MOLECULAR PROPERTIES AND STRUCTURE OF CONNEXINS IN HEART

  • Nicholson, Bruce J (PI)

Project: Research project

Project Details

Description

In heart, gap junctions serve the crucial role of electrically coupling
muscle fibers to ensure rapid propagation of synchronous contractions.
However, the identification of multiple connexins in heart (i.e.,
Cx37,40,43,45,46) clearly indicates a greater complexity. We and others
have demonstrated that intracellular channels comprised of some of these
connexins show distinctive gating properties and conductance.
Heterotypic interactions between connexins can further modify these
properties. This diversity of connexins in heart could then confer
subtle (and possibly regional) regulation of electrical conductance or
could reflect specializations for additional roles such as metabolic
coupling or second messenger transmission. We propose to analyze the
nature of the specialized properties conferred by these different
connexins and their potential role in cardiac function. Initially, the distribution of these different junctional proteins will
be established using both in situ hybridization with highly specific
nucleotide probes and antibodies raised to peptides from the deduced
sequences in complementary studies on rat and bovine heart sections.
Concurrent analyses using the Xenopus oocyte expression system will be
aimed at defining the specific properties of intercellular channels
comprised of different connexins. Using paired Xenopus oocytes injected
with cRNAs of the connexins of interest, two major groups of experiments
are proposed. First, the gating properties of the various connexins in
response to voltage, pH and Ca++ will be determined using dual cell
voltage clamps. Possible heteromeric interactions between connexins and
their effects on channel properties will also be investigated. Relating
these results to the in vivo distribution of connexins may indicate the
manner in which gap junctions modulate the flow of current in the heart.
A second series of studies will investigate the passage of larger
molecules through junctions. These studies have significance for second
messenger responses in heart. In the laboratory of Dr. David Triggle,
we will develop a graded series of probes which will allow the
determination of exclusion limits and selectivity of junctions comprised
of each of the cardiac connexins and their potential hybrid forms. By
modifying these probes with respect to surface charge and
hydrophobicity, and incorporating photoactivatable cross-linking groups,
the nature of the residues lining the channel which may contribute to
specificity will also be studied. Site-directed mutagenesis of channel
residues will further refine our understanding of the structure of these
channels and the molecular basis of any selectivity filters that may be
detected. These studies will define the properties of intercellular
communication in the heart at a molecular level, so that the roles of
junctional proteins in electrical conductance and as regulators of
excitability can be addressed.
StatusFinished
Effective start/end date8/1/9212/31/96

Funding

  • National Institutes of Health
  • National Institutes of Health: $175,525.00
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)

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