Kinetics of the functional loss of different muscarinic receptor isoforms in xenopus oocytes

Native Xenopus oocytes express two isoforms of muscarinic receptors that mediate qualitatively different physiological responses. Oocytes of the majority of donors (common) express M₃-like receptors (M₃Rs) at comparable densities at both the animal and vegetal hemispheres of the cell. Rare (variant) donors possess oocytes that express mainly M₁-like receptors (M₃Rs), localized predominantly at the animal hemisphere. We have investigated the apparent degradation of these two isoforms and its relationship to their hemispheric distribution. Cycloheximide (CHX) caused a time-dependent decrease in receptor-mediated responses and [³H]quinuclidinyl benzylate (QNB) binding in oocytes from both types of donors. The t( 1/2 ), values ranged between 3 and 7 h. Removal of CHX resulted in rapid recovery of the response. This implied rapid degradation and turnover of both types of receptors. The loss of M₁Rs was more rapid than that of M₁Rs. Moreover, the decrease was more rapid and more extensive on the animal hemisphere in both types of donors. Injection of oocytes expressing either receptor isoform with specific antisense oligonucleotides complementary to either m₁ or m₃ muscarinic receptors (from mouse) showed receptor loss at approximately the same rate as that calculated from experiments with CHX. Furthermore, oocytes of variant donors express M₁Rs exclusively on the animal hemisphere, while the residual activity round on the vegetal hemisphere of the cell was mediated by M₃Rs. Inhibition of putative receptor glycosylation with tunicamycin caused a rapid decrease in receptor-mediated responses and radioligand binding on M₁Rs, but had virtually no effect on M₃Rs. The expression of cloned m₁ muscarinic receptors, however, was not affected by tunicamycin, suggesting that glycosylation is not a general prerequisite for the functional expression of muscarinic receptors.