Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Neuroscience

Supervisor

Donglin Bai

Abstract

Gap junction (GJ) channels directly connect the cytoplasm of neighboring cells, and allow for the transfer of ions and small molecules between two cells. GJ channels are known to be gated by the transjunctional voltage (Vj, the voltage difference between the interiors of adjoining cells), which is referred to as Vj-dependent gating. GJ channels show varying degrees of sensitivity to Vj, depending on the type of connexin(s) comprising the GJ channel. GJ channels formed by different connexins also show unique unitary conductance ranging from ~ 10 pS to ~300 pS. However, the molecular structures determining these unique channel properties are not clear.

Our objective is to investigate the role of the amino terminal (NT) domain in determining the differences in the Vj-dependent gating and unitary conductance of the Cx36 and Cx50 GJ channels. We generated a series of mutants by exchanging the amino-terminus (NT) domains or individual amino acid residues in the NT, between Cx36 and Cx50. Dual whole-cell patch-clamp technique was applied to characterize these channel properties in neuroblastoma N2A cells transfected with these connexin mutants.

First we demonstrated that the NT domain of Cx36 engineered to replace the corresponding domain of Cx50 imparts the Cx50 GJ channel similar but not identical Vj­-dependent gating and unitary conductance to the Cx36 GJ channels. This suggests that the NT is crucial, although it may not be the only domain that determines these properties. We then focused on some charged residues in the NT of Cx36 and Cx50. We found that introducing the positively charged Arg (9th position of Cx36 NT) into the corresponding site of Cx50 resulted in functional GJ channels with remarkably modified Vj-dependent gating and reduced unitary conductance (by 30%). When the negatively charged Asp3 of Cx50 was replaced by the negatively charged Glu from the corresponding position of Cx36, the mutant GJ channel also showed drastic changes in Vj-dependent gating. Finally, in order to find out the relative contribution of each hemichannel during Vj-dependent gating process, we examined heterotypic GJ channels formed by Cx50 and Cx50-mutants. Our study suggests that complex interactions exist between the two hemichannels during Vj-dependent gating process, which therefore inducing novel properties to heterotypic GJ channels.

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