Single anion channels reconstituted from cardiac mitoplasts

Abstract

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. The appearance of anion rather than cation channels could be promoted by exposing the bilayers to osmotic gradients formed by Cl- salts of large, relatively impermeant, cations at a pH of 8.8. Two distinct activities were identified. These comprised a multisubstate anion channel of intermediate conductance (approximately 60 pS in 300 vs. 50 mM choline Cl, approximately 100 pS in symmetric 150 mM KCl), and a lower-conductance anion channel (approximately 25 or approximately 50 pS in similar conditions), which only displayed two well-defined substates, at approximately 25 and approximately 50% of the fully open state. The larger channels were not simple multiples of the lower-conductance channels, but both discriminated poorly, and to a similar extent, between anions and cations (PCl-/Pcholine+ approximately 12, PCl-/PK+ approximately 8). The lower-conductance channel was only minimally selective between different anions (PNO3-(1.0) = PCl- > PBr- > PI- > PSCN-(0.8)), and its conductance failed to saturate even in high (> 1.0 M) activities of KCl. The channels were not obviously voltage dependent, and they were unaffected by 0.5 mM SITS, H2O2, propranolol, quinine or amitriptyline, or by 2 mM ATP, or by variations in pH (5.5-8.8). Ca2+ and Mg2+ did not alter single channel activity, but did modify single current amplitudes in the lower-conductance channel. This effect, together with voltage-dependent substate behavior, is described in the following paper.