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 (Piñeros & Tester, 1995. Planta 195:478-488)]. Concentrations as low as 0.5 microM 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 V0.5. 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 Kd(0) of 24 to 26 microM. This results in a Kd at -100 mV of 2 microM. Methoxyverapamil had qualitatively the same effects. This intra-pore binding site can be accessed directly from the extracellular side of the rca channel, but apparently not from the cytosolic side.
Piñeros
M
M
Department of Botany, University of Adelaide, S.A., Australia.
Tester
M
M
D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D005711:Gallopamil / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018517:Plant Roots; D014908:Triticum / Q000378:metabolism*; D014700:Verapamil / Q000378:metabolism*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005711:Gallopamil; D014700:Verapamil