Characterization of a chloride channel reconstituted from cardiac sarcoplasmic reticulum

Abstract

We have characterized a voltage-sensitive chloride channel from cardiac sarcoplasmic reticulum (SR) following reconstitution of porcine heart SR into planar lipid bilayers. In 250 mm KCl, the channel had a main conductance level of 130 pS and exhibited two substrates of 61 and 154 pS. The channel was very selective for Cl⁻ over K⁺ or Na⁺ ($P_{{\text{K}}^{\text{ + }} } /P_{{\text{Cl}}^{\text{ - }} } = 0.012$ and $P_{{\text{Na}}^{\text{ + }} } /P_{{\text{Cl}}^{\text{ - }} } \sim 0.040$). It was permeable to several anions and displayed the following sequence of anion permeability: SCN⁻ > I⁻ > NO ⁻₃ ∼ Br⁻ > Cl⁻ > f⁻ > HCOO⁻. Single-channel conductance saturated with increasing Cl⁻ concentrations (K _m= 900 mm and γ_max = 488 pS). Channel activity was voltage dependent, with an open probability ranging from ∼1.0 around 0 mV to ∼0.5 at +80 mV. From −20 to +80 mV, channel gating was time-independent. However, at voltages below −40 mV the channel entered a long-lasting closed state. Mean open times varied with voltage, from ∼340 msec at −20 mV to ∼6 msec at +80 mV, whereas closed times were unaffected. The channel was not Ca²⁺-dependent. Channel activity was blocked by disulfonic stilbenes, arylaminobenzoates, zinc, and cadmium. Single-channel conductance was sensitive to trans pH, ranging from ∼190 pS at pH 5.5 to ∼60 pS at pH 9.0. These characteristics are different from those previously described for Cl⁻ channels from skeletal or cardiac muscle SR.