, Volume 146, Issue 2, pp 211-224

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

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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.

We thank Ian Jennings for writing and implementing some of the software used in this study and Anna J. Bate for technical assistance. The work was supported by grants from the Biotechnology and Biological Sciences Research Council to E.J. (PDF/14) and DS (PG87/529).