Functional expression and biophysical properties of polymorphic variants of the human gap junction protein connexin37.
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-fs254Delta293) 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-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) 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-fs254Delta293 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.
Kumari
S S
SS
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
Varadaraj
K
K
Valiunas
V
V
Ramanan
S V
SV
Christensen
E A
EA
Beyer
E C
EC
Brink
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017630:Connexins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D000939:Epitopes; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000378:metabolism; D011110:Polymorphism, Genetic; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000097006:Gap Junction alpha-4 Protein
D017630:Connexins; D018076:DNA, Complementary; D000939:Epitopes; D000097006:Gap Junction alpha-4 Protein