STRUCTURE-FUNCTION STUDIES OF GAP JUNCTIONS

The mechanisms underlying gating of ion channels are not yet fully
understood. The application of molecular genetic and biophysical
techniques should lead to the description of the properties, organization
and primary sequence of the protein domains that are responsible for the
voltage dependence of gap junctions. The regulation of intercellular
communication by voltage dependent gap junctions has been postulated to
play a role in development, neural signalling and integration, and control
of secretion. In vertebrates, gap junction proteins are known to be
encoded by a small gene family that shares no extensive sequence homology
with other ion channels. Several gap junction proteins (connexins) for
which cloned DNA are available have been shown to form channels with
different voltage sensitivities and kinetics, but the divergence in their
primary protein sequence is sufficient to prevent the identification of
regions that are required for the expression of voltage dependence. We
will focus our initial investigations on two vertebrate connexins Cx26 and
Cx32. Gap junctions formed from homopolymers of Cx26 differ markedly in
the form and types of voltage dependence when they are compared to
junctions formed by homopolymers of the closely related protein, Cx32.
Heterotypic channels resulting from the union of Cx26 hemichannels with
Cx32 hemichannels are unique in that they display junctional currents that
rectify with a fast time course when transjunctional voltages, Vj, are
applied. This fast Vj dependent rectification is similar to that described
for some electronic synapses formed by gap junctions in the nervous system.
We have developed a new procedure for the formation of gene chimeras that
is not dependent on the existence of sequence homology between the two
domains. We will use this procedure to determine the protein sequences
that are responsible for the differences in voltage dependence of Cx26 and
Cx32 and the fast rectification of heterotypic channels by examining the
properties of channels formed by the expression these chimeras in pairs of
Xenopus oocytes. The role of identified protein domains in the process
voltage dependent gating can be inferred from biophysical analyses and
tested lines the channel lumen and forms a gate that regulates ion flow.
If this hypothesis is verified, the relationship between this domain and
other regions of the molecule that function in the expression of voltage
dependence will be explored. In the long term these studies should provide
an account of the molecular mechanisms that underlie the process of voltage
gating of gap junctions. The descriptions of such molecular mechanisms may
have applicability to gating of other voltage dependent of ion channels and
the should provide information concerning the relationship between protein
structure and its function.