Identification of a Zn²⁺ binding site on the marine gABA_A receptor complex: Dependence on the Second transmembrane domain of β subunits

Abstract

Whole‐cell currents were recorded from Xenopus laevis oocytes expressing wild‐type and mutant recombinant GABAA receptors to locate a binding site for Zn2+ ions in the β3 subunit. The Cl−‐selective current, spontaneously gated by β3 subunit homomers, was enhanced by pentobarbitone and inhibited by picrotoxinin. The potencies of these agents were minimally affected by mutating histidine (H) 292 to alanine (A) in the second transmembrane domain (TM2). Zn2+ inhibited the β3 subunit‐gated conductance (IC50, 0.31 μm); the inhibition was voltage insensitive. The H292A mutation in β3 subunits caused a 1000‐fold reduction in Zn2+ potency (IC50, 307 μm). GABA‐activated responses recorded from heteromeric α1β3 GABAA receptors were also inhibited by Zn2+ (IC50, 0.11 μm). This inhibition was reduced by mutating H292A in the β3 subunit (IC50, 22.8 μm). H292 in TM2 of the β3 subunit is an important determinant of a Zn2+ binding site on the GABAA receptor. Its location in the presumed ion channel lining suggests that Zn2+ can penetrate into an anion‐selective channel and that the ionic selectivity filter and channel gate are located beyond H292.

Identification of Residues within GABAAReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

“…1), as defined by FLAG staining in the absence of permeabilization, consistent with the results seen with (9E10) ␣6S and also with the ability of ␤3 to form functional homomeric receptors ( Fig. 1; Connolly et al, 1996;Wooltorton et al, 1997, Taylor et al, 1999. In contrast, the wild-type ␣1 subunit can readily assemble with ␤3 to form functional GABA-gated channels (MacDonald and Olsen, 1994;Rabow et al, 1995).…”

“…C ells were used 24 -48 hr after transfection and voltage-clamped at Ϫ40 mV with membrane currents. Drugs and Krebs' solution were rapidly applied (exchange rate, ϳ30 msec) to single cells using a modified U-tube (Wooltorton et al, 1997). GABA equilibrium concentration -response curve data were fitted to the following equation:…”

“…2 B). Homomeric ␤3 receptors also exhibit a degree of spontaneous gating in the absence of any ligand, which can be inhibited by Zn 2ϩ (Wooltorton et al, 1997). Although in some cells (n ϭ 3 of 8) 10 M Zn 2ϩ induced a small outward current, the majority of expressing cells failed to demonstrate any response to Zn 2ϩ (Fig.…”

“…Expression of ␣ and ␤ subunits produces GABA-gated currents, but coexpression with a ␥ subunit is essential in conferring benzodiazepine sensitivity on expressed receptors (MacDonald and Olsen, 1994;Rabow et al, 1995). Interestingly, the ␤3 subunit, and to a lesser extent the ␤1 subunit, can assemble into homomeric channels that gate spontaneously in a number of heterologous expression systems (Sigel et al, 1989;Krishek et al, 1996;Wooltorton et al, 1997). Recently, four N-terminal amino acids within the ␤3 subunit have been identified that control homo-oligomerization and cell surface expression of this subunit compared to ␤2 (Taylor et al, 1999).…”

“…Given that the ␣1S subunit is retained within the ER, the cell surface ( Fig. 1) expressed receptors are likely to be composed of predominantly ␤3 subunits forming homomeric receptors (Davies et al, 1997;Wooltorton et al, 1997), which would account for the direct action of pentobarbitone (Fig. 2 B).…”