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The role of different isoforms of IP₃ receptor in activation of store-operated Ca²⁺ channels in liver cells

In most animal cells a decrease in the concentration of Ca²⁺ in the endoplasmic reticulum (ER) and possibly other intracellular stores results in activation of store-operated Ca²⁺ channels (SOCs) in the plasma membrane. Physiologically, the empting of Ca²⁺ stores occurs through inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)-operated Ca²⁺ channels. In addition to being a major component in the mechanism responsible for Ca²⁺ release from the ER in non-excitable cells, it has been suggested that IP₃Rs play a direct role in activation of SOCs by conformational coupling. To elucidate the role of IP₃Rs in activation of SOCs in liver, we used short interfering RNA (siRNA) to reduce specifically the expression of the genes encoding each type of IP₃R in H4IIE liver cells. Whole cell patch clamping was used to measure the SOC current (I_SOC) initiated by the SERCA inhibitor thapsigargin or IP₃. Immunofluorescence and Western blotting were employed to verify the effectiveness of siRNA and the time course of the knock down of IP₃Rs. We found that all 3 types of IP₃Rs are expressed in H4IIE cells, and were able to knock each type down using specific siRNAs. The amplitude of thapsigargin-initiated I_SOC in cells transfected with siRNA against each type of IP₃R was the same as that in control cells. These results indicate that, in contrast to considerable published data for other cell types, IP₃Rs are unlikely to activate SOCs through a conformational coupling mechanism. Using IP₃ in the pipette revealed that knocking down IP₃ R1, but not the other types of IP₃R, is sufficient to prevent activation of the I_SOC by IP₃. This indicates that Ca²⁺ stores linked to SOCs predominantly express type 1 IP₃R.