Asn-Linked Glycosylation Contributes to Surface Expression and Voltage-Dependent Gating of Cav1.2 Ca<SUP>2+</SUP> Channel

초록

The Ca(v)1.2 Ca2+ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca(v)1.2 into Gln (Q) to explore the effects of N-glycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca(v)1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca(v)1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca(v)1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca(v)1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca(v)1.2/WT. Tunicamycin treatment of oocytes expressing Ca(v)1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that N-glycosylation contributes to the surface expression and voltage-dependent gating of Ca(v)1.2.

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