Characterization of the high-affinity verapamil binding site in a plant plasma membrane Ca²⁺-selective channel

Despite biochemical evidence for the existence of high-affinity phenylalkylamine receptors in higher plants, their effects on channel activity have only been demonstrated at relatively high concentrations. We have performed a quantitative single-channel analysis of the changes induced by extracellular verapamil in the rca channel [a wheat root plasma membrane Ca²⁺-selective channel (Pineros and Tester, 1995. Planta 195:478-488)], Concentrations as low as 0.5 μM verapamil induced a blockade of the inward current, with no evident reduction of the single-channel current amplitude. Blockade by verapamil was concentration and voltage dependent. Preliminary analysis suggested the blockade was due to a reduction in the maximum open state probability rather than a change in V_0.5. Further analysis of the association and dissociation rate constants revealed a binding site located 56 to 59% down the voltage drop from the extracellular face of the channel, with a K(d)(0) of 24 to 26 μM. This results in a K(d) at -100 mV of 2 μM. Methoxyverapamil had qualitatively the same effects. This intra-pore binding site can be accessed directly from the extracellular side of the I rca channel, but apparently not from the cytosolic side.