Functional expression and biophysical properties of polymorphic variants of the human gap junction protein connexin37

被引:32
作者
Kumari, SS
Varadaraj, K
Valiunas, V
Ramanan, SV
Christensen, EA [1 ]
Beyer, EC
Brink, PR
机构
[1] SUNY Stony Brook, Dept Physiol & Biophys, Stony Brook, NY 11794 USA
[2] Univ Chicago, Dept Pediat, Chicago, IL 60637 USA
关键词
intercellular communication; connexin; truncation; frame shift; GFP tag; T7; tag; conductance;
D O I
10.1006/bbrc.2000.3054
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254 Delta 293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254 Delta 293 had large unitary conductances (similar to 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254 Delta 293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254 Delta 293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics. (C) 2000 Academic Press.
引用
收藏
页码:216 / 224
页数:9
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