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

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 gamma -aminobutyric acid A (GABA(A)) receptor. We analysed the actions of PX on single-channel currents from recombinant GABA(A) receptors formed from alpha1, beta2 and gamma 2L subunits. 2. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 muM) and a saturating coneentration (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 alpha1 subunit had a major effect on the rate of block. Mutation of the residue alpha1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the beta2 subunit (beta2 A252S) had no effect, nor did a complementary mutation in the gamma 2L subunit (gamma 2LS266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of for ming part of the binding site for PS.