High pH accelerates GABA deactivation on perch-ρ1B receptors

Abstract

The ionotropic GABA C receptor, formed by GABA ρ subunits, is known to be modulated by a variety of endogenous compounds, as well as by changes in pH. In this study, we explore the proton sensitivity of the GABA ρ subunits cloned from the perch retina, and report a novel action of high pH on the homomeric receptor formed by one of the GABA ρ subunits, the perch-ρ 1B subunit. Raising extracellular pH to 9.5 significantly accelerated GABA deactivation responses elicited from oocytes expressing the perch-ρ 1B subunit, and reduced its sensitivity to GABA. The change in the kinetics of the GABA-offset response occurred without altering the maximum response amplitude, and the reduced GABA sensitivity was independent of membrane potential. Although acidification of the extracellular solution also accelerated GABA deactivation for all other GABA ρ receptors examined in this study, the effects of high pH were unique to the homomeric receptor formed by the perch-ρ 1B subunit. In addition, we found that, unlike the effects on the response to the naturally occurring full agonist GABA, the responses elicited by partial agonists (imidazole-4-acetic acid (I4AA) and β-alanine) in the presence of the high pH solution showed a significant reduction in the maximum response amplitude. When considered in terms of a model describing the activation of GABA C receptors, in which pH can potentially affect either the binding affinity or the rate of channel closure, the results were consistent with the view that external alkalization reduces the gating efficiency of the receptor. To identify the proton sensitive domain(s) of the perch-ρ 1B receptor, chimeras were constructed by domain swapping with other perch-ρ subunits. Analysis of the pH sensitivities of the various chimeric receptors revealed that the alkaline-sensitive residues are located in the N-terminal region of the perch-ρ 1B subunit.