Tonoplast ion channels from sugar beet cell suspensions : inhibition by amiloride and its analogs

The properties of the vacuolar membrane (tonoplast) ion channels of sugar beet (Beta vulgaries) cell cultures were studied using the patch-clamp technique. Tonoplast currents displayed inward rectification in the whole vacuole and isolated outside-out patch configurations and permeability ratios P_K+/P_Na+ = 1 and P_K+/P_Cl− = 5. Amiloride and two of its analogs, 5-(N-methyl-N-isobutyl)-amiloride and benzamil, inhibitors of Na⁺ channels in animal systems, blocked inward currents by reducing single-channel openings. Concentrations for 50% inhibition of vacuolar currents of 730 nanomolar, 130 nanomolar, and 1.5 micromolar for amiloride, benzamil, and 5-(N-methyl-N-isobutyl)-amiloride, respectively, were obtained from whole-vacuole recordings. The high inhibitory action (affinity) of amiloride and its analogs for the tonoplast cation channel suggests that these compounds could be used for the isolation and biochemical characterization of this protein.