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Solubilization of muscarinic receptor subtypes from baculovirus infected Sf9 insect cells
1Department of Medical and Physiological Chemistry, Uppsala University, Sweden.
Abstract:
Five different subtypes (human m1, m2, m5 and rat m3, m4) of muscarinic acetylcholine receptors (mAChR) were produced in insect Sf9 cells by infection with recombinant baculoviruses. N-[3H]methylscopolamine ([3H]NMS) has a similar affinity to each of these mAChR subtypes in cell membranes, while pirenzepine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyri do- (2,3-b)(1,4)benzo-diazepin-6-on (AF-DX 116) and (+/-)-p-fluoro-hexahydrosiladifenidol (p-F-HHSiD) have a higher affinity for m1, m2 and m3, respectively, than for the other subtypes, indicating the maintenance of subtype specificity of mAChR in this system. Digitonin (1%, w/w) with sodium cholate (0.1%, w/w) solubilized 51% of m1, 36% of m2, 3% of m3, 28% of m4 and 17% of m5 mAChR from these cell membranes with retention of the [3H]NMS binding activity. Optimization of cholate concentrations resulted in solubilization of up to 50-60% for m1, m2 and m4, but up to 25% for m5 and 7% for m3. Optimal concentrations of cholate differed from one subtype to another. Sucrose monolaurate solubilized 21-43% of m1, m2 and m4, but only up to 12% for m5 and 2% for m3. 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS) was practically ineffective in mAChR solubilization from Sf9 cell membranes for all subtypes investigated. Solubilization with digitonin and cholate had little influence on [3H]NMS affinity for m2 and m4, but decreased m1 and m5 affinity by 10-fold and that of m3 by more than 50-fold. These results indicate that the solubility and stability of mAChR in detergents differ among the subtypes, in spite of their structural similarities. These differences should be taken into account when comparing the five subtypes, particularly when determining the proportion of each subtype in a given tissue by precipitating the solubilized mAChR with subtype-specific antibodies.
Insights
This study explored muscarinic acetylcholine receptor (mAChR) subtypes in insect cells, finding that detergents solubilize them differently. These differences impact binding affinity and must be considered for subtype analysis.
Area of Science:
- Pharmacology
- Molecular Biology
- Biochemistry
Background:
- Muscarinic acetylcholine receptors (mAChRs) are crucial G protein-coupled receptors involved in various physiological processes.
- Five subtypes (m1, m2, m3, m4, m5) exist, exhibiting distinct pharmacological profiles and tissue distributions.
- Understanding mAChR subtype behavior is vital for developing targeted therapeutics.
Purpose of the Study:
- To investigate the solubilization and binding characteristics of five mAChR subtypes expressed in insect Sf9 cells.
- To assess the impact of different detergents on mAChR subtype stability and ligand affinity.
- To provide insights into the differential behavior of mAChR subtypes for accurate quantification and comparison.
Main Methods:
- Production of human (m1, m5) and rat (m3, m4) mAChR subtypes in Sf9 cells using recombinant baculoviruses.
- Radioligand binding assays using N-[3H]methylscopolamine ([3H]NMS) to assess affinity for various mAChR subtypes.
- Solubilization of cell membranes using detergents like digitonin, sodium cholate, sucrose monolaurate, and CHAPS.
- Evaluation of detergent effects on [3H]NMS binding affinity post-solubilization.
Main Results:
- All five mAChR subtypes maintained subtype-specific ligand affinities in cell membranes.
- Digitonin and cholate effectively solubilized m1, m2, and m4 mAChRs, with varying efficiencies for m3 and m5.
- Solubilization significantly reduced ligand affinity for m1, m3, and m5 mAChRs, while m2 and m4 showed minimal changes.
- Different detergents exhibited varying efficacies and impacts on mAChR subtype solubility and stability.
Conclusions:
- mAChR subtypes display distinct solubility and stability profiles in detergents, despite structural similarities.
- The choice of detergent and its concentration is critical for effective mAChR solubilization while preserving binding activity.
- These subtype-specific differences in detergent interaction are important for accurate comparative studies and quantification of mAChR subtypes in biological samples.