Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate |
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Authors: | A Eliassi L Garneau G Roy R Sauvé |
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Institution: | (1) Department of Physiology, Membrane Transport Research Group, University of Montréal, C.P. 6128, Succ. Centre-ville, Montréal Canada H3C 3J7, CA |
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Abstract: | We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes
of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with
a mean conductance of 164 ± 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl− than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with
an open probability value ranging from 0.9 at −20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could
also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease
of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis
of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern
was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability
sequence for test halides determined from reversal potentials was Br− > Cl− > I−≈ F−. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br− and Cl− have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel
activity was not affected by 50 μm DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl−. Our results suggest that channel block involves the interaction of HPO2−
4 with a site located at 70% of the membrane span.
Received: 10 January 1997/Revised: 29 May 1997 |
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Keywords: | : Endoplasmic reticulum — Cl− channel — Phosphate — Bilayer — Vesicles — Cl− Channel in ER |
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