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Thesis Format

Integrated Article

Degree

Master of Science

Program

Physiology and Pharmacology

Supervisor

Bai, Donglin

Abstract

Connexins form intercellular communication channels known as gap junctions (GJs), which are found throughout the vertebrate species. GJs formed by different connexins harbor unique channel properties that have not been fully defined. High-resolution structures of native Cx46 and Cx50 GJs from sheep (sCx46 and sCx50) were recently resolved. Molecular dynamics studies identified the NT domains, especially the 9th position, as key determinants in the differences of energetic barrier to K+ permeation in sCx46 and sCx50 GJs. We studied functional properties of GJs formed by sCx46, sCx50, NT domain swapped chimeras (sCx46-50NT and sCx50-46NT), and point variants at the 9th residue (sCx46-R9N and sCx50-N9R) in GJ-deficient N2A cells. We found that these variants formed functional GJs except sCx46-50NT. Altered single channel conductance, open dwell time, and Vj-gating in these variants indicate that that the NT-domain is important for the rate of ion permeation, open stability, and Vj-gating of these GJs.

Summary for Lay Audience

The lens of the eye is devoid of blood vessels. As a result, the circulation of substrates in the lens is dependent on gap junction channels. These specialized channels connect neighboring cells directly to allow intercellular transport of ions, nutrients, signaling molecules, and metabolic wastes. Connexins are the building blocks for gap junction channels. Gap junctions composed of different connexins showed distinct channel properties, but the underlying molecular/structural mechanisms are not fully defined. Lens fiber cells express connexin46 and connexin50, and mutations of each of these connexin genes were found to be associated with inherited cataract. Recently, high-resolution structure models of sheep lens connexin46 and connexin50 gap junctions were obtained. The structures and computer simulations of these gap junctions indicate that the amino terminal domains of these connexins are in such a position likely to determine the channel properties, especially at the 9th amino acid residue position. To better understand the functional role of the amino terminal domain and at the 9th residue, we studied variants with switched amino terminal domains or the 9th residues of these two lens connexins. We found that most of our generated variants were functional and showed altered channel properties, such as rate of ion permeation, open stability, and voltage control of opening/closing of these gap junction channels. Our study combines structural simulation and functional study to reveal the mechanisms of how the amino terminal domain determines channel properties in these lens gap junction channels. These findings may also help us understand gap junctions of other connexins.

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Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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