Characterization of an anion transporter in the plasma membrane of barley roots

To examine the relationship between H⁺-ATPase and the transport of anions, we investigated the effects of various inhibitors on the activity of the H⁺-ATPase, the transport of protons, and the transport of Cl^- ions using plasma membrane vesicles prepared from barley roots. Some inhibitors, namely, 4,4-diisothiocyano-2,2-stilbene disulfonate (DIDS) and Zn²⁺ ions markedly inhibited H⁺-ATPase activity. Other compounds, such as phenylglyoxal (PGO) and niflumic acid (NIF), inhibited H⁺-ATPase activity by 20-30%, while anthracene-9-carboxylate (A-9-C) and tetraethylammonium chloride (TEA-Cl) had little effect on this activity. The ATP-dependent acidification of the interior of vesicles was strongly dependent on the presence of permeant anions, such as chloride (Cl^-) and nitrate (NO₃/^-), and it was completely inhibited by 0.2 mM NIF. Other compounds, namely, A-9- C of 0.1 mM and TEA-Cl of 10 mM, did not affect H⁺-transport activity. The inhibition of H⁺-transport activity by NIF was observed even when the activity was assayed in the presence of KCl, KNO₃, or bis-tris-propane (BTP)-Cl. Using ³⁶Cl^-, we quantified Cl^--transport activity by measuring the uptake of Cl^- ions into the plasma membrane vesicles. The uptake depended on the potential difference across the membrane that was generated by H⁺-ATPase; it was enhanced by an inside-positive potential gradient. At 0.1 mM, NIF completely blocked the voltage-dependent Cl^--transport activity. From these properties of the Cl^- transporter and the inhibition of H⁺transport activity by NIF, we suggest that H⁺-transport activity across the plasma membrane might be modulated by the transport of anions via a NIF- sensitive anion-permeable transporter that acts to collapse the inside- positive potential generated by H⁺-ATPase.