Opening of Ca²⁺ channels in isolated red beet root vacuole membrane by inositol 1,4,5-trisphosphate

Abstract

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 cells^1–5 by acting on a specific receptor⁶. There are also reports that InsP₃ is important in plant cells^7–12. Using patch-clamp techniques^13–15, 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.