Homomeric rho 1 GABA channels: activation properties and domains.
Expression of human rho 1 subunits in Xenopus laevis oocytes forms homomeric GABA-activated chloride channels with activation and pharmacological properties distinct from those of typical heteromeric GABA channels (e.g., alpha 1 beta 2 gamma 2). Here, we describe these rho 1 activation features and use site-directed mutagenesis to identify amino acids involved in GABA-mediated activation. In comparison to heteromeric GABA channels (alpha 1 beta 2 gamma 2), GABA channels comprised of rho 1 subunits are approximately 40-fold more sensitive to GABA, activate 8.3-fold more slowly (at a GABA concentration equal to their respective EC50S), do not desensitize with maintained agonist application, and close approximately 8-fold more slowly after agonist removal. Site-directed mutagenesis of rho 1 GABA channels has identified five amino acids (Y198, Y200, Y241, T244, and Y247) located between the N-terminal extracellular cysteine loop and first membrane spanning domain, that when conservatively mutated, significantly impaired GABA-mediated activation. These five residues are grouped in two domains that correspond in position to the putative agonist-binding domains previously identified for the beta 2 subunit of alpha 1 beta 2 gamma 2 GABA channels. Y198, T244, and Y247 correspond directly to crucial amino acids identified in the beta 2 subunit; Y200 and Y241 do not. These differences may account, in part, for the unique activation and pharmacological features of homomeric rho 1 GABA channels.
Amin
J
J
Department of Physiology and Biophysics, University of South Florida College of Medicine, Tampa 33612-4799.
Weiss
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018118:Chloride Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003437:Crotonates / Q000494:pharmacology; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D008969:Molecular Sequence Data; D009118:Muscimol / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011487:Protein Conformation; D018075:RNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012160:Retina / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D018118:Chloride Channels; D003437:Crotonates; D005136:Eye Proteins; D018075:RNA, Complementary; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; C055331:4-aminocrotonic acid; D009118:Muscimol; D005680:gamma-Aminobutyric Acid