OPENING OF CA-2+ CHANNELS IN ISOLATED RED BEET ROOT VACUOLE MEMBRANE BY INOSITOL 1,4,5-TRISPHOSPHATE

INOSITOL 1,4,5-trisphosphate (InsP3) is often implicated in pathways that couple extracellular signals to cellular calcium-dependent effectors. Various experiments have demonstrated that InsP3 can release calcium from intracellular stores in animal cells1-5 by acting on a specific receptor6. There are also reports that InsP3 is important in plant cells7-12. Using patch-clamp techniques13-15, we have now obtained direct measurements of a Ca2+ current in the isolated red beet root vacuole. This current was dependent on the InsP3concentration (Michaelis constant = 2.2 x 10-7 M at +80 mV) as well as the voltage across the vacuolar membrane. It was only partially inhibited by the calcium antagonists verapamil16 (2 x 10-5 M) and TMB8 (3,4,5-trimethoxybenzoic acid 8-diethyl-aminooctylester; 10-4M)10-11. Other inositides tested (inositol 1,4-bisphosphate; inositol 1,3,4- and 2,4,5-trisphosphates; inositol 1,3,4,5-tetrakisphosphate) did not produce a significant current even at concentrations up to 2 x 10-5 M. Single channels, opened by 10-6 M InsP3 in isolated patches of the vacuole membrane, had conductances of 30 pS with 5 x 10-3M Ca2+in the vacuole and 10-3 M Ca2+ outside. They were voltage-dependent and opened only on depolarization of the vacuoles. Their open state showed extensive 'flickering9, which did not depend on the Mg2+concentration. These results show that InsP3 releases calcium through an intact intracellular plant membrane by activating a Ca2+ channel, and that the opening of this channel is voltage-dependent. © 1990 Nature Publishing Group.