The Journal of Neuroscience, August 15, 2001, 21(16):5885-5892
On the Contribution of the First Transmembrane Domain to
Whole-Cell Current through an ATP-Gated Ionotropic P2X Receptor
William R.
Haines,
Mark M.
Voigt,
Keisuke
Migita,
Gonzalo
E.
Torres, and
Terrance M.
Egan
Department of Pharmacological and Physiological Science, St. Louis
University School of Medicine, St. Louis, Missouri 63104
Scanning cysteine mutagenesis was used to identify potential
pore-forming residues in and around the first transmembrane domains of
ionotropic P2X2 receptor subunits. Twenty-eight unique
cysteine-substituted mutants (R28C-Y55C) were individually expressed in
HEK293 cells by lipofection. Twenty-three of these were functional as
assayed by application of ATP to transfected voltage-clamped cells.
Individual mutants varied in their sensitivity to ATP; otherwise,
currents through functional mutant receptors resembled those of the
homomeric wild-type (WT) receptor. In five (H33C, R34C, I50C, K53C, and S54C) of 23 functional mutants, coapplication of 30 µM
ATP and 500 nM Ag+ irreversibly
inhibited inward current evoked by subsequent applications of ATP
alone. These inhibitions did not result in a lateral shift in the
agonist concentration-response curve and are unlikely to involve a
modification of the agonist binding site. Two (K53C and S54C) of the
five residues modified by Ag+ applied in the
presence of ATP when the channels were gating were also modified by 1 mM (2-aminoethyl)methanethiosulfonate applied
in the absence of ATP when the channels were closed. These data suggest
that domains near either end of the first transmembrane domain
influence ion conduction through the pore of the P2X2 receptor.
Key words:
ATP; scanning cysteine mutagenesis; purinergic; ion
channel; ligand-gated; methanethiosulfonate
Copyright © 2001 Society for Neuroscience 0270-6474/01/21165885-08$05.00/0
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