A CHANNEL THAT ALLOWS INWARDLY DIRECTED FLUXES OF ANIONS IN PROTOPLASTS DERIVED FROM WHEAT ROOTS

An anion channel that only allows outward current flow (anion influx) has been identified in protoplasts derived from wheat (Triticum aestiv um L., Triticum turgidum L.) roots. The anion outward rectifier (anion OR) measured by patch-clamp of whole cells activated very quickly, us ually reaching a steady-state level in less than 100 ms and was easily distinguished from the cation outward rectifier (cation OR) which act ivated more slowly during membrane depolarisation. The anion OR is per meable to NO3- and Cl-, moderately permeable to I-, and relatively imp ermeable to H2PO4- and ClO4-. An anomalous mole-fraction effect betwee n ClO4- and Cl- was observed on the outward current, indicating that t he 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 eq uilibrium potential for the permeant anion. It activated at more negat ive membrane potentials when NO3- was substituted for Cl- in the exter nal medium, indicating that the channel may function to allow NO3- inf lux under luxuriant external NO3- concentrations. For most experiments , K+ and Cl- were the main cation and anion in solution, and under the se conditions it appeared likely that the anion OR functioned in membr ane-potential regulation by facilitating a Cl- influx at membrane pote ntials more positive than the chloride reversal potential (E(Cl)). If E(Cl) was more negative than the K+ reversal potential (E(K)) then the anion OR dominated but both the anion and cation ORs occurred togethe r when the membrane potential difference (V(m)) was positive of both E (Cl) and E(K). 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'-Diisothiocyanostilben e-2,2'-disulfonic acid (DIDS) irreversibly blocked about 34% of the cu rrent when applied extracellularly at a concentration of 25 muM, and a bout 69% at a concentration of 200 muM. However, DIDS (200 muM) also o ccasionally acted as an irreversible blocker of the cation OR. Perchlo rate blocked irreversibly 75% of the current at an external concentrat ion 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 intracellu lar calcium concentration significantly increased the occurrence of th e fast outward current in whole cells (P < 0.005, X2 test). With appro ximately 10 Nm calcium inside the cell the anion outward current was o bserved in 64% (n = 45) of cells and with 50 nM calcium inside the cel l the anion current was observed in 88% (n = 69) of cells. Single-anio n 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 observe d in whole cells. The significance of the anion OR as a likely route f or Cl uptake in high salinities is discussed.