Asymmetric block of the plant vacuolar Ca2+-permeable channel by organic cations

In this work we have analysed the voltage-dependent block of the slow activ ating channel from red beet vacuoles by Tris, quaternary ammonium ions and the natural polyamines putrescine, spermidine and spermine. All these organ ic cations when applied from the cytosolic side blocked the channel by bind ing apparently deep (z delta values in the range of 0.65-1.35) within the p ore. Tetraethylammonium ion did not pass the selectivity filter, whereas th e cations with a smaller cross-section and Tris could pass across the entir e pore, as evidenced by a relief of block at high positive voltages. Voltag e dependence of the establishment of block from cytosolic side and of its r elief was anomalously strong in the sense that the total charge moved acros s the pore for all blockers tested, with a notable exception of spermine, w as in excess of their actual valence. This behaviour is consistent with the existence of multiple binding sites within a long pore, their simultaneous occupancy and interaction between different ions. In contrast, binding of blockers from the vacuolar (lumenal) side appears to Follow a single-ion ha ndling rule, with a common binding site for all amines located at approxima tely 30% of the electrical distance from the lumenal side.