Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform

Abstract

Sarcoplasmic reticulum (SR) K + channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (γ max = 250pS; K m = 33mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (ΔG i) and the effective gating charge (Z s): ΔG i = 0.27kcal/mol andZ s = −1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K + channel shares several biophysical properties with the cardiac isoform: g = 180pS, ΔG i = 0.75kcal/mol, Z s = −1.45 in 150 mM K-gluconate, and a similar sigmoid P o/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K + channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca 2+ (0.1 μM–1 mM) or Mg 2+ (0.25–1 mM) as well as by cGMP (25–100 μM), lemakalim (1–100 μM), glyburide (up to 10 μM) or charybdotoxin (45–200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K + channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.