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The use of ER targeted aequorin to study store-operated Ca²⁺ inflow in a liver cell line

Authors Chan, Man Yee
Issue Date 2002
Summary 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 IP3Rs (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.
Note Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2002
Language English
Format Thesis
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