Cooperative block of the plant endomembrane ion channel by ruthenium red

Effects of ruthenium red (RR) on the slow Ca(2+)-activated Ca(2+)-permeable vacuolar channel have been studied by patch-clamp technique. Applied to the cytosolic side of isolated membrane patches, RR at concentrations of 0.1-5 microM produced two distinct effects on single channel kinetics, long lasting closures and a flickering block of the open state. The first effect was largely irreversible, whereas the second one could be washed out. The extent of flickering block steeply increased (zdelta = approximately 1.35) with the increase of cytosol-positive voltage, dragging RR into the channel pore. At least two RR ions are involved in the block according to Hill coefficient n = approximately 1.30 for the dose response curves. The on-rate rate of the drug binding linearly depended on the RR concentration, implying that one RR ion already plugged the pore. The blocked state was further stabilized by binding of the second RR. This stabilization was in excess of that predicted by independent binding as the dependence of unblocking rate on RR concentration revealed. A cooperative model was therefore employed to describe the kinetic behavior of RR binding. At zero voltage the half-blocking RR concentration of 36 microM and the bimolecular on-rate constant of 1.8 x 10(8) M(-1) s(-1) were estimated.