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KMID : 0545120150250081371
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Journal of Microbiology and Biotechnology
2015 Volume.25 No. 8 p.1371 ~ p.1379
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Asn-Linked Glycosylation Contributes to Surface Expression and Voltage-Dependent Gating of Cav1.2 Ca2+ Channel
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Park Hyun-Jee
Min Se-Hong
Won Yu-Jin
Lee Jung-Ha
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Abstract
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The Cav1.2 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 Cav1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Cav1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Cav1.2/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 Cav1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Cav1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Cav1.2/WT. Tunicamycin treatment of oocytes expressing Cav1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Cav1.2.
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KEYWORD
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N-glycosylation, Cav1.2 Ca2+ channel, point mutation, Xenopus oocyte, voltage clamping
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