The broad relevance of gap junctional communication to human health has been graphically illustrated over the last few years by the direct association of no less than 5 very distinct diseases with defects in connexin genes, indicating that the connexin phenotype of a gap junction may impart specialized properties that lead to diverse functions. This contention is strongly supported by the even greater variety of phenotypes that result from genetic ablation of connexins in mice. Although the variability in connexin sequences in the cytoplasmic domains has led to significant interest in their differential regulation, the major function that the gap junctions subserve is the diffusion of low MW metabolites between cells. Specificity in the filtering of these signals by different connexins would certainly impose a whole new layer of complexity to the topic of intercellular communication. This proposal seeks to build on our previous demonstration of selectivity among connexins for ions, larger traces, and natural metabolites by defining the structure of the pore, its gating elements and selectivity filters, and to use this to develop models to explain the mechanism of selectivity. This information is central to understanding the physiological role of gap junctions, and how quite subtle defects in their structure can lead to a diverse array of disease states in man. Specific steps proposed are:(1) Use SCAM, a form of cysteine scanning mutagenesis in which sulfhydryl reagents are introduced and tested for ability to block the channel, to map the residues of connexins that form the pore; (2) Test the accessability of the SCAM channel blocking reagents to different sites in the channel during different gating states to define the physical locations of the channel gate(s) (3) Further characterize the permselectivity properties of connexins with quantitative assays of families of larger probes that vary in specific properties, and, through mutagenesis of the connexin(s), define the sites that confer selecytivity properties for both artificial and natural permeants (4) Develop a 3-dimensional model of diffusion through the gap junction pore using Poisson- Nernst-Planck theory, incorporating structural information on the pore gleaned in Aims 1 and 2.
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