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 (M1Rs), 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 t1/2 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 than that of M3Rs. 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 found 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.