LUMENAL CALCIUM MODULATES UNITARY CONDUCTANCE AND GATING OF A PLANT VACUOLAR CALCIUM-RELEASE CHANNEL

The patch clamp technique has been used to investigate ion permeation and Ca2+-dependent gating of a voltage-sensitive Ca2+ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential m easurements in bi-ionic conditions revealed a sequence for permeabilit y ratios of Ca2+ approximate to Sr2+ approximate to Ba2+ > Mg2+ much g reater than K+ which is inversely related to the size of the unitary c onductances K+ much greater than Mg2+ approximate to Ba2+ > Sr2+ appro ximate to Ca2+, suggesting that ion movement is not independent. In th e presence of Ca2+, the unitary K+ current is reduced in a concentrati on- and voltage-dependent manner by Ca2+ binding at a high affinity si te (K-0.5 = 0.29 mM at 0 mV) which is located 9% along the electric fi eld of the membrane from the vacuolar side. Comparison of reversal pot entials obtained under strictly bi-ionic conditions with those obtaine d in the presence of mixtures of the two ions indicates that the chann el forms a multi-ion pore. Lumenal Ca2+ also has an effect on voltage- dependent channel gating. Stepwise increases of vacuolar Ca2+ from mic romolar to millimolar concentrations resulted in a dramatic increase i n channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials . The steepness of the concentration dependence of channel activation by Ca2+ at -41 mV predicts that two Ca2+ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.