Bilayer channels, sucrose-permeable

The studies outlined in this section describe the ways we have addressed the foregoing problems of connexin reconstitution by utilizing connexin-32, the predominant form of connexin in rat liver. Our goals were to establish unambiguously that connexin-32 formed channels in liposome membranes, to identify connexin channels in planar bilayers, and to study their properties. Two methods were used to identify reconstituted channels formed by connexin-32. In one method, protein was solubilized from preparations of junctional membrane and incorporated into unilamellar liposomes. Connexin-32 was identified as a channel-forming protein by its specific enrichment in liposomes that were permeable to sucrose. In the other method, connexin-32 was affinity-purified (with a monoclonal antibody directed specifically against connexin-32) directly from octylglucoside-solubilized plasma membranes. Liposomes formed with such material were permeable to sucrose and Lucifer Yellow. Sucrose-permeable liposomes from each method were fused with planar bilayers to study the properties of connexin channels. 

Bilayer Channels from Sucrose-Permeable Liposomes (87, 101, 102). Sucrose-permeable liposomes from the foregoing studies were fused with planar phospholipid bilayers (110, 111). The data from the liposomes that contain connexin from isolated junctions differed in some respects from the data obtained with the affinity-purified connexin. At the present time, the data from the affinity-purified material is not fully characterized. Therefore, most of the data described in the following text are from liposomes that contain connexin-32 from isolated junctions exceptions are noted. 

Figure 7. Bilayer conductance induced by affinity-purified connexin-32. A, Sucrose-permeable liposomes formed with affinity-purified connexin-32 were fused with planar phospholipid bilayers as described. Highly filtered (5-Hz comer frequency) currents show unstable conductances, but large, rapid fluctuations that cluster around multiples of about 125 pS may be discerned (arrowheads). The bilayer voltage was 50 mV. B, Higher resolution recording of channels from affinity-purified connexin-32. Records show discrete gating conductance transitions, but with a high rate and amplitude of current fluctuations through the open channels. Unitary conductance is difficult to determine, but is near 200 pS. The bilayer voltage was 100 mV.

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