Store-Operated Ca²⁺ Channels (SOCs) are present in the plasma membrane of liver cells, all other "non-excitable" cells and many "excitable" animal cells. One of the main physiological roles of SOCs is to maintain an adequate Ca²⁺ concentration in the endoplasmic reticulum (ER). Despite their almost universal function, little is known about their molecular nature and the signalling pathways that activate SOCs. It is clear, however, that the activation of SOCs is triggered by a decrease in the concentration of Ca²⁺ in the ER. The aim of my project was to use aequorin, a Ca²⁺ -sensitive photoprotein, specifically targeted to the ER (ERAEQ) to further investigate the role of ER Ca²⁺ and the part played by (Ca²⁺ + Mg ²⁺) ATPases (SERCAs) in the activation of SOCs. Stably transfected ERAEQ- e...[ Read more ]

Store-Operated Ca²⁺ Channels (SOCs) are present in the plasma membrane of liver cells, all other "non-excitable" cells and many "excitable" animal cells. One of the main physiological roles of SOCs is to maintain an adequate Ca²⁺ concentration in the endoplasmic reticulum (ER). Despite their almost universal function, little is known about their molecular nature and the signalling pathways that activate SOCs. It is clear, however, that the activation of SOCs is triggered by a decrease in the concentration of Ca²⁺ in the ER. The aim of my project was to use aequorin, a Ca²⁺ -sensitive photoprotein, specifically targeted to the ER (ERAEQ) to further investigate the role of ER Ca²⁺ and the part played by (Ca²⁺ + Mg ²⁺) ATPases (SERCAs) in the activation of SOCs. Stably transfected ERAEQ- expressing H4-IIE liver cells were generated and immunofluorescence results showed that the aequorin had been successfully targeted to the ER.

When extra-cellular Ca²⁺ was added to cells depleted of their ER Ca²⁺ by a brief pre-treatment with 2,5-di-tert-butyl benzohydroquinone (BHQ), an increase in ERAEQ- generated luminescence (and hence [Ca²⁺]_er) was recorded using a photo-multiplier tube. This increase in [Ca²⁺]_er) was reduced in the presence of SERCA inhibitors (thapsigargin and BHQ); an ER Ca²⁺ chelator (Tetrakis-(2-pyridymethyl) ethylenediamine); a cytoplamsic Ca²⁺ chelator ( 5,5' Dibromo BAPTA-AM; DBB-AM); an agonist of ryanodine receptors (caffeine); an agonist of IP₃R_s (ATP); and blocked completely by an antagonist of SOCs (Gd³⁺). Transient increases in aequorin-generated luminescence induced by Ca²⁺ addition were also imaged in single H4-IIE cells using a Photon Imaging Microscope. These data suggest that: 1) ERAEQ reproducibly and reliably reflects changes in [Ca²⁺]_er; 2 ) Ca²⁺ inflow to the ER requires SOCs; 3) Thapsigargin/BHQ-sensitive SERCAs are responsible for the bulk of ER Ca²⁺ refilling; 4) The ER of H4-IIE cells also contain thapsigargin/BHQ- insensitive SERCAs that also contribute to ER refilling; and 5 ) The degree of ER-refilling that occurs in the presence of the cytoplasmic Ca²⁺ chelator DBB-AM, suggests that Ca²⁺ entering the cell via SOCs passes into the ER by a more direct route through the subplasmalemmal space, rather than through the deeper cytoplasmic space.