Heterogeneity of solubilized muscarinic cholinergic receptors: Binding and hydrodynamic properties

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