Calcium-regulated anion channels in the plasma membrane of Lilium longiflorum pollen protoplasts

Currents through anion channels in the plasma membrane of Lilium longiflorum pollen grain protoplasts were studied under conditions of symmetrical anionic concentrations by means of patch-clamp whole-cell configuration. With Cl(-)-based intra-and extracellular solutions, three outward-rectifying anion conductances, I(Cl1), I(Cl2) and I(Cl3), were identified. These three activities were discriminated by differential rundown behaviour and sensitivity to 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), which could not be attributed to one or more channel types. All shared strong outward rectification, activated instantaneously and displayed a slow time-dependent activation for positive potentials. All showed modulation by intracellular calcium ([Ca(2+)](in)), increasing intensity from 6.04 nM up to 0.5 mM (I(Cl1)), or reaching a maximum value with 8.50 mu M (I(Cl2) and I(Cl3)). After rundown, the anionic currents measured using NO(3)(-)-based solutions were indistinguishable, indicating that the permeabilities of the channels for Cl(-) and NO(3)(-) are similar. Additionally, unitary anionic currents were measured from outside-out excised patches, confirming the presence of individual anionic channels. This study shows for the first time the presence of a large anionic conductance across the membrane of pollen protoplasts, resulting from the presence of Ca(2+)-regulated channels. A similar conductance was also found in germinated pollen. We hypothesize that these putative channels may be responsible for the large anionic fluxes previously detected by means of self-referencing vibrating probes.