The significance of gap junction mediated communication to human health is increasingly evident from the human diseases caused by expression of defective gap junction proteins (connexins Cx), including: peripheral neuropathies, cardiac developmental malformations, deafness, cataracts and skin diseases. The diverse phenotypes in mice arising from Cx gene ablation and substitution lend further support to the importance of gap junctions to development and function of the cardiovascular system, in particular, and to the animal in general. The primary function of gap junctions is to mediate the intercellular exchange of the signaling molecules that result in coordinated tissue function in health and disease. The selectivity of this exchange pathway is apparently Cx specific and acutely regulated. The long-term goal of the current proposal is to identify the functional consequences of sequential and simultaneous expression of Cx40 and Cx43. In particular, the selectivity of the junctions formed by Cx43, Cx40 and both connexins, and the mechanisms and structural basis for regulation of junctional selectivity by phosphorylation-dependent mechanisms will be examined.
In Aim 1 the consequences of phosphorylation/dephosphorylation on the selectivity of Cx43 or Cx40 comprised channels and junctions are explored. Selectivity will be quantified using state-of-the-art electrophysiology, fluorescence microscopy and mathematical modeling techniques.
In Aim 2 the structural bases for the responses delineated in Aim 1 are determined. The amino acid residues targeted by specific kinases in each Cx are identified by the powerful combination of electrospray ionization (ES), which allows for rapid and sensitive analyses of proteins and peptides, coupled to an HPLC (LC), which allows for on-line separation of peptide mixtures, followed by tandem mass spectrometry (MS/MS). The functional significance of identified phosphorylation sites in each Cx will be explored using site-directed mutants of those sites.
In Aim 3 the consequences of mixed channel formation on junctional selectivity, the regulation thereof by phosphorylation-dependent mechanisms, and the structural bases for observed regulation are examined. The results of the proposed studies should provide new insights on the functional consequences of differential Cx expression, which is essential information for understanding the structural basis for gap junction channel selectivity and regulation thereof, and necessary data for the development of testable hypotheses regarding the rules of Cx interaction and their potential consequences in the in vivo setting.
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