Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

Abstract

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 measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca²⁺ ≈ Sr²⁺ ≈ Ba²⁺ > Mg²⁺ ≫ K⁺ which is inversely related to the size of the unitary conductances K⁺ ≫ Mg²⁺ ≈ Ba²⁺ > Sr²⁺ ≈ Ca²⁺, suggesting that ion movement is not independent. In the presence of Ca²⁺, the unitary K⁺ current is reduced in a concentration- and voltage-dependent manner by Ca²⁺ binding at a high affinity site (K _0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca²⁺ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca²⁺ from micromolar to millimolar concentrations resulted in a dramatic increase in 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.