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First published on March 16, 2009; DOI: 10.1124/mol.108.054510

0026-895X/09/7506-1400-1411$20.00
Mol Pharmacol 75:1400-1411, 2009

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Activation and Modulation of Concatemeric GABA-A Receptors Expressed in Human Embryonic Kidney Cells

Gustav Akk, Ping Li, John Bracamontes, and Joe Henry Steinbach

Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri

We have employed whole-cell and single-channel electrophysiology to examine the kinetic and pharmacological properties of GABA-A receptors consisting of {gamma}2L-β2-{alpha}1 and β2-{alpha}1 subunit concatemeric constructs expressed in human embryonic kidney cells. Concatemeric receptors activated by GABA exhibited the same single-channel conductance, channel opening rate constant, and basic open- and closed-time properties as receptors containing free subunits. However, the whole-cell GABA dose-response and the single-channel effective opening rate curves were shifted to higher GABA concentrations, suggesting that the concatemeric receptors have a lower affinity to GABA. Pharmacological tests demonstrated that the concatemeric receptors were potentiated by pentobarbital, diazepam, and the neurosteroid (3{alpha},5{alpha})-3-hydroxypregnan-20-one (3{alpha}5{alpha}P), and were insensitive to Zn2+. Selective introduction of the {alpha}1Q241L mutation, previously shown to abolish {alpha}1β2{gamma}2L channel potentiation by neurosteroids, into one of the two concatemeric constructs had a relatively small effect on receptor activation by GABA or macroscopic potentiation by the neurosteroid 3{alpha}5{alpha}P. Single-channel measurements showed that the kinetic mechanism of action of the steroid is unchanged when the mutation is introduced to the {gamma}2L-β2-{alpha}1 concatemer. We infer that a single wild-type {alpha} subunit is capable of mediating the full set of kinetic effects in the presence of steroids. Introduction of the {alpha}1Q241W mutation, previously shown to mimic the effect of the steroid on {alpha}1β2{gamma}2L channels, selectively into either concatemeric construct altered the mode of activity elicited by P4S, but the presence of mutations in both {alpha} subunits was required to affect open-time distributions. The data indicate that the {alpha}1Q241W mutation acts as a partial steroid modulator.

Received for publication December 31, 2008.

Accepted for publication March 16, 2009.

Address correspondence to: Gustav Akk, Dept. of Anesthesiology, Campus Box 8054, 660 S. Euclid Ave, St. Louis, MO 63110. E-mail: akk{at}morpheus.wustl.edu






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