Residues in the first transmembrane domain of the Caenorhabditis elegans GABA(A) receptor confer sensitivity to the neurosteroid pregnenolone sulfate.
The GABA(A) receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABA(A) receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC-49 GABA receptor, a close homolog of the mammalian GABA(A) receptor. The UNC-49 locus encodes two GABA receptor subunits, UNC-49B and UNC-49C. UNC-49C is sensitive to PS but UNC-49B is not sensitive. By analyzing chimeric receptors and receptors containing site-directed mutations, we identified two regions required for PS inhibition. Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general.
Wardell
Bryan
B
Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, Salt Lake City, UT 84112, USA.
Marik
Purba S
PS
Piper
David
D
Rutar
Tina
T
Jorgensen
Erik M
EM
Bamber
Bruce A
BA
D000595:Amino Acid Sequence; D000818:Animals; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005260:Female; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D009154:Mutation / Q000235:genetics; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D010852:Picrotoxin / Q000494:pharmacology; D011284:Pregnenolone / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D029742:Caenorhabditis elegans Proteins; D050506:Mutant Chimeric Proteins; D010852:Picrotoxin; D011284:Pregnenolone; D011963:Receptors, GABA-A; C018370:pregnenolone sulfate