Capacitative calcium entry (CCE) is the process by which depletion of intracellular calcium stores leads to the activation of calcium influx at the plasma membrane. It has been proposed that inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) sense the reduction of calcium within calcium stores and control the opening of channels at the plasma membrane via direct physical interaction. Likely candidates for the CCE channels are members of the transient receptor potential (TRPC) family. Using heterologous expression of the proposed interacting domains of TRP and InsP3Rs, and manipulation of intracellular InsP3 levels, we examined the necessity for InsP3Rs in the activation of CCE.
Intracellular calcium levels were monitored in HEK-293 cells using fura-2 (30 min loading with acetoxymethyl ester followed by a 30 min de-esterification period) at room temperature. CCE was activated by depletion of intracellular stores with thapsigargin (1 µM) in calcium-free medium. The peak calcium rise following re-addition of calcium to the extracellular medium was taken as the index of CCE activation. Heterologous expression of eGFP-tagged proteins was achieved using standard transfection procedures. Data are presented as means ± S.E.M. Statistical significance was calculated using Student’s unpaired t test. For the heterologous expression experiments, control and test cells were imaged in the same fields of view, with the transfected cells identified by eGFP fluorescence.
Expression of the domain of TRP3 (amino acids 742 to 797) proposed to interact with InsP3Rs decreased the peak CCE signal from 477 ± 34 to 307 ± 23 nM (n = 20). In contrast, the domain of InsP3R3 (amino acids 638 to 710) that has been suggested to interact with TRP3 did not alter peak CCE levels. Manipulation of intracellular InsP3 levels was achieved using expression of type 1 InsP3 5Ô-phosphatase or a ligand binding ‘sponge’ domain of the InsP3R. Both moieties completely inhibited calcium release via the InsP3-linked agonist ATP (100 µM), without changing the size of the intracellular calcium store. 5Ô-Phosphatase expression reduced CCE from 463 ± 22 nM (n = 47 cells) to 369 ± 23 nM (n = 25 cells) (P < 0.05). Similarly, the InsP3 sponge decreased CCE from 432 ± 38 nM (n = 15 cells) to 301 ± 27 nM (n = 18 cells) (P < 0.05). InsP3 production was inhibited using the phospholipase C antagonist U73122 (10 µM), which completely blocked ATP-induced calcium mobilisation. Treatment with U73122 resulted in a decreased peak CCE signal from 989 ± 116 nM (n = 25 cells) to 210 ± 16 nM (n = 23 cells) (P < 0.05).
These data are consistent with a requirement for both InsP3 and InsP3Rs in the activation of CCE.