This proposal is focused on elucidating the gating mechanisms of connexins 43 and 37 under defined ionic and expression constraints.
The aims are: 1) Determine the gating characteristics of a non-transfected population of connexin43 derived gap junction channels using dual whole cell batch clamp (DWCP). Human vascular smooth muscle-cell pairs (HVSM) derived from corpora cavernosa, which are known to contain connexin43, will be used as well as NRK cell pairs which contain rat connexin43. Gap junction channel gating will be monitored by measurement of the mean open time(s), mean closed time(s), open probabilities and voltage sensitivity. Further, we ascertain whether there is cooperative gating (non-independent gating between gap junction channels. 2a) Use DWCP in N2A cells transfected with rCx43 or humanCx37 and determine the behavior of heterotypic Cx43/Cx37 gap junction channels. Homotypic behavior for Cx37 is in the literature and we present data in the preliminary results for rCx43 transfected cells. We will continue to study the homotypic forms and determine their gating characteristics for comparison with non-transfected rCx43. Homotypic refers to two identical hemichannels linked to form a gap junction channel (Cx43/Cx43) and a heterotypic refers to two non-identical hemichannels linked together (Cx43/Cx37) where each hemichannel is composed of only one connexin type. 2b) Co-transfect Cx43 and Cx37 into N2A cells (a potential model for endothelial cell intercellular communication) and monitor the multichannel behavior. The low number of channels formed in the N2A cells in combination with our analysis will reveal any gating differences between homotypic and heterotypic gap junction channels as well as gap junctions made of heteromers (heteromers=hemichannels of two different forms,Cx43 and Cx37 found in the same hemichannel). Our assumption is that heteromeric and heterotypic forms are distinguishable from each other and homotypic forms in terms of parameters such as open probabilities, mean open times, mean closed times, cooperative gating, voltage dependence and unitary conductance. 3.) Use direct patch clamp on HVSM cells and transfected N2A cells to identify gap junction channels on the surface of freshly isolated cells. 4) Elucidate gate-to-gate interactions (contingent gating) for single gap junction channels using DWCP data where large deltaVj steps are applied and modeling both macroscopic currents and unitary channel activity. We will also use osmotic loading (Zimmerberg and Parsegian, 1986) to distinguish volume changes between different conductance states of gap junction channels.
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