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Substituted cysteine accessibility mutagenesis of P2X2 receptors suggests the position of the gate with structural implications for closed‐open transitions
Author(s) -
Khakh Baljit S.,
Kracun Sebastian
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.lb584
Subject(s) - biophysics , chemistry , ion channel , mutagenesis , transmembrane domain , helix (gastropod) , intracellular , cysteine , transmembrane protein , receptor , biochemistry , biology , mutation , gene , enzyme , ecology , snail
P2X receptors are trimeric ligand‐gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor reveals that the ion‐conducting pathway is formed by three transmembrane domain 2 (TM2) alpha‐helices. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In the present study we used site‐directed mutagenesis (SDM) and electrophysiology to quantify gated access to Cd 2+ at substituted cysteines in TM2 of P2X2. Our closed state data are consistent with the zfP2X4.1 structure, with I332 and T336 positioned one helical turn apart lining the channel wall on approach to the gate. Our open state data reveal gated access to deeper parts of the pore (T339, V343, D349 and L353), suggesting the closed channel gate is between T336 and T339. We also found unexpected interactions between native C348 and D349C that result in tight Cd 2+ binding deep within the intracellular vestibule in the open state. Our data suggest that the channel gate opens near T336/T339 and is accompanied by movement of the pore‐lining regions, which narrow towards the cytosolic end of TM2. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP. This research was supported by the NIH.