The Hydrophobic Residues in Amino Terminal Domains of Cx46 and Cx50 Are Important for Their Gap Junction Channel Ion Permeation and Gating.

Lens gap junctions (GJs) formed by Cx46 and Cx50 are important to keep lens transparency. Functional studies on Cx46 and Cx50 GJs showed that the Vj-gating, single channel conductance (γj), gating polarity, and/or channel open stability could be modified by the charged residues in the amino terminal (NT) domain. The role of hydrophobic residues in the NT on GJ properties is not clear. Crystal and cryo-EM GJ structures have been resolved, but the NT domain structure has either not been resolved or has showed very different orientations depending on the component connexins and possibly other experimental conditions, making it difficult to understand the structural basis of the NT in Vj-gating and γj. Here, we generated missense variants in Cx46 and Cx50 NT domains and studied their properties by recombinant expression and dual whole-cell patch clamp experiments on connexin-deficient N2A cells. The NT variants (Cx46 L10I, N13E, A14V, Q15N, and Cx50 I10L, E13N, V14A, N15Q) were all able to form functional GJs with similar coupling%, except Cx46 N13E, which showed a significantly reduced coupling%. The GJs of Cx46 N13E, A14V and Cx50 E13N, N15Q showed a reduced coupling conductance. Vj-gating of all the variant GJs were similar to the corresponding wild-type GJs except Cx46 L10I. The γj of Cx46 N13E, A14V, Cx50 E13N, and N15Q GJs was reduced to 51%, 82%, 87%, and 74%, respectively, as compared to their wild-type γjs. Structural models of Cx46 L10I and A14V predicted steric clashes between these residues and the TM2 residues, which might be partially responsible for our observed changes in GJ properties. To verify the importance of hydrophobic interactions, we generated a variant, Cx50 S89T, which also shows a steric clash and failed to form a functional GJ. Our experimental results and structure models indicate that hydrophobic interactions between the NT and TM2 domain are important for their Vj-gating, γj, and channel open stability in these and possibly other GJs.

Differential Domain Distribution of gnomAD- and Disease-Linked Connexin Missense Variants.

Twenty-one human genes encode connexins, a family of homologous proteins making gap junction (GJ) channels, which mediate direct intercellular communication to synchronize tissue/organ activities. Genetic variants in more than half of the connexin genes are associated with dozens of different Mendelian inherited diseases. With rapid advances in DNA sequencing technology, more variants are being identified not only in families and individuals with diseases but also in people in the general population without any apparent linkage to Mendelian inherited diseases. Nevertheless, it remains challenging to classify the pathogenicity of a newly identified connexin variant. Here, we analyzed the disease- and Genome Aggregation Database (gnomAD, as a proxy of the general population)-linked variants in the coding region of the four disease-linked α connexin genes. We found that the most abundant and position-sensitive missense variants showed distinct domain distribution preference between disease- and gnomAD-linked variants. Plotting missense variants on topological and structural models revealed that disease-linked missense variants are highly enriched on the structurally stable/resolved domains, especially the pore-lining domains, while the gnomAD-linked missense variants are highly enriched in the structurally unstable/unresolved domains, especially the carboxyl terminus. In addition, disease-linked variants tend to be on highly conserved residues and those positions show evolutionary co-variation, while the gnomAD-linked missense variants are likely on less conserved residue positions and on positions without co-variation. Collectively, the revealed distribution patterns of disease- and gnomAD-linked missense variants further our understanding of the GJ structure-biological function relationship, which is valuable for classifying the pathogenicity of newly identified connexin variants.

The Complex and Critical Role of Glycine 12 (G12) in Beta-Connexins of Human Skin.

Glycine is an amino acid with unique properties because its side chain is composed of a single hydrogen atom. It confers conformational flexibility to proteins and conserved glycines are often indicative of protein domains involving tight turns or bends. All six beta-type connexins expressed in human epidermis (Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx32) contain a glycine at position 12 (G12). G12 is located about halfway through the cytoplasmic amino terminus and substitutions alter connexin function in a variety of ways, in some cases altering protein interactions and leading to cell death. There is also evidence that alteration of G12 changes the structure of the amino terminus in connexin- and amino acid- specific ways. This review integrates structural, functional and physiological information about the role of G12 in connexins, focusing on beta-connexins expressed in human epidermis. The importance of G12 substitutions in these beta-connexins is revealed in two hereditary skin disorders, keratitis ichthyosis and erythrokeratodermia variabilis, both of which result from missense mutations affecting G12.

Crucial motifs and residues in the extracellular loops influence the formation and specificity of connexin docking.

Most of the early studies on gap junction (GJ) channel function and docking compatibility were on rodent connexins, while recent research on GJ channels gradually shifted from rodent to human connexins largely due to the fact that mutations in many human connexin genes are found to associate with inherited human diseases. The studies on human connexins have revealed some key differences from those found in rodents, calling for a comprehensive characterization of human GJ channels. Functional studies revealed that docking and formation of functional GJ channels between two hemichannels are possible only between docking-compatible connexins. Two groups of docking-compatible rodent connexins have been identified. Compatibility is believed to be due to their amino acid residue differences at the extracellular loop domains (E1 and E2). Sequence alignment of the E1 and E2 domains of all connexins known to make GJs revealed that they are highly conserved and show high sequence identity with human Cx26, which is the only connexin with near atomic resolution GJ structure. We hypothesize that different connexins have a similar structure as that of Cx26 at the E1 and E2 domains and use the corresponding residues in their E1 and E2 domains for docking. Based on the Cx26 GJ structure and sequence analysis of well-studied connexins, we propose that the E1-E1 docking interactions are staggered with each E1 interacting with two E1s on the docked connexon. The putative E1 docking residues are conserved in both docking-compatible and -incompatible connexins, indicating that E1 does not likely serve a role in docking compatibility. However, in the case of E2-E2 docking interactions, the putative docking residues are only conserved within the docking-compatible connexins, suggesting the E2 is likely to serve the function of docking compatibility. Docking compatibility studies on human connexins have attracted a lot of attention due to the fact that putative docking residues are mutational hotspots for several connexin-linked human diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.