Existence of Sites for Anions and Divalent Cations in the Solubilized γ‐Aminobutyric Acid/Benzodiazepine Receptor Complex

Abstract: This study evaluated the ability of γ‐aminobutyric acid (GABA), baclofen, monovalent anions, divalent cations, and various combinations thereof to protect solubilized benzodiazepine (BZ) receptors of types 1 and 2, when contained together on the complex, against heat inactivation. Neither anions, cations, nor GABA alone provided significant protection of solubilized BZ receptors against heat, but inclusion of monovalent anions or divalent cations together with 500 μM GABA did afford protection. Monovalent anions combined with GABA (500 μM) provided 50% to full protection. Divalent cations, such as CaCl₂ (2.5 mM) or MgCl₂ (2.5 mM) in the presence of GABA (500 μM) yielded 45% and 24% protection, respectively. Other divalent cations tested (Zn²⁺, Hg²⁺, Co²⁺, and Ni²⁺) were poor protectors, even when combined with GABA. Monovalent anions (200 mM NaCl) and divalent cations (5 mM CaCl₂) when tested together provided no protection. Similarly, baclofen (the GABA‐B agonist) provided no protection, either alone or together with anions or divalent cations. These results indicate that the independent but interacting recognition sites of GABA, BZ, anions, and divalent cations, previously detected in the membrane‐bound state, are retained in the solubilized state.