Molecularly diverse connexin subunits assemble to form gap junctions that differ in their functional properties, and it is presumed that these channels subserve different physiological functions. The nervous system expresses at least seven different connexins. The overall goal of Project 3 is to determine which combinations of nervous system connexins form channels, how these connexins interact and how channel properties differ. We will use both cell-cell channels and hemichannels that operate in isolation without an apposed hemichannel. Electrophysiological studies at macroscopic and single channel levels will be used to characterize gating of junctional conductance, g-j, by transjunctional voltage (v-j) and permeation in wild type and mutated recombinant channels. We will also determine whether cytoplasmic Ca2+ and H+, well-known blockers of junctional communication, act directly on the channel protein. Mutational analysis combined with domain exchange will be used to localize protein domains critical to these processes.
Specific Aim 1. Characterization of v-j dependence of g-j of homotypic and heterotypic junctions. We will determine the combinations of nervous system connexins that will form functional heterotypic junctions and characterize the V-j dependence of these junctions. We will establish whether multiple connexins expressed by the same cell interact in junction formation.
Specific Aim 2. Identification of molecular components involved in voltagegating. Different hemichannels are closed by opposite polarities of V-j. We will use chimeras and point mutations to localize amino acid residues within, as well as outside of the N-terminus that contribute to the gating charge. We will use cell- cell junctions between oocytes and isolated hemichannels to characterize gating properties at the single channel level. These studies will indicate the conditions under which isolated connexin hemichannels may have a physiological or pathophysiological role.
Specific Aim 3. Characterization of permeability of isolated hemichannels. We will determine relative ion permeabilities of Cx46 and Cx43 hemichannels and undertake a mutational analysis to identify components of the selectivity filter. Excised patches containing hemichannels permit sequential application of multiple solutions to the exposed face of the hemichannel, facilitating determination of ionic selectivity. Mutational analysis is facilitated as well, because recombinant connexins can be quickly expressed and characterized in Xenopus oocytes.
Specific Aim 4. Characterization of permeability of cell-cell channels. We will transfect Cx46 and Cx43 into Neuro-2a cells and determine relative ion permeabilities of Cx43/Cx43 and Cx46/Cx46 homotypic channels and Cx43/Cx46 heterotypic channels.
Specific Aim 5. Characterization of Ca2+ and H+ actions on gap junctions. We will directly apply Ca2+ and H+ to outside-out patches containing Cx46 (or Cx43) hemichannels and determine efficacy and mode of action of these agents. Rapid exchange of Ca2+- and H+-containing solutions will provide high time resolution. We hypothesize that these agents act by binding to cause a conformational change that closes the channels. X-linked Charcot-Marie-Tooth disease and visceroatrial heterotaxia are human genetic disorders that have been linked to mutations of connexins. Our studies should shed light on the pathogenesis of these diseases.
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