Properties of channels from rat liver gap junction membrane fractions incorporated into planar lipid bilayers

RESUMO

Rat membrane fractions highly enriched for gap junctions can be incorporated into planar lipid bilayers exhibiting channel currents with both voltage-dependent and independent components. Voltage dependence, however, is only one of the characteristics of liver gap junction channels. Other features include poor ionic selectivity and sensitivity to calcium, pH, octanol and to some intracellularly applied antibodies. To further test the junctional nature of channels from membrane fractions highly enriched in gap junctions incorporated into lipid bilayers we studied the sensitivity of these channels to uncoupling agents and determined channel selectivity properties. We found the incorporated channels to be insensitive to calcium and octanol, and in most cases to pH in the range of 5-7, suggesting that either these agents do not interact directly with the junctional channels or that the corresponding gating regions are inactivated during the isolation and reconstitution procedures. Attempts to block channel activity using polyclonal and monoclonal connexin 32 antibodies were generally unsuccessful, although one antibody (a monoclonal directed against the carboxy terminus portion of connexin32) blocked channel activity. Selectivity measurements indicated that the incorporated channels were slightly cation selective (PNa=Pk > PCl) and were permeable to large ions. These results further support the idea that functional connexin32 gap junction channels are present in channel activity recorded from rat liver junctional membranes incorporated into planar bilayers