A Point Mutation in the Coding Sequence of the β-Hexosaminidase α Gene Results in Defective Processing of the Enzyme Protein in an Unusual GM2-Gangliosidosis Variant

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-3H]methylscopolamine ( 3H]NMS) and 3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 11-22-(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido2,3-b]1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited 3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with 3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.