ANALYSIS OF A NOVEL DOUBLE-BARRELED ANION CHANNEL FROM RAT-LIVER ROUGH ENDOPLASMIC-RETICULUM

The presence of anionic channels in stripped rough endoplasmic reticulum membranes isolated from rat hepatocytes was investigated by fusing microsomes from these membranes to a planar lipid bilayer. Several types of anion-selective channels were observed including a voltage-gated Cl- channel, the activity of which appeared in bursts characterized by transitions among three distinct conductance levels of 0 pS (0 level), 160 pS (O-1 level), and 320 pS (O-2 level), respectively, in 450 mM (cis) 50 mM (trans) KCI conditions. A chi(2) analysis on current records where interburst silent periods were omitted showed that the relative probability of current levels 0 (baseline), 0,, and 0, followed a binomial statistic. However, measurements of the conditional probabilities W(level 0 at pi/level O-2 at 0) and W(level O-2 at pi/level 0 at 0) provided clear evidence of direct transitions between the current levels 0 and O-2 without any detectable transitions to the intermediate level O-1, It was concluded on the basis of these results that the observed channel was controlled by at least two distinct gating processes, namely 1) a voltage-dependent activation mechanism in which the entire system behaves as two independent monomeric channels of 160 pS with each channel characterized by a simple Open-Closed kinetic, and 2) a slow voltage-dependent process that accounts for both the appearance of silent periods between bursts of channel activity and the transitions between the current levels O and 0,. Finally, an analysis of the relative probability for the system to be in levels 0, O-1, and O-2, showed that our results are more compatible with a model in which all the states resulting from the superposition of the two independent monomeric channels have access at different rates to a common inactivated state than with a model where a simple Open-Closed main gate either occludes or exposes simultaneously two independent 160-pS monomers.