MOLECULAR GENETICS APPROACH TO GAP JUNCTIONS

Gap junctions are found in virtually all animal cells and have evolved to comprise a multigene family, with a multiplicity of properties and regulatory mechanisms that are expressed in a tissue specific manner. The characterization of the genetic elements that underlie gap junction expression may aid in the definition of the differences, as well as similarities, in gap junction properties, regulation and function of the various members of the connexin gene family. The goal of this proposal is to apply a genetic approach to the study of gap junctions by using two organisms particularly amenable to genetic and molecular manipulations; the fruit fly, Drosophila melanogaster and the nematode, Caenorhabditis elegans. These organisms by virtue of their strengths in classical and molecular genetics provide numerous alternate strategies for the identification and isolation of gap junction mutations. Since fundamental molecular and biophysical descriptions of gap junctions are lacking in these organisms, we will generate the necessary immunological and molecular probes and undertake electrophysiological analyses to identify the type, distribution and biophysical properties of gap junctions in different tissues and developmental stages. Due to the inaccessibility of many of the cell types in both organisms to standard electrophysiological recording techniques, the biophysical characterizations will be accomplished by the incorporation of isolated gap junctions into planar lipid bilayers and by the expression of purified mRNA in pairs of Xenopus oocytes. Antibodies directed against connexins and agents known to modulate gap junction gating will be used to confirm the identity of the channels recorded in the artificial systems. The information obtained from the immunological and biophysical approaches provides a means of defining gap junction phenotypes and consequently identifying and recovering potential gap junction mutants. Our strategies for the recovery of mutants include the study of phenotypes that are likely to result as a consequence of changes in the structure or regulation of gap junctions and the construction of segmental aneuploids to identify regions of the Drosophila genome that display dosage sensitive regulation. The recovery of genetic munitions affecting gap junction mediated communication should lead to the cloning of connexin genes from C. elegans and Drosophila and will help to define the role gap junctions play in complex physiological and behavioral functions. The combination of immunological, biophysical, molecular and genetic approaches in Drosophila and C. elegans offers the opportunity to address questions related to the roles of gap junction that are not possible in other organisms lacking powerful genetic tools. Also, since it appears that there is strong conservation between ion channels in vertebrates and invertebrates, studies in Drosophila and C. elegans are likely to provide information applicable to gap junction communication in general.