Received for publication December 12, 2006.
Revised February 20, 2007.
Accepted for publication March 6, 2007.

NATURAL AND ENANTIOMERIC ETIOCHOLANOLONE INTERACT WITH DISTINCT SITES ON THE RAT 122L GABA_A RECEPTOR

Abstract

We have studied the ability of the androgen etiocholanolone and its enantiomer (ent-etiocholanolone) to modulate the rat 122L GABA_A receptor function transiently expressed in HEK 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 while 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.

Key words: GABAA, GABAC, Single channel kinetics

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