Pregnenolone sulfate block of GABA_A receptors: mechanism and involvement of a residue in the M2 region of the α subunit

Abstract

1. Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the y-aminobutyric acid A (GABA A ) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA A receptors formed from a1, b2 and y2L subunits.2. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 µM) and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel activity at either concentration of GABA.3. PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules.4. The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field.5. Change in a specific residue near the intracellular end of the channel lining portion of the a1 subunit had a major effect on the rate of block. Mutation of the residue a1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the b2 subunit (b2 A252S) had no effect, nor did a complementary mutation in the y2L subunit (y2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

Mechanisms of neurosteroid interactions with GABAA receptors

“…Thus, the presence of 3α5αP does not interfere with the ability of pregnenolone sulfate to reduce cluster duration nor does the presence of pregnenolone sulfate interfere with the ability of 3α5αP to prolong channel open time durations (unpublished observations). This has also been seen for the steroid analogue, ACN, and pregnenolone sulfate (Akk et al, 2001). …”

“…A mutation to the 2′ residue in the M2 transmembrane domain in the α1 subunit (α1V256S) specifically reduces the ability of inhibitory steroids to modulate receptor function but is ineffective on the actions of potentiating steroids (Akk et al, 2001). Although the 2′ residue is unlikely to constitute a binding site for inhibitory steroids, its preferential action on the effects of inhibitory steroids suggests that the effects of the two types of steroids follow separate transduction pathways.…”

“…Thus, at least part of the action of sulfated steroids may be to enhance receptor accumulation in these non-conducting states and thereby truncate IPSCs. In single-channel recordings, coapplication of an inhibitory steroid, e.g., pregnenolone sulfate with GABA, results in a briefer mean cluster duration without affecting intracluster open or closed time distributions (Akk et al, 2001). …”

Steroid modulation of GABAA receptor-mediated transmission in the hypothalamus: Effects on reproductive function

“…These compounds are believed to act as non-competitive antagonists at site(s) separate from those that bind the positive neurosteroid modulators. These steroid modulators promote a reversible inhibition of peak GABA-elicited currents by enhancing the rate of slow desensitization and by decreasing channel opening frequency, while having negligible effects on open or burst durations (Mienville and Vicini, 1989;Shen et al, 2000;Akk et al, 2001;Wang et al, 2003; for review, Henderson and Jorge, 2004).…”

“…Fewer studies have directly assessed the subunit-specificity of negative neurosteroid modulators, although mutations in the α 1 , but not homologous mutations in the β 2 or γ 2S subunits, reduce the rate of block elicited by pregnenolone sulfate by 30-fold (Akk et al, 2001). A recent report by Rahman et al (2006) indicates that substitution of an α 5 for an α 1 subunit in recombinant α x β 2 γ 2L receptors results in enhanced efficacy, but no effect on potency, for a panel of inhibitory modulators including pregnenolone sulfate, DHEAS, and a number of 3β-hydroxypregnane steroids.…”