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Gap junctions were initially believed to be constituted by the same connexin until gap junction- enriched sample from different tissues displayed different bands on SDS-PAGE (48-51). Subsequently, more and more evidence came out to indicate that gap junction proteins harbor a big family. Until now, 19 connexin genes in the mouse genome and 21 connexin genes in the

human genome have been deposited in the mouse and human genomic databases. The widest acceptance of nomenclature used to distinguish connexin is word connexin abbreviation connexin followed by the predicted molecular mass of connexins in kilodaltons (52,53). Additionally, a prefix is added to label the species that connexins originate from. An alternative nomenclature was created to clarify some confusion met when homologous connexins have completely different molecular masses in distinct species (e.g., hCx46, bCx44 and cCx56). Connexins are sorted into subgroups (α, β and γ) based on amino acid sequence homology and evolutionary relationships.

Figure 1.6 A molecular phylogenetic tree for human connexin protein (HCx) and rat connexin33 (rCx33).

The divergent sequence portions (the intracellular loop and the carboxyl-terminal domain) were removed from the multiple alignments. The connexin family can be broadly separated into three main subgroups, the historical, α (green) and β (blue) and a more divergent γ branch (54).

Compared with α and β family, γ family is well known and widely divergent. On the above phylogenetic tree (Fig. 1.6), connexins in the γ family show larger evolutionary distances. Sequence alignment of hCx45, hCx26 and hCx50 result indicates that γ family Cx45 does not have the “MDW” amino acid at the beginning of N-termini. Besides, the CL is much longer in Cx45 than that of Cx26 and Cx50. Compared with the β-type connexins, the α-type connexins have a larger M2–M3 cytoplasmic loops and a larger cytoplasmic C-terminal tails. Besides, the second amino acid “G” is absent in beta family amino acid. Finally, the initiation of assembly occurs in distinct cellular organelles (Figure 5); one of the beta family members Cx32 assembly occurs in the ER/ERGIC (55). In contrast, Cx43 (56)and Cx46 (57) which belong to the alpha family start to assemble in the trans-Golgi network (Fig. 1.7).

Figure 1.7 The life cycle of connexin.

Connexins were synthesized in the ER first, following by oligomerization in different intracellular department. The initiation of assembly of Cx32 and Cx43 are from ER and trans-Golgi network, respectively. The hexamers were transported along the microtubules to the cell surface, where plasma membrane insertion occurred. Hemichannels could undergo lateral diffusion in plasma membrane and form gap junction plaque eventually by docking with hemichannels from the other cell. Microtubes participated in the stabilization of gap junction plaque. The degradation of gap junction protein were achieved in lysosome or proteasome (55).

Connexins universally distribute in vertebrate tissues with the exception of several highly differentiated cell types: red blood cell, mature skeletal muscle cell and spermatocytes. The connexin expression pattern is complicated in tissues. One or even more connexins may express in a given tissue and a given connexin can be present in more than one tissues. The cDNA probes significantly contribute to the screening of connexins in different tissues. A list of connexins

expressed in various tissue types are listed below (Table 1.1). Cx26 can be observed in liver, skin, liver and cochlea, but Cx46 and Cx50 expression is exclusively in the eye. Furthermore, in the different stages of one given tissue, the types of connexins vary and the expression amount displays a great variety. Take Cx43 as an example, abundant mRNA of Cx43 and Cx38 had been identified in Xenopus Oocytes before ovulation and meiotic maturation, while Cx43 gradually disappears during the maturation of oocytes. Then Cx38 is replaced with Cx30 by the early gastrula stage (58,59). In summary, the regulation of connexin expression is both temporal and spatial.

Table 1.1 Connexin gene expression pattern in human.

Gene

Expression Patterns

GJB2(Cx26)

GJB1(Cx32)

GJA1(Cx43)

GJCa(Cx45)

GJA3(Cx46)

GJA8(Cx50)

Among all these characterized connexins, it is worthwhile to note that Cx26 usually expresses in conjunction with other connexins in the same family (60). The co-existence of different connexins in one cell dramatically increases the structure combinations of both connexon and GJ channels (Fig. 1.8). Homomeric or heteromeric connexons can be formed by six identical connexins or different connexins, respectively. Another complexity is generated during the docking of connexons from two adjacent cells. GJ channels can be either formed by two identical or different hemichannels, leading the formation of homotypic or heterotypic GJ.

Figure 1.8 Scheme presentation of connexin and gap junction channel.

(a) Connexin is compromised by four transmembrane helices, two extracellular loops, one cytoplasmic loop, N- and C-domains. (b) Six connexins oligomerize to form hemichannels called “connexons”, which then align in the extracellular space to complete the formation of gap junction channels. Different connexins can selectively interact with each other to form homomeric, heteromeric, and heterotypic channels, which differ in their content and spatial arrangement of connexins subunits (61).

It is not certain to what extent the formation of heterotypic gap junction channels will alter the cell communication. The heterogeneous gap junction channels are conducive to cells from the following aspects: 1) expand the diversity of molecules exchanged between cells since different gap junctions exhibit unique permeability; 2) increase the options for fine regulation of gap junctions; 3) provide control over the assembly of hemichannels and gap junction channels (62). It is notable that a given connexin is only compatible to certain connexins. The heterogeneous gap

junction communication had been assessed using the xenopus oocyte expression system (63,64). So far, the compatibility of connexins has been depicted. Bruzzone and his colleagues fused one fragment from the N-terminus to the second transmembrane of Cx32 to Cx43 from the cytoplasmic loop to the C-terminus and overexpressed this mutant in xenopus oocytes. By measuring the gap junction conductance, they found that this mutant only paired functionally with Cx43, but not Cx32. The result indicates that the middle cytoplasmic loop and the C-terminus are portions influence the interaction between different connexins (65).