Gap junctions, since their first description, have been proposed as generic mediators of relative non-specific intercellular communication through the direct exchange of ions and small metabolites between neighboring cells. More recently, the identification of a diverse family of proteins (connexins) which form these channels in different tissues has been taken to indicate a degree of specificity or regulation of this pathway. In support of this, different connexins have been shown to form channels with unique gating properties, single channel conductance & permeabilities. In addition overall level of intercellular conductance is modulatable through phosphorylation events. Recently, l have shown that the active modulation of conductance by phosphorylation in the case of Cx43, is a product of changes in single channel conductance. This raises the possibility that the permeability of juctional channels is not only subject to regulation at the transcriptional level (phenotypic region) but also regulatable transiently at the posttranslational level (physiological region). This proposal seeks to rigorously test this hypothesis while also exploring the specific molecular mechanisms which underly the gating of Cx43 gap Juctional channels by phosphorylation. Firstly, two well characterized cell lines for connexin expression (SKHep1 & N2A lines) will be further refined to eliminate endogenous coupling (through selection, or antisense approaches if necessary), and to produce differential levels of connexin expression (via the inducable tet promoter, or through selection) suitable for either single channel analysis or biochemistry. Secondly, both intact & perfused cell pairs will be utilized to define the effects of specific kinase pathways (ie, PKA,PKC,PTK's) on gap junction function. Emphasis will be placed on limiting cross-talk between transduction pathways, either pharmacologically (in the intact system), or addition of specific reagents (in the perfused system), and defining the nature of any conductance changes (i.e. open probability of the channels, their single channel conductance, etc.) Thirdly, the same perfused system will also allow a series of quantitative assessments of the permeability of the gap junctional channels to a series of larger dyes of differing change & steric character. The precise quantitation possible with this system will allow a direct test of the hypothesis that physiologically critical parameters of the gap junction (i.e. permeability to selected metabolites) is subject to acute regulation. Finally, both point mutated & chimeric constructs of Cx43 will be introduced into the cells system to directly test whether kinases operate directly on the Connexin itself, & if so, to define their sites of action. This will also allow us to test alternative models of the molecular basis of channel gating & conductance regulation. It is projected that these studies will significantly advance our understanding of the interplay, between gap junctional coupling & the signal transduction pathways with which they interact, & will allow us to test the exciting possibility of an acute means for modulating the nature of intercellular metabolic flow.
