Solubilization and sedimentation analysis of muscarinic acetylcholine receptors

To investigate if G-protein-receptor interactions can be characterized using sucrose density gradients (SDG) we have determined the experimental conditions for muscarinic acetylcholine receptor (mAChR) solubilization and analysis on SDG. Solubilization of 65-80% of [3H]QNB bound mAChR was accomplished with 1% of detergent. Analysis of solubilized receptors on SDG containing 0.4 M KCl and 0.1% detergent demonstrated that the physical properties of the receptor-detergent complexes are influenced by the solubilizing detergent as well as detergents included in the SDG. Neither GTP gamma S nor NaF and AlCl3 altered the sedimentation properties of mAChR, suggesting that the solubilized mAChR is no longer associated with G-protein under these conditions. Receptors bound to [3H]oxotremorine and [3H]QNB had similar sedimentation properties, suggesting that, once solubilized, mAChRs do not remain associated with G-proteins. Covalent labeling with [3H]PrBCM followed by solubilization and analysis on SDS-gel electrophoresis demonstrated the presence of intact receptor molecule. These observations suggest that the changes in the sedimentation properties of detergent-receptor complexes are independent of G-protein interactions and are influenced by the nature of the detergent associated with the mAChR during analysis.