Characterization of a voltage-dependent Ca2+-selective channel from wheat roots

A new mechanism for calcium flux in wheat (Triticum aestivum L.) root cells has been characterized. Membrane vesicles were enriched in plasma membrane using aqueous-polymer two-phase partitioning and incorporated into artificial lipid bilayers, allowing characterization of single channels under voltage-clamp conditions. Membrane marker activities showed 74% and 83% purity in plasma membrane when expressed in terms of membrane area and activity, respectively. Since membrane vesicles obtained by aqueous-polymer two-phase partitioning yield a population of membrane vesicles of regular orientation, and vesicle fusion into planar lipid bilayers occurs in a defined manner, the orientation of the channel upon vesicle incorporation could be determined. Thus ionic activities and potentials could be controlled appropriately on what we propose to be the cytosolic (trans) and extracellular (cis) faces of the channel. The unitary conductance in symmetrical 1 mM CaCl₂ was 27±0.4 (pS). The correlation between the theoretical and observed reversal potentials in asymmetrical conditions showed that the channel was highly selective for Ca²⁺ over Cl^–. Experiments simulating physiological ionic conditions showed a P_Ca ²⁺/P_K ⁺ of 17–26, decreasing in this range as the extracellular CaCl₂ concentration increased from 0.1 to 1 mM. The channel was also permeable to the essential nutrient ions, Mg²⁺ and Mn²⁺. The open probability of the channel was strongly dependent on the membrane potential. Inactivation with time was observed at more negative membrane potentials, and was immediately reversed as soon as the membrane potential was decreased. At membrane potentials more negative than -130mV, the channel remained mainly in the closed state, suggesting that in vivo the channel would remain largely closed and would open only upon membrane depolarization. The channel was blocked by micromolar concentrations of extracellular verapamil and trivalent cations, Al³⁺ being the most effective of those tested. Exposure of the cytosolic and extracellular sides of the channel to inositol 1,4,5-trisphosphate had no effect on the channel activity. We suggest a plasma-membrane origin for the channel as shown by biochemical and electrophysiological evidence, and discuss possible physiological roles of this channel, both in Ca²⁺ uptake into roots and in signal transduction.Abbreviations IP₃ 1,4,5-trisphosphate - PM plasma membrane We wish to thank Dr. Christa Niemietz, Dr. Robert Reid and Prof. Andrew Smith for valuable discussions. This work was supported by the Australian Research Council and an OPRS award to M.P.