InsP₃ and Ins(1,3,4,5)P₄ act in synergy to stimulate influx of extracellular Ca²⁺ in Xenopus oocytes

To investigate the role of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P₄] in the regulation of Ca²⁺ influx, we injected inositol phosphates into Xenopus oocytes and measured Ca²⁺-gated Cl^- current to assay intracellular free Ca²⁺ concentration ([Ca²⁺](i)). To assess Ca²⁺ influx, we removed extracellular Ca²⁺ or added the inorganic Ca²⁺ channel blocker Mn²⁺ to the extracellular bath and measured the resulting change in Cl^- current. Ins(1,3,4,5)P₄ did not cause Ca²⁺ influx when injected alone or when preceded by an injection of Ca²⁺. In contrast, Ins(1,3,4,5)P₄ stimulated Ca²⁺ influx when injected after the poorly metabolized inositol trisphosphate (InsP₃) analogues D-myo-inositol 1,4,5-trisphosphorothioate [Ins(1,4,5)P₃S₃] or D-myo-inositol 2,4,5-trisphosphate [Ins(2,4,5)P₃]. These results indicate that Ins(1,3,4,5)P₄ is not sufficient to stimulate Ca²⁺ influx but acts in synergy with InsP₃s to cause Ca²⁺ influx. We also studied the effect of Ca²⁺ influx on the immediate metabolism of D- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in single oocytes. Ca²⁺ influx shunted the metabolism of Ins(1,4,5)P₃ toward the formation of Ins(1,3,4,5)P₄ and away from D-myo-inositol 1,4-bisphosphate [Ins(1,4)P₂]. These results suggest that there is a positive feedback regulatory mechanism in which Ca²⁺ influx stimulates Ins(1,3,4,5)P₄ production and Ins(1,3,4,5)P₄ stimulates further Ca²⁺ influx.