved in the voltage-gated cation channel superfamily, that comprises a helix bundle formed by the intracellular ends of transmembrane helix six of each subunit. For several K(+) and TRPM family channels, activation gate opening requires the presence of phosphatidylinositol-bisphosphate (PIP(2)) in the inner membrane leaflet. Most TRPM family channels inactivate upon prolonged stimulation in inside-out patches; this "rundown" is due to PIP(2) depletion. TRPM2 currents also run down within minutes, but the molecular mechanism of this process is unknown. Here we report that high-affinity PIP(2) binding regulates Ca(2+) sensitivity of TRPM2 activation. Nevertheless, TRPM2 inactivation is not due to PIP(2) depletion; rather, it is state dependent, sensitive to permeating ions, and can be completely prevented by mutations in the extracellular selectivity filter. Introduction of two negative charges plus a single-residue insertion, to mimic the filter sequence of TRPM5, results in TRPM2 channels that maintain unabated maximal activity for over 1 h, and display altered permeation properties but intact ADP ribose/Ca(2+)-dependent gating. Thus, upon prolonged stimulation, the TRPM2 selectivity filter undergoes a conformational change reminiscent of that accompanying C-type inactivation of voltage-gated K(+) channels. The noninactivating TRPM2 variant will be invaluable for gating studies.
authors: Department of Medical Biochemistry,  Semmelweis University, Budapest H-1094,  Hungary.
authors: Balázs
authors: B
authors: Tóth
authors: László
authors: L
authors: Csanády
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002412:Cations; D002463:Cell Membrane Permeability; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D042541:Intracellular Space / Q000378:metabolism; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D016062:Porosity; D011485:Protein Binding; D016415:Sequence Alignment; D055672:Static Electricity; D050053:TRPM Cation Channels / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D002412:Cations; D019269:Phosphatidylinositol 4,5-Diphosphate; D050053:TRPM Cation Channels; D000246:Adenosine Diphosphate Ribose; D002118:Calcium