A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted cysteine accessibility method.
P2X receptors are a family of ATP-gated ion channels thought to have intracellular N and C termini and two transmembrane segments separating a large extracellular domain. We examined the involvement of the second putative transmembrane domain (TM2) of the P2X2 subunit in ion conduction, using the substituted cysteine accessibility method (SCAM). This method tests the ability of hydrophilic reagents such as Ag+ or the methanethiosulfonates to modify covalently the sulfhydryl side chains exposed to aqueous environments. ATP-gated current was measured in HEK293 cells transiently expressing either wild-type or functional mutant P2X2 receptors containing a cysteine substitution in or around TM2. Application of Ag+ to gating channels had no sustained effect on wild-type P2X2 (WT) but irreversibly altered whole-cell currents in 15 mutants. By contrast, bath application of (2-aminoethyl)methanethiosulfonate (MTSEA) to closed channels inhibited 8 of the 15 residues affected by Ag+ when the channel was gating. Inhibition of the closed channel was prevented in seven of eight mutants when membrane-permeant MTSEA was scavenged by 20 mM intracellular cysteine, indicating that these seven mutants lie on the intracellular side of the channel gate. Further, MTSEA inhibited current through G342C in the absence of intracellular cysteine but augmented the current when cysteine was present, suggesting that this residue may be part of the gate. Taken together, the data help to the identify a functional domain of the channel pore by mapping residues on either side of the channel gate.
Egan
T M
TM
Department of Pharmacological and Physiological Sciences, St. Louis University Health Sciences Center, St. Louis, Missouri 63104, USA.
Haines
W R
WR
Voigt
M M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003545:Cysteine; D004305:Dose-Response Relationship, Drug; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics*; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000235:genetics*; D058476:Receptors, Purinergic P2X2; D012834:Silver / Q000494:pharmacology; D014867:Water / Q000737:chemistry
D007202:Indicators and Reagents; D009479:Neuropeptides; C546401:P2RX2 protein, human; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; C093171:methanethiosulfonate ethylammonium; D014867:Water; D012834:Silver; D000255:Adenosine Triphosphate; D005020:Ethyl Methanesulfonate; D003545:Cysteine