Reconstitution of vacuolar ion channels into planar lipid bilayers

Abstract

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l −1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16–25 pS (28 experiments), 30–42 pS (26), 49–56 pS (15) and 64–81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca 2+-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl −, NO − 3, SO 2− 4 and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.