Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits

被引:101
作者
Cassidy, John S. [1 ]
Ferron, Laurent [1 ]
Kadurin, Ivan [1 ]
Pratt, Wendy S. [1 ]
Dolphin, Annette C. [1 ]
机构
[1] UCL, Dept Neurosci Physiol & Pharmacol, London WC1E 6BT, England
基金
英国惠康基金; 英国医学研究理事会;
关键词
BETA-SUBUNIT; TRAFFICKING; IDENTIFICATION; EXPRESSION; PROTEIN; ALPHA-2-SUBUNIT; MODULATION; CA(V)2.1; CURRENTS; SITE;
D O I
10.1073/pnas.1403731111
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Ca(V)1 and Ca(V)2 voltage-gated calcium channels are associated with beta and alpha(2)delta accessory subunits. However, examination of cell surface-associated Ca(V)2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged Ca(V)2 channels. Here we report the development of fully functional Ca(V)2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although alpha(2)delta subunits copurify with Ca(V)2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether alpha(2)delta subunits are associated with these channels at the cell surface. We found that, whereas alpha(2)delta-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with Ca(V)2.2/beta 1b, despite our finding that alpha(2)delta-1 increases plasma-membrane Ca(V)2.2 expression. However, this was due to occlusion of the antigenic epitope by association with Ca(V)2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between alpha(2)delta-1 and the alpha 1 subunit at the plasma membrane. We further show that, although Ca(V)2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type alpha(2)delta-1 (but not a gabapentin-insensitive alpha(2)delta-1 mutant), the interaction between Ca(V)2.2 and alpha(2)delta-1 is not disrupted by gabapentin. Altogether, these results demonstrate that Ca(V)2.2 and alpha(2)delta-1 are intimately associated at the plasma membrane and allowus to infer a region of interaction.
引用
收藏
页码:8979 / 8984
页数:6
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