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. 

A significant fraction of the liposomes that contain junctional protein were sucrose-permeable (Figure 2D and E). This result shows a correlation between protein from junctional membrane and sucrose permeability, but does not, by itself, permit the conclusion that connexin-32 forms the sucrose-permeable pathway. 

The argument against sucrose permeability being due to a nonconnexin protein is as follows If the sucrose permeability was due to a hypothetical nonconnexin protein, and the proteins were distributed independently of one another in the liposomes, then connexin-32 and its fragment would be present in the same ratio in the two liposome populations, each distributed independently of sucrose permeability. Because they are not, it is reasonable to conclude that connexin-32 causes a permeability to sucrose. The presence of some fragment in the sucrose-permeable liposomes is accounted for by its presence in liposomes that also contained full-length (functional) connexin. The conclusion that connexin-32 forms a pore does not require that all nonconnexin protein be excluded from the sucrose-permeable liposomes or that connexin-32 account for all of the sucrose permeability. It relies on the positive and specific correlation between sucrose permeability and enrichment for full-length connexin-32. 

The affinity-purified connexin-32 was incorporated into unilamellar liposomes as before. The connexin-32 induced a sucrose permeability in liposomes, as assayed by the density-shift technique, and gave results essentially identical to those in Figure 2. Liposomes that were sucrose-permeable did not retain the dye Lucifer Yellow (retained by the sucrose-impermeable liposomes), which is near the upper size-permeability limit for gap junction channels (19, 108, 109). The fraction of the liposomes that were permeable to sucrose decreased by a factor of 4 when the pH in the gradients was changed from 7.5 to 6.0. This effect was partially reversible. 

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. 

A 20-pS channel was occasionally observed along with these channels, but because it was closed at zero membrane voltage, it was unlikely to mediate the density shift. Because it was also found in sucrose-impermeable liposomes, the 20-pS channel could not be assigned an identity based on sucrose permeability. This channel may be a minor contaminant or a partially denatured form of connexin-32. 

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.

Erlacher, L, Wyatt, J., Pflugbeil, S., Roller, M., Ullrich, R., Vogelsang, H., et al.. Sucrose permeability as a marker for NSAID-induced gastroduodenal injury, Clin Exp Rheumatol,1998,16 iy.69-Tl. 

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