A channel that allows inwardly directed fluxes of anions in protoplasts derived from wheat roots

Abstract

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestivum L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, usually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which activated more slowly during membrane depolarisation. The anion OR is permeable to NO 3 − and Cl−, moderately permeable to I−, and relatively impermeable to H2PO4/− and ClO4/−. An anomalous mole-fraction effect between ClO4/− and Cl− was observed on the outward current, indicating that the channel is a multi-ion pore. The anion OR is gated by both voltage and external anion concentration such that it activates near to the equilibrium potential for the permeant anion. It activated at more negative membrane potentials when NO 3 − was substituted for Cl− in the external medium, indicating that the channel may function to allow NO 3 − influx under luxuriant external NO 3 − concentrations. For most experiments, K+ and Cl− were the main cation and anion in solution, and under these conditions it appeared likely that the anion OR functioned in membrane-potential regulation by facilitating a Cl− influx at membrane potentials more positive than the chloride reversal potential (ECl). If ECl was more negative than the K+ reversal potential (EK) then the anion OR dominated but both the anion and cation ORs occurred together when the membrane potential difference (Vm) was positive of both ECl and EK. The cation OR was inhibited by increasing external Cl− concentrations, but the anion OR was not affected by external K+ or Na+ concentration. The anion-transport inhibitors, zinc and phenylglyoxal were ineffective in blocking the anion OR. 4,4′-Di-isothiocyanostilbene-2, 2′-disulfonic acid (DIDS) irreversibly blocked about 34% of the current when applied extracellularly at a concentration of 25 μM, and about 69% at a concentration of 200 μM. However, DIDS (200 μM) also occasionally acted as an irreversible blocker of the cation OR. Perchlorate blocked irreversibly 75% of the current at an external concentration of 10 mM and did not block the cation OR. Whole-cell currents also indicated that the anion OR was insensitive to external pH (pH=5–7) and calcium concentration ([Ca2+]=0.1–10 mM). Increasing intracellular calcium concentration significantly increased the occurrence of the fast outward current in whole cells (P < 0.005, X2 test). With approximately 10 nM calcium inside the cell the anion outward current was observed in 64% (n = 45) of cells and with 50 nM calcium inside the cell the anion current was observed in 88% (n = 69) of cells. Single-anion OR channels observed in outside-out patches had a conductance in 300 mM KCl (external) of about 4 pS. When voltage pulses were applied to outside-out patches the average currents were similar to those observed in whole cells. The significance of the anion OR as a likely route for Cl uptake in high salinities is discussed.