Received for publication May 1, 2008.
Revised May 29, 2008.
Accepted for publication June 10, 2008.

Mutations of the GABA-A receptor 1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids

Abstract

Neuroactive steroids are among the most efficacious modulators of the mammalian GABA-A receptor. Previous work has proposed that receptor potentiation is mediated by steroid interactions with a site defined by the residues 1N407/Y410 in the M4 transmembrane domain, and residue 1Q241 in the M1 domain. We examined the role of residues in the 1 subunit M1 domain in the modulation of the rat 122L GABA-A receptor by neuroactive steroids. The data demonstrate that the region is critical to the actions of potentiating neuroactive steroids. Receptors containing the 1Q241W or 1Q241L mutations were insensitive to (3,5-3-hydroxypregnan-20-one (35P), albeit with different underlying mechanisms. The 1Q241S mutant was potentiated by 35P but the kinetic mode of potentiation was altered by the mutation. Interestingly, the 1Q241L mutation was without effect on channel potentiation by (3,5-3-hydroxymethylpregnan-20-one, but mutation of the neighboring residue, 1S240, prevented channel modulation. A steroid lacking a H-bonding group on C3 (5-pregnan-20-one) potentiated the wild-type receptor but not the 1Q241L mutant. The findings are consistent with a model where the 1S240 and 1Q241 residues shape the surface to which steroid molecules bind.

Key words: GABAA, GABAC, Mutagenesis/Chimeric approaches