GABArho 1/GABAAalpha 1 receptor chimeras to study receptor desensitization

Abstract

␥-Aminobutyrate type C (GABAC) receptors are ligand-gated ion channels that are expressed preponderantly in the vertebrate retina and are characterized, among other things, by a very low rate of desensitization and resistance to the specific GABAA antagonist bicuculline. To examine which structural elements determine the nondesensitizing character of the human homomeric 1 receptor, we used a combination of gene chimeras and electrophysiology of receptors expressed in Xenopus oocytes. Two chimeric genes were constructed, made up of portions of the 1-subunit and of the ␣1-subunit of the GABAA receptor. When expressed in Xenopus oocytes, one chimeric gene (1͞␣1) formed functional homooligomeric receptors that were fully resistant to bicuculline and were blocked by the specific GABAC antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and by zinc. Moreover, these chimeric receptors had a fast-desensitizing component, even faster than that of heterooligomeric GABAA receptors, in striking contrast to the almost nil desensitization of wild-type 1 (wt 1) receptors. To see whether the fast-desensitizing characteristic of the chimera was determined by the amino acids forming the ion channels, we replaced the second transmembrane segment (TM2) of 1 by that of the ␣1-subunit of GABAA. Although the ␣1-subunit forms fast-desensitizing receptors when coexpressed with other GABA A subunits, the sole transfer of the ␣1TM2 segment to 1 was not sufficient to form desensitizing receptors. All this suggests that the slow-desensitizing trait of 1 receptors is determined by a combination of several interacting domains along the molecule.Xenopus oocytes ͉ ␥-aminobutyrate type C receptor ͉ (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid S everal years ago a new type of ␥-aminobutyrate (GABA) receptor, now called GABA C , was identified clearly by expressing retina mRNA in Xenopus oocytes (1). In sharp contrast to the previously known GABA A receptors, the GABA C receptors show very little desensitization, are not blocked by the specific GABA A receptor antagonist bicuculline, and are not modulated by pentobarbital or steroids (1-3). GABA C is now recognized as a new family of receptors of which, so far, three members have been cloned (1-3). Moreover, there is a larger diversity of GABA C receptors because of alternative splicing (4). In contrast to the heteromeric nature of GABA A receptors, which are formed by a combination of several types of subunits, the three GABA C genes form functional homomeric receptors when expressed in Xenopus oocytes (5-9).The electrical responses to GABA, which have been recorded from various types of retinal cells, are due to activation of both GABA A and GABA C receptors (10-14), and, except for a bicuculline-resistant GABA current seen in the golden perch retina (15), all other GABA C responses desensitize very slowly. For example, the GABA currents elicited in oocytes expressing GABA1 or its alternative spliced form (GABA1⌬51) decay only about 8% or less even after 10 min of exposur...

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