Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational modification, and unitary channel conductance (gamma j). Despite a 10-fold variation in gamma j, whether connexin-specific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that gamma j is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel gamma j (and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2',7'-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na+ and K+ channels), implying similar mechanisms for selectivity.

We examined the expression and function of gap junctions in two rat osteoblastic cell lines, ROS 17/2.8 and UMR 106‐01. The pattern of expression of gap junction proteins in these two cell lines was distinct: ROS cells expressed only connexin43 on their cell surface, while UMR expressed predominantly connexin45. Immunoprecipitation and RNA blot analysis confirmed the relative quantitation of these connexins. Microinjected ROS cells passed Lucifer yellow to many neighboring cells, but UMR cells were poorly coupled by this criterion. Nevertheless, both UMR and ROS cells were electrically coupled, as characterized by the double whole cell patch‐clamp technique. These studies suggested that Cx43 in ROS cells mediated cell‐cell coupling for both small ions and larger molecules, but Cx45 in UMR cells allowed passage only of small ions. To demonstrate that the expression of different connexins alone accounted for the lack of dye coupling in UMR cells, we assessed dye coupling in UMR cells transfected with either Cx43 or Cx45. The UMR/Cx43 transfectants were highly dye coupled compared with the untransfected UMR cells, but the UMR/Cx45 transfectants demonstrated no increase in dye transfer. These data demonstrate that different gap junction proteins create channels with different molecular permeabilities; they suggest that different connexins permit different types of signalling between cells.

Gap junction channels have long been viewed as static structures containing a large-diameter, aqueous pore. This pore has a high permeability to hydrophilic molecules of approximately 900 daltons in molecular weight and a weak ionic selectivity. The evidence leading to these conclusions is reviewed in the context of more recent observations primarily coming from unitary channel recordings from transfected connexin channels expressed in communication-deficient cell lines. What is emerging is a more diverse view of connexin-specific gap junction channel structure and function where electrical conductance, ionic selectivity, and dye permeability vary by one full order of magnitude or more. furthermore, the often held contention that channel conductance and ionic or molecular selectivity are inversely proportional is refuted by recent evidence from five distinct connexin channels. The molecular basis for this diversity of channel function remains to be identified for the connexin family of gap junction proteins.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins. (J. Clin. Invest. 1993. 91:997-1004