Identification of a β subunit TM2 residue mediating proton modulation of GABA type A receptors

Abstract

GABA type A (GABAA) receptors are functionally regulated by external protons in a manner dependent on the receptor subunit composition. Although H+ can regulate the open probability of single GABA ion channels, exactly what residues and receptor subunits are responsible for proton-induced modulation remain unknown. This study resolves this issue by using recombinant α1/βi subunit GABAA receptors expressed in human embryonic kidney cells. The potentiating effect of low external pH on GABA responses exhibited PKa in accord with the involvement of histidine and/or cysteine residues. The exposure of GABAA receptors to the histidine-modifying reagent DEPC ablated regulation by H+, implicating the involvement of histidine residues rather than cysteines in proton regulation. Site-specific substitution of all conserved external histidines to alanine on the β subunits revealed that H267 alone, in the TM2 domain, is important for H+ regulation. These results are interpreted as a direct protonation of H267 on α1/βi receptors rather than an involvement in signal transduction. The opposing functional effects induced by Zn2+ and H+ at this single histidine residue most likely reflect differences in charge delocalization on the imidazole rings in the mouth of the GABAA receptor ion channel. Additional substitutions of H267 in β subunits with other residues possessing charged side chains (glutamate and lysine) reveal that this area of the ion channel can profoundly influence the functional properties of GABAA receptors.