Identification of K⁺, Cl⁻, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary

K⁺, Cl⁻, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl⁻. The Cl⁻ channel opened at positive vacuolar membrane potentials for cytoplasmic Cl⁻ influx had a high conductance of 110pS in symmetrical 100mM Cl⁻. When K⁺ and Cl⁻ channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺∶Sr²⁺∶Ca²⁺∶Mg²⁺=1∶0.6∶0.6∶0.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl⁻, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.