GABAA receptor subunit γ2 and δ subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts

Abstract

1. To determine their contributions to the rapid kinetic properties of GABAA receptor (GABAR) currents, alpha1 and beta3 subunit subtypes without or with delta or gamma2L subtypes were transiently coexpressed in mouse L929 fibroblasts to produce alpha1beta3, alpha1beta3delta, or alpha1beta3gamma2L GABAR isoforms. 2. Brief (2-3 ms) applications of 1 mM GABA to outside-out membrane patches containing alpha1beta3, alpha1beta3delta, or alpha1beta3gamma2L isoforms elicited currents that activated rapidly with monophasic time courses and deactivated rapidly with biphasic time courses. alpha1beta3gamma2L currents exhibited a slower mean deactivation rate (76.1 ms) than alpha1beta3 (34.1 ms) or alpha1beta3delta currents (42.8 ms). 3. During 1 mM GABA applications, alpha1beta3gamma2L currents activated more rapidly (0.46 ms) than alpha1beta3 currents (1.7 ms) or alpha1beta3delta currents (2.4 ms). During 4000 ms GABA applications, alpha1beta3 and alpha1beta3gamma2L currents desensitized with triphasic time courses to similar extents (alpha1beta3, 94.6 %; alpha1beta3gamma2L, 92.4 %) and with similar mean rates (alpha1beta3, 352 ms; alpha1beta3gamma2L, 462 ms). In contrast, alpha1beta3delta currents desensitized only 55.6 % with a biphasic time course and slower mean rate (1260 ms). 4. These experiments demonstrated that the alpha1beta3 heterodimer formed a GABAR channel with rapid deactivation and rapid and nearly complete desensitization. Addition of the delta subunit did not alter the activation rate, but produced a receptor with slower and less complete desensitization. Addition of the gamma2L subtype increased activation rate, prolonged deactivation and changed the pattern of rapid desensitization. 5. Rapid kinetic and steady-state single-channel data were used to construct kinetic models that predicted the behaviour of the alpha1beta3gamma2L and alpha1beta3delta currents. These models represent a reconciliation of macroscopic and steady-state single-channel data for GABARs and provide a framework for systematically assessing the functional significance of different GABAR isoforms.