Title

A study of connexin43 cell-to-cell channels in cultured cells

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Physiology and Biophysics

First Committee Member

Birgit Rose, Committee Chair

Abstract

The formation of cell-to-cell channels and their clusters, the gap junction plaques, was studied in a transformed cell line, rat Morris hepatoma cells (MHD1), using microinjection of fluorescent tracers to detect open channels, Northern blots to quantify the mRNA for channel protein, immunoprecipitation and Western blots to identify and characterize biochemically the channel protein, and immunostaining to pinpoint subcellular localization of the channel protein.A low level of the channel protein connexin43 was detected in the cells, but open cell-to-cell channels were not. Increasing intracellular cAMP concentration induced open channels, raised the level of connexin43 mRNA and protein and brought about connexin43 phosphorylation. To force abundant expression (overexpression) of connexin43, cells were transfected with connexin43 cDNA. Overexpression did not per se result in open channels or in the channel clusters of gap junction plaques, nor was connexin43 phosphorylated; however, when overexpression was combined with inhibition of glycosylation, open channels and phosphorylated connexin43 were induced but clusters were not. Clustering ensued after subsequent activation of protein kinase C. In contrast to protein kinase C, activation of protein kinase A by cAMP induced open channels and caused clustering in the absence of glycosylation inhibition. Disruption of microfilaments did not affect already existing channels but prevented all clustering and aided protein kinase C in closing channels. Expression of cadherin promoted channel formation in MHD1 cells but not in another cell line. I conclude that intercellular communication in MHD1 cells is mediated by connexin43 channels; that channel formation and channel clustering are impeded by cell surface carbohydrates and that they are independently regulated processes, channel clustering involving protein kinase A and C and requiring intact microfilaments; and that cell-cell adhesion molecules are not required for channel formation but that they can promote or hinder it, depending on cell type.

Keywords

Biology, Cell; Biology, Animal Physiology; Biophysics, General

Link to Full Text

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