Background: GABAC receptors are part of the ligand-gated ion channel family of receptors that share some functional and structural features: e.g., they have four putative transmembrane domains (TM1-TM4) and the TM2-segment is presumed to form the ion-channel. GABAC receptors open chloride channels and do not desensitize even after long exposures to GABA. These receptors are highly expressed in vertebrate retina, where they may play a unique role due to their unusual biophysical and pharmacologic characteristics.
Methods: To determine whether the TM2 domain plays a role in the process of desensitization of GABAC receptors, we used site-directed mutagenesis to produce several permutations within the leucine (L9') residue of the TM2 domain of the human GABArho1 subunit. Recombinant receptors were expressed in Xenopus laevis oocytes and their functional and pharmacologic properties were studied by using a two-microelectrode, voltage-clamp.
Results: Several amino acid changes led to receptors that did not generate GABA-currents, whereas an Asp for Leu mutation in the well-conserved L9' position of the rho1 subunit (L301D-rho1) generated a fast-desensitizing, bicuculline-resistant receptor that was antagonized by TPMPA, a specific GABAC receptor antagonist. Moreover, in contrast with wild-type rho1 receptors, which are practically not gated by taurine, L301D-rho1 mutant receptors generated substantial taurine-currents.
Conclusions: Substitution of L9' residue in the TM2 region of GABArho1 receptor for an amino acid residue with an acidic lateral chain greatly accelerates its desensitization rate and increases taurine-agonism. This mutant will be useful to study mechanisms involved in gating and desensitization of GABAC receptors in particular, and of neurotransmitter receptors in general.