Solubilization of A1 adenosine receptor from pig brain: Characterization and evidence of the role of the cell membrane on the coexistence of high‐ and low‐affinity states

Abstract

The present solubilization strategy recognizes the important role of detergent cocktails in the solubilization and subsequent stability of adenosine A₁, receptors from pig brain cortical membranes. The 3‐[3‐(cholamidopropyl) dimethylammonio]‐1‐propane‐sulfonate‐digitonin mixture produced the extraction of up to 52% of the receptor with an enrichment of 1.2‐fold with respect to crude membranes. The binding activity of the soluble extract was very stable even in the absence of glycerol. In crude membranes the existence of high‐ and low‐affinity states was detected, but in the soluble extract and in the detergent‐treated membranes only the high‐ affinity state was detected. Association‐dissociation curves showed that in crude membranes no interconversion between high‐ and low‐affinity sites is produced by the association of the ligand [³H]R‐N⁶ ‐phenylisopro‐pyladenosine. These results suggest that the high‐ and the low‐affinity states are different conformations induced by the structure of the membrane. The modulation of the binding activity by (Gpp(NH)p)5′ ‐guanylylimidodiphosphate and Mg²⁺ was studied. In crude membranes Gpp(NH)p shifted the high‐affinity state to the low‐affinity state, whereas the contrary occurred when Mg²⁺ was used. The effect of both Mg²⁺ and Gpp(NH)p was also assayed with the soluble extact and with the detergent‐treated membranes. In addition to a decrease of the overall binding capacity, Gpp(NH)p promoted a conversion to all low‐affinity states in the detergent‐treated membranes or to all very‐low‐affinity sites in the soluble extract. Mg²⁺ and Gpp(NH)p counteracted their effects in intact membranes, whereas Mg²⁺ could not reverse the uncoupling effect of Gpp(NH)p with solubilized or detergent‐treated membranes. Thus, it is suggested that Mg²⁺ acts at sites other than guanine‐nucleotide sites. If high‐affinity states correspond to receptor/G protein complexes and low‐affinity states correspond to the uncoupled receptor, we should conclude that Mg²⁺, as well as the loss of membrane integrity, favours the interaction of A₁ receptor molecule with G protein.