Molecular Components and Nomenclature of Gap Junctions

Abstract

After the confirmation of the existence of gap junction channels, it was imperative to know their molecular composition. In the beginning, it was regarded that all the gap junctions are made of same kind of protein. However, further studies showed that there exist differences in the protein components of the gap junction. For example, the proteins obtained from various gap junction-enriched preparations showed different electrophoretic mobilities in the range of 21–70 kDa, when detected by SDS-PAGE. This concept was further established after performing micro-sequencing of the amino-terminal regions of these proteins that revealed the differences in the primary sequence of the gap junction proteins. Based on the primary sequence information, oligonucleotide probes were synthesized to screen libraries for the existence of other gap junction proteins. Moreover, generation of antibodies also proved instrumental for the isolation of different gap junction proteins. Based on these techniques, a gap junction protein of 32 kDa was isolated from the liver of rat and human cDNA clones. Similarly, a cDNA encoding a related but a different polypeptide of 43 kDa was isolated from rat heart gap junctions. In the following years, many different gap junction proteins were isolated from various cells and tissues. Thus, it became evident that there exists a family of gap junction proteins. Presently we now know that there are about 20 different gap junction proteins existing in the mouse and human genome. After the discovery of different gap junction proteins, their biochemical characterization was performed, and it was found that the basic principal component of gap junctions is a membrane protein called connexin (Cx). The gap junctions are assembled from the connexin proteins, and this assembly is hierarchical in nature. Connexins assemble together to form a basic unit of structure called the connexon, which is a hexameric structure with a torrid appearance. An individual connexon from one cell docks or associates with a corresponding connexon on a neighbouring cell to form a gap junction channel. Usually, multiple channels cluster or aggregate in the plane of the membrane to form what is called as gap junction plaques. The question arises whether any other proteins, besides connexins, are part of gap junctions. However, the evidences indicate that the gap junctions are purely made of connexin proteins. For example, reconstitution of purified connexins into artificial membranes yields functional channels. Moreover, expression of connexin cDNAs in heterologous systems (including yeast) yields not only functional gap junction channels but also gap junctions that are ultrastructurally identical to those occurring naturally in vivo. Further sequence and structural elucidation of connexins demonstrated that each connexin protein is composed of nine main domains. These include four transmembrane domains, intracellular N-terminal and C-terminal domains, two extracellular loops that are stabilized by intramolecular disulfide bonds, and a cytoplasmic loop (Fig. 3.1). The N-terminus, the two extracellular loops, and the four transmembrane domains are highly conserved among different connexin isoforms. In contrast, the cytoplasmic loop and the C-terminal domain are divergent and variable in length and sequence, thus accounting for the functional differences between the different connexins and the connexon types.