A single amino acid in the second transmembrane domain of GABA rho subunits is a determinant of the response kinetics of GABAC receptors.
The rho subunits that constitute the gamma-aminobutyric acid (GABA)C receptors of retinal neurons form a unique subclass of ligand-gated chloride channels that give rise to sustained GABA-evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA rho1B and rho2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site-directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABAC receptors. When the site contains a proline residue, as in wild-type rho1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild-type rho2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA rho receptors, e.g., when the serine residue of the rho2A receptor was changed to proline, the response of the mutant receptor to imidazole-4-acetic acid (I4AA) mimicked that of the rho1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various rho receptor subunits. It is noteworthy that the proline residue in native rho1 subunits and the serine residue of rho2 subunits are well conserved in all species, a good indication that the presence of multiple GABA rho subunits serves to generate GABAC receptors that display the wide range of response kinetics observed on various types of retinal neurons.
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 West Taylor Street, Chicago, Illinois 60612, USA.
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D017931:DNA Primers; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D010471:Perches; D017433:Protein Structure, Secondary; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000166:cytology; D017948:Retinal Rod Photoreceptor Cells / Q000187:drug effects / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D017931:DNA Primers; C087200:GABA-C receptor; D046911:Macromolecular Substances; D018079:Receptors, GABA; D011993:Recombinant Fusion Proteins; D005680:gamma-Aminobutyric Acid