Depletion of calcium from intracellular stores results in the activation of calcium entry across the plasma membrane (SOCE). The mechanism by which the ER signals its state of filling to the plasma membrane however remains unclear. A direct conformational coupling between the InsP₃R and plasma membrane influx channels as well as a diffusible messenger have both been hypothesised to be involved. In keeping with the conformational-coupling hypothesis, a role for both InsP₃ and the InsP₃R has been reported. However, such studies are compromised by the poor pharmacology of the InsP₃ signalling cassette. Here, utilising molecular techniques to manipulate InsP₃ and InsP₃R levels we show that both InsP₃ and InsP₃Rs are required for maximal activation of SOCE.

Intracellular calcium levels were monitored in HEK293 cells using Fura-2. SOCE was activated by depletion of intracellular stores with thapsigargin (2 µM) in calcium-free medium. The peak calcium rise following re-addition of calcium to the extracellular medium was taken as the index of SOCE activation. Heterologous expression of proteins was achieved using standard transfection procedures. Data are presented as mean ± S.E.M. Statistical significance was calculated using unpaired Student’s t test. Fluorescence of expressed GFP-tagged proteins or of a co-transfected plasmid was used to identify transfected cells allowing control and test cells to be simultaneously imaged.

Increasing phospholipase C (PLC) activity by agonist application (ATP 10 µM) or overexpression of the calcium sensitive PLCδ resulted in a significant enhancement of SOCE by 58.9 ± 5.2 %.(n = 103, P < 0.005) and 22.7 ± 8.53 % (n = 38, P < 0.05) respectively. Inhibition of PLC by U73122 (10 µM) reduced SOCE by 78.8 ± 1.6 % (n = 23, P < 0.005). The modulation of SOCE by PLC was due to its regulation of InsP₃ production. Increasing InsP₃ levels by overexpression of phosphoinositol (PI) 4-OH kinase significantly increased SOCE by 24.0 ± 9.0 % (n = 45, P < 0.02). In addition, decreasing intracellular InsP₃ levels by overexpression of an InsP₃ sequestering ‘sponge’ or the InsP₃ metabolising 5′-phosphatase or a catalytically inactive PI4-OH kinase resulted in a significant decrease in SOCE (30.3 ± 6.3 % (n = 18, P < 0.005), 20.3 ± 5.0 % (n = 25, P < 0.05) and 28.8 ± 6.8 % (n = 35, P < 0.005) respectively of control values). To test whether the InsP₃R was also involved, short interfering RNA (RNAi) was used to knockdown InsP₃R expression. Stable transfection of cells with RNAi directed against the type 3 InsP₃R resulted in an almost complete inhibition of its expression without affecting the expression of other calcium handling proteins SERCA and calreticulin. In these cells the magnitude of SOCE was significantly decreased by 41.2 ± 1.8 (n = 88, P < 0.005) compared to control cells. We therefore conclude that activated InsP₃Rs are required for maximal activation of SOCE.