Characterization of an Anion Transporter in the Plasma Membrane of Barley Roots

Abstract

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-C1) 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-C1 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 KC1, KNO3, or bis-tris-prop ane (BTP)-CI. Using 36C1, 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~-lransport 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.