Abstract

Gap junctions play a crucial role in embryogenesis, especially of the heart where connexin43 is thought to be important for its formation as well as synchronized contraction. Several reports have shown that connexin43 protein is phosphorylated on serine residues in vitro and in vivo.

Studies performed in our laboratory have demonstrated that cell-to-cell communication in cells expressing connexin43 can be controlled rapidly and reversibly by microinjection of active protein kinases or phosphatases that target serine or threonine residues. Phosphorylation by cAMP-dependent protein kinase (PKA) seems to favor channel opening, whereas the effect of protein kinase C (PKC) on channel gating depends on the existing phosphorylation state of connexin43 protein. The cytoplasmic carboxyl terminus of connexin43 contains several consensus sites for phosphorylation by PKA and PKC, especially residues 359 to 376, which contain three tandem Arg-X-Ser-Ser sequences.

In this study we demonstrate that this domain of connexin43 is a target for phosphorylation, which then raised the question of how these modifications are able to affect channel opening and closing. We proposed a model for gating of connexin43 channels in which phosphorylation of the serines allows them to interact with positively charged lysine residues in the cytoplasmic loop in an antiparallel coil-coil motif. This interaction moves the cytoplasmic tail away from the pore and may favour channel opening. To test this hypothesis site-directed mutagenesis was used to construct expression plasmids for connexin43 in which a) serines 364, 368 and 372 of the cytoplasmic tail were replaced by fixed negatively charged aspartates or b) lysines 102, 105, 108, and 109 of the cytoplasmic loop were replaced with neutrally charged glutamines or negatively charged glutamates. These plasmids were then transfected into a communication-deficient cell line. Both of the cytoplasmic loop mutants showed reduced levels of communication as established by dye transfer. In addition their single channel conductance was predominantly seen to be 36 pS, which is the smallest conductance substate of connexin43 channels.

Based on these results I conclude that gating of connexin43 channels is intricately regulated by phosphorylation and that the C-loop and C-tail domains are probably involved in this mechanism.

LLU Discipline

Physiology

Department

Physiology

School

Graduate School

First Advisor

William H. Fletcher

Second Advisor

Lora M. Green

Third Advisor

Lee M. Graves

Fourth Advisor

Subburaman Mohan

Fifth Advisor

Lawrence B. Sandberg

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1999

Date (Title Page)

9-1999

Language

English

Library of Congress/MESH Subject Headings

Heart -- anatomy & histology; Heart -- physiology; Gap Junctions; Embryo Transfer; Cloning Organism; Connexin 43; Phosphorylation; Myocardial Contraction

Type

Dissertation

Page Count

xii; 240

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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