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The Journal of Neuroscience, May 15, 2002, 22(10):3873-3880
Mutational Analysis of the Conserved Cysteines of the Rat
P2X2 Purinoceptor
J. Dylan
Clyne,
Lin-Fang
Wang, and
Richard I.
Hume
Department of Molecular, Cellular, and Developmental Biology,
University of Michigan, Ann Arbor, Michigan 48109-1048
P2X receptors are ATP-gated cation channels that are widely
expressed in the brain. The extracellular domains of all seven P2X
receptors contain 10 conserved cysteines, which could form disulfide
bonds or binding sites for transition metals that modulate P2X
receptors. To test whether these cysteines are critical for receptor
function, we studied wild-type rat P2X2 receptors and 10 mutant P2X2 receptors, each containing an alanine
substituted for a cysteine. Nine mutants were functional but had
reduced maximum currents compared with wild-type P2X2
expressed in either Xenopus oocytes or human embryonic
kidney (HEK) 293 cells. The 10th mutant (C224A) did not respond
to ATP when expressed in oocytes and gave very small
currents in HEK 293 cells. Seven mutants (C113A, C124A, C130A, C147A,
C158A, C164A, and C214A) showed rightward shifts (9- to 30-fold) in
their ATP concentration-response relationships and very little
potentiation by zinc. In contrast, C258A and C267A had EC50
values similar to those of wild-type P2X2 and were
potentiated by zinc. Acidic pH potentiated wild-type and all mutant
receptor currents. Despite the loss of zinc potentiation in seven
mutants, these cysteines are unlikely to be exposed in the zinc-binding site, because [2-(trimethylammonium)ethyl] methanethiosulfonate bromide did not prevent zinc potentiation of wild-type
receptor currents. On the basis of correlations in the maximum current, EC50, zinc potentiation, and pH potentiation, we
suggest that the following cysteine pairs form disulfide bonds:
C113-C164, C214-C224, and C258-C267. We also suggest that C124,
C130, C147, and C158 form two disulfide bonds, but we are unable to
assign specific cysteine pairs to these two bonds.
Key words:
P2X; purinergic; mutagenesis; disulfide bonds; DTT; zinc; pH
Copyright © 2002 Society for Neuroscience 0270-6474/02/22103873-08$05.00/0
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