Programme Contents

Molecular components of store-operated Ca²⁺ entry in liver cells

To maintain Ca²⁺ homeostasis and to deliver Ca²⁺ to the right place at the right time living cells use a complex array of Ca²⁺ pumps, transporters and ion channels. It has been generally accepted that in most animal cells replenishment of Ca²⁺ lost due to the activity of plasma membrane Ca²⁺-ATPase is mediated through a store-operated Ca²⁺ entry (SOCE) mechanism. According to this theory, release of Ca²⁺ from the intracellular Ca²⁺ stores caused by hormone binding to G-protein- or tyrosine kinase- coupled receptors activates store-operated Ca²⁺ channels (SOCs) in the plasma membrane. Recent evidence suggests that the Ca²⁺ release activated Ca²⁺ channel expressed in hematopoietic cells, which is the best characterised SOC, is composed of Orai1 polypeptides that interact with STIM1 (Stromal interaction molecule 1), an EF hand containing, Ca²⁺-binding polypeptide which senses the decrease in Ca²⁺ in the ER. Depletion of intracellular Ca²⁺ stores causes STIM1 to accumulate in areas of junctional ER located in close proximity to the plasma membrane, while Orai1 accumulates in the areas of plasma membrane apposed to STIM1 puncta. Utilizing siRNA mediated knockdown we have shown that STIM1 and Orai1 proteins are major components of SOCE in liver cells. However, there is evidence that several other proteins are also involved. Knockdown of PLC-γ1 in H4-IIE cells substantially decrease the amplitude of I_SOC initiated by either IP₃ or thapsigargin. No interaction between PLC-γ1 and STIM1 is detected in immunoprecipitation experiments. This suggests that PLC-γ1 is required to couple ER Ca²⁺ release to the activation of SOCs independently of any PLC-γ1-mediated generation of IP₃ and independently of a direct interaction between PLC-γ1 and STIM1. The likelihood that there are additional to STIM1 and Orai1 proteins that are involved in SOCE is supported by our recent results of the ectopic expression of STIM1 and Orai1 in liver cells. We have found that some biophysical properties of the Orai1/STIM1 current vary significantly between transfected cells and depend on the levels of expression of STIM1 and Orai1.