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Natural and Enantiomeric Etiocholanolone Interact with Distinct Sites on the Rat 122L GABA_A Receptor

Departments of Anesthesiology (P.L., J.B., J.H.S., G.A.) and Molecular Biology and Pharmacology (B.W.K., D.F.C.), Washington University School of Medicine, St. Louis, Missouri

We have studied the ability of the androgen etiocholanolone and its enantiomer (ent-etiocholanolone) to modulate rat 122L GABA_A receptor function transiently expressed in human embryonic kidney cells. Studies on steroid enantiomer pairs can yield powerful new information on the pharmacology of steroid interactions with the GABA_A receptor. Both steroids enhance currents elicited by GABA, but ent-etiocholanolone is much more powerful than etiocholanolone at producing potentiation. At a low GABA concentration (0.5 µM, <EC₅), the presence of 10 µM ent-etiocholanolone potentiates whole-cell currents by almost 30-fold, whereas 10 µM etiocholanolone merely doubles the peak response. At higher GABA concentration (5 µM, EC₂₅), the potentiation curve for ent-etiocholanolone is positioned at lower concentrations than that for etiocholanolone. Single-channel kinetic analysis shows that exposure to etiocholanolone has a single effect on currents: the relative frequency of long openings is increased in the presence of steroid. But exposure to ent-etiocholanolone produces two kinetic effects: an increase in the relative frequency of long openings and a decrease in the frequency of long closed times. The presence of etiocholanolone does not inhibit potentiation by ent-etiocholanolone, suggesting that etiocholanolone is unable to interact with the sites through which ent-etiocholanolone modifies receptor function. The double mutation 1(N407A/Y410F) prevents potentiation by etiocholanolone but not by ent-etiocholanolone, and the 1(Q241A) and 1(I238N) point mutations fully abolish potentiation by etiocholanolone but not by ent-etiocholanolone. We conclude that etiocholanolone and its enantiomer interact with distinct sites on the 122L GABA_A receptor.

Received for publication December 11, 2006.

Accepted for publication March 6, 2007.

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

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