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

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

Complex channel activity recorded from rat liver GAP junctional membranes incorporated into lipid bilayers

Channels from isolated liver junctional membranes were incorporated into lipid bilayers and studied under voltage clamp conditions. Detergent treatment of junctional membrane fragments greatly increased the incidence of channel incorporation but did not noticeably alter the properties of the incorporated channels. Incorporation resulted in channel activity displaying an approximately symmetric voltage dependence in which conductance was decrease with imposed transmembrane voltage exceeding ñ 20 mV. A residual coltage-independent conductance was also detected in membranes in which liver junctional membranes were incorporated. The magnitude of this voltage-insensitive component varied from less than 20% to more than 75% of the total conductance. These results are generally similar to those described by Young, Chn and Gilula(Cell, 48:733-743, 1987) in incorporation experiments following detergent treatment of isolated gap junction membranes. However, we interpret these data as indicating the existence of distint channel populations in the incorporated membrane fractions. Our results suggest that a population of larger conductance channels (* 150 pS) contributes the voltage-dependent component of the membrane conductance, while smailler channels (unitary conductance abouth 50-150 pS) contribute the voltage-independent component. The biophysical proprieties of the larger channel are comparable to those seen in communication-deficient cells transfected with connexin32, confirming a report describing conductance of bilayers in which electroeluted 27-kDa liver gap junction protein was inserted. These findings indicate that connexin32 comprises the larger, voltage-dependent channels seen in the bilayer experiments in which liver junctional membranes are incorporated