Heterogeneity of solubilized muscarinic cholinergic receptors: Binding and hydrodynamic properties

Abstract

Previous studies have described the conversion, after detergent solubilization, of the multiple populations of membrane-bound muscarinic agonist binding sites to a population of uniform affinity. This paper describes the solubilization of at least two receptor species, distinct in their agonist binding characteristics, which are capable of interconversion by transition metal ions. This finding enabled a more detailed examination of the molecular properties and regional differences of brain muscarinic receptors than was previously possible. Muscarinic receptors (mAChR) obtained from the rat cerebral cortex or medulla pons were solubilized using digitonin or the zwitterion detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). The equilibrium binding of the antagonist [ 3H]-4- N-methylpiperidyl benzilate ([ 3H]4NMPB) to detergent-solubilized receptors resembled binding to neural membranes and exhibited subnanomolar affinity, saturability, and simple mass action kinetics. Agonist binding to soluble preparations was measured by competition of [ 3H]4NMPB binding sites. Saturation isotherms for agonist binding to digitonin- and Chapssolubilized mAChR obtained from various brain regions appear flattened and have Hill coefficients in the range 0.52–0.78. Computerized modelling techniques indicate that the best fit to the experimental data is provided by a model specifying two soluble muscarinic agonist binding sites with differing dissociation constants, K H and K l , respectively. Solubilization of cerebral cortex membranes with Chaps or digitonin resulted in a population with a composition of high- and low-affinity sites similar to that found in the membrane-bound state. In contrast, solubilization of the medulla pons resulted in an approximately 40% loss of high-affinity sites. Solubilized receptors retained the sensitivity to transition metals ions, but were insensitive to guanine nucleotides. Density gradient centrifugation indicated that Chaps-solubilized mAChR are composed of two molecular forms with S 20,w to 9.9 S and 14.9 S. The 14.9 S species comprises approximately 30% of the total binding activity in the cortex and approximately 40% in the medulla. We identify the 14.9 S species as being associated with a guanylnucleotide binding protein because treatment of medulla membranes with guanylylimidodiphosphate prior to solubilization results in disappearance of 14.9 S with 9.9 S unchanged. Sedimentation of cortical mAChR in the presence of Cu 2+ leads to an increase in 14.9 S to almost 50% of the total binding activity. Digitoninsolubilized mAChR sediment as a single 11.3 S species, but in the presence of Cu 2+ or Co 2+ a 16.6 S species is evident.