Intercellular communication mediated by gap junctions is believed to underlie crucial cell behaviors such as the spread of electrical excitation in smooth and cardiac muscle. However, gap junctions also selectively connect cells in many non-excitable tissues. In these cases, the biological significance of communication is not clear. A novel method to define the role of communication between nonexcitable cells will be used: We have created connexin mutations that function as dominant- negative inhibitors of communication. These mutants, which do not form active channels, can interact with normal connexins and suppress their ability to form active channels. To analyze the importance of communication in embryogenesis, organ homeostasis/physiology and tumor suppression, we will express inhibiting connexin mutants in early Xenopus development and during rodent mammary gland morphogenesis and lactation. We have discovered that a human hereditary disorder, X-linked Charcot- Marie-Tooth disease (CMTX), is associated with mutations in the gene encoding connexin32. CMTX is a demyelinating peripheral neuropathy. We have shown that Cx32 is abundant in Schwann cells which is consistent with the presentation of this disease. However, it is not known how the CMTX mutations affect Cx32 channel activity, nor how the activity of Cx32, or other connexins that may be present, influence myelination. To investigate these issues, we will use the paired oocyte system to test identified Cx32 mutations for activity. The cellular location of other connexins that may be present in Schwann cells will be determined. An animal model will be developed to permit the early phases of this disease to be studied. Gap junction channels can be gated by pH, Ca++, transjunctional voltage and, as we have recently shown, by phosphorylation. V-src induced tyrosine phosphorylation of Cx43 profoundly inhibits the activity intercellular channels containing it, consistent with a role for communication in transformation by v-src. We propose to test a) if other connexins can be regulated in this fashion b) if kinases other than v-src can exert a similar effect and c) if gating of Cx43 occurs in vivo in the absence of v-src. The latter will be assessed by producing an antibody specific for Cx43 phophorylated at tyrosine-265.
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