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The Journal of Neuroscience, April 1, 1998, 18(7):2350-2359
A Domain Contributing to the Ion Channel of ATP-Gated
P2X2 Receptors Identified by the Substituted Cysteine
Accessibility Method
Terrance M.
Egan,
William R.
Haines, and
Mark M.
Voigt
Department of Pharmacological and Physiological Sciences, St. Louis
University Health Sciences Center, St. Louis, Missouri 63104
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.
Key words:
ATP receptor; P2X subtype; scanning cysteine mutagenesis; sulfhydryl-modifying reagents; ion channel; transmembrane domain
Copyright © 1998 Society for Neuroscience 0270-6474/98/1872350-10$05.00/0
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