Biophysical properties of rat Connexin37 stably expressed in N2A cell lines*

Abstract

Gap junctions have been identified as the conduit for electrical and metabolic coupling between adjacent cells providing modifiable cytoplasmic continuity. Gap junctions consist of subunits called connexins which are members of a gene family with as many as 17 connexin genes or cDNA's found in various species where their expression is tissue specifically and developmentally regulated.;We have assayed the N2A cell line to determine the level of endogenous coupling (which was sparse and mediated by Connexin45) in anticipation of utilizing these cells for exogenous expression of novel connexin gene products for electrophysiological analysis. We co-transfected N2A cells with a vector (pSG5) into which we had subcloned the cDNA encoding rat Cx37, and with a vector containing the gene for neomycin resistance. We isolated seven clones which expressed rat Connexin37 (rCx37) and these have been assayed to various degrees using dual whole cell voltage clamp and immunocytochemistry.;Pairs of these cells express macroscopic currents which relax with time to nonzero steady state values when transjunctional voltage (V{dollar}\sb{lcub}\rm j{rcub}{dollar}) gradients are imposed. Unitary conductance has been shown to have a mainstate and substate. The unitary conductance of the mainstate at low V{dollar}\sb{lcub}\rm j{rcub}{dollar} is large--{dollar}\sp{lcub}\sim{rcub}{dollar}260 pS in 135 mM CsCl internal solution--while the substate has a conductance of {dollar}\sp{lcub}\sim{rcub}{dollar}80 pS with the same internal solution.;We assayed selectivity and permeability of the gap junctions formed by rCx37 by substituting cations or anions of larger size in the internal solution of whole-cell patch pipettes. We report here that rCx37 forms a channel which is markedly cation selective, and which permits passage of cations of different size with differing mobilities. Additionally, we have made an observation consistent with the ion substitution results using confocal microscopy and Indo-1 for Ca{dollar}\sp{lcub}++{rcub}{dollar} imaging: calcium transients were seen to spread from cell to cell even as Lucifer Yellow (an anionic dye) failed to demonstrate coupling. Finally, we have found that the channels are saturated at relatively low ionic strength. ftn{dollar}\sp*{dollar}Originally published in DAI Vol. 57, no. 2. Reprinted with corrected title.