Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 33, Pages 13440-13445Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1204702109
Keywords
C-type inactivation; cation permeation; PI(4,5)P-2; electrostatic effect; gating kinetics
Categories
Funding
- Orszagos Tudomanyos Kutatasi Alapprogramok Grant [F 68143]
- Howard Hughes Medical Institute
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The Ca2+-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 Ca2+ 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 (PIP2) in the inner membrane leaflet. Most TRPM family channels inactivate upon prolonged stimulation in inside-out patches; this rundown is due to PIP2 depletion. TRPM2 currents also run down within minutes, but the molecular mechanism of this process is unknown. Here we report that high-affinity PIP2 binding regulates Ca2+ sensitivity of TRPM2 activation. Nevertheless, TRPM2 inactivation is not due to PIP2 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/Ca2+-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.
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