Cooperative block of the plant endomembrane ion channel by ruthenium red

Effects of ruthenium red (RR) on the slow Ca2+-activated Ca2+-permeable vac uolar channel have been studied by patch-clamp technique. Applied to the cy tosolic side of isolated membrane patches, RR at concentrations of 0.1-5 mu M produced two distinct effects on single channel kinetics, long lasting c losures and a flickering block of the open state. The first effect was larg ely irreversible, whereas the second one could be washed out. The extent of flickering block steeply increased (z delta = similar to 1.35) with the in crease of cytosol-positive voltage, dragging RR into the channel pore. At l east two RR ions are involved in the block according to Hill coefficient n = similar to 1.30 for the dose response curves. The on-rate rate of the dru g binding linearly depended on the RR concentration, implying that one RR i on already plugged the pore. The blocked state was further stabilized by bi nding of the second RR, This stabilization was in excess of that predicted by independent binding as the dependence of unblocking rate on RR concentra tion revealed. A cooperative model was therefore employed to describe the k inetic behavior of RR binding. At zero voltage the half-blocking RR concent ration of 36 mu M and the bimolecular on-rate constant of 1.8 x 10(8) M-1 s (-1) were estimated.