CA2(-CALMODULIN MODULATES ION-CHANNEL ACTIVITY IN STORAGE PROTEIN VACUOLES OF BARLEY ALEURONE CELLS())

Many plant ion channels have been identified, but little is known abou t how these transporters are regulated. We have investigated the regul ation of a slow vacuolar (SV) ion channel in the tonoplast of barley a leurone storage protein vacuoles (SPV) using the patch-clamp technique . SPV were isolated from barley aleurone protoplasts incubated with Ca Cl2 in the presence or absence of gibberellic acid (GA) or abscisic ac id (ABA). A slowly activating, voltage-dependent ion channel was ident ified in the SPV membrane. Mean channel conductance was 26 pS when 100 mM KCI was on both sides of the membrane, and reversal potential meas urements indicated that most of the current was carried by K+. Treatme nt of protoplasts with GA(3) increased whole-vacuole current density c ompared to SPV isolated from ABA- or CaCl2-treated cells. The opening of the SV channel was sensitive to cytosolic free Ca2+ concentration ( [Ca2+](i)) between 600 nM and 100 mu M, with higher [Ca2+](i) resultin g in a greater probability of channel opening. SV channel activity was reduced greater than 90% by the calmodulin (CaM) inhibitors W7 and tr ifluoperazine, suggesting that Ca2+ activates endogenous CaM tightly a ssociated with the membrane. Exogenous CaM partially reversed the inhi bitory effects of W7 on SV channel opening. CaM also sensitized the SV channel to Ca2+. In the presence of similar to 3.5 mu M CaM, specific current increased by approximately threefold at 2.5 mu M Ca2+ and by more than 13-fold at 10 mu M Ca2+. Since [Ca2+](i) and the level of Ca M increase in barley aleurone cells following exposure to GA, we sugge st that Ca2+ and CaM act as signal transduction elements mediating hor mone-induced changes in ion channel activity.