Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Bai, Donglin

Abstract

Arginine76 (R76) or the equivalent residue is highly conserved in most connexins, mutations on this residue in six connexins have been linked to five different inherited diseases, indicating its functional importance. Here we examined the functional status and properties of gap junctions (GJs) containing R76 mutations in Cx50 (R76H and R76C), Cx45 (R75H) and Cx43 (R76H/S/C) with an emphasis on GJ function by pairing the mutants with corresponding wildtype or another docking compatible connexin. Cx50 R76H, R76C and Cx45 R75H failed to form functional homotypic or heterotypic GJs. Cx43 R76H and R76S formed functional GJs by themselves or paired with wildtype Cx43, but with altered properties. However, Cx43 R76C failed to form functional GJs by itself or with Cx43. Interestingly all three Cx43 mutants formed functional GJs when paired with Cx45. Our findings and homology structure models provide molecular insights into the possible pathogenic mechanism of R76 mutants in these connexins.

Summary for Lay Audience

Most tissue cells communicate directly with each other to maintain synchronized activities via protein channels known as gap junctions (GJs). Each GJ is made up of two hemichannels and each hemichannel is formed by the oligomerization of six connexin proteins. Tissue cells typically express more than one connexin type and different tissues often express different types of connexins. Thus, different types of GJs can be formed to synchronize cells in the same tissue or to propagate signals between different tissues to meet physiological demands. The arginine residue at the 76th position (R76) is at a critical structural junction in the GJ structure and this residue never changes during the evolutionary process of different species and among different types of connexins. Mutations at the R76 or equivalent residue in six different connexins have been associated with five inherited diseases, although the molecular mechanisms have not been fully studied, especially on GJs formed by mutant expressing cells paired with wildtype connexins. Here, we studied the functional status and properties of R76 mutations in connexin 50 (Cx50), Cx45, and Cx43 with an emphasis on mutant expressing cells paired with cells expressing wildtype connexin or another connexin normally capable of forming functional GJs. We found that Cx50 R76H, R76C and Cx45 R75H were unable to form functional GJs in any of our tested pairings. Cx43 R76H and R76S formed functional GJs as well as forming functional GJs when paired with wildtype Cx43, but with altered channel properties. Cx43 mutants (R76H, R76S, and R76C) all formed functional GJs when paired with Cx45. Our study indicated that most of the R76 mutations in these studied connexins are loss-of-function mutations for GJ function, even when paired with wildtype or different compatible connexins. We also developed molecular structure models to see possible changes associated with each mutant on this residue. We revealed that mutations on R76 or equivalent residue led to various degrees of loss in local molecular interactions within the same connexin or between neighbouring connexins in a hemichannel, which could lead to impaired GJ function and disease state for patients who carry mutations on this residue.

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