Exogenous expression of mRNAs provides opportunities for comparison of different proteins in constant environments for evaluation of changes in properties as a result of mRNA modification, and for utilization of systems in which particular properties of the expressed proteins are more accessible to study. The Xenopus oocyte is a well established system for expression of channel proteins including connexins. It will be used to express connexin mRNAs from tissue sources and cloned gap junction cDNAs. The macroscopic current properties of the junctions will be compared to their tissue counterparts to establish the fidelity of expression and possible effects of the different membrane and cytoplasmic environment. To correlate structure and function mRNAs will be modified in specific ways and changes in channel physiology evaluated. Known connexin structures suggest sites for functionally significant changes with respect to conductance, voltage dependence, block by H+ and control by phosphorylation. The oocyte because of its large size permits injection of enzymes and other reagents and this experimental convenience will be exploited to study the effects of kinases on different connexins including modified ones. We have obtained a stable transfectant of a gap junction gene into a cell line that did not express any gap junction gene. The transfected cells form junctions whose properties will be compared to junctions in the cells of origin at both macroscopic and single channel levels. We will study this and other transfectants with respect to such behavioral parameters as growth rate and metastatic potential, to further analyze the functional roles of gap junctions. Cells will be transfected with modified mRNAs that appear of particular interest from results with the oocyte system. We will use transfected cells to study homotypic and heterotypic junctions at the single channel level, and we will evaluate the possibility of heteromeric hemichannels with double transfectants of a single cell line. We will continue to incorporate or reconstitute gap junctions into bilayer membranes and compare their channel properties to those of in situ junctions. Antibodies shown to block gap junctions in situ should provide unequivocal identification of gap junction channels in the bilayers. We will directly study the effects of kinases on channel kinetics without interference by cytoplasmic constituents. We will examine effects of altering the lipid environment. We will also attempt to incorporate fusion proteins or connexins encoded by modified cDNAs. This study provides a three faceted approach that utilizes oocyte, transfection and bilayer systems. Each system provides unique advantages for study of channel physiology and structure-function relations. In addition transfection enables a long term, multigenerational assessment of the role of gap junctions in cell behavior.
