A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorganic phosphate

Precise calcium (Ca2+) regulation is essential to most cellular functions and cell survival, while Ca2+ dystasis can lead to cell death [1]. Eukaryotic cells regulate intracellular Ca2+ concentration and distribution by transport across membranes into organelles or the extracellular environment using a complex system of ion pumps, exchangers, channels, and binding proteins [2,3]. Both the extracellular and total cellular Ca2+ concentration is typically 2 mM, while the free concentration in the cell cytosol at rest is maintained at 100 nM - four orders of magnitude lower than the extracellular concentration [4]. This high electrochemical gradient makes Ca2+ an ideal second messenger, with small local cytosolic changes in concentration representing large fractional changes. The endoplasmic reticulum (ER) is a major intracellular store of second messenger Ca2+, and this laboratory established a technique to quantify 45Ca2+ accumulation in ER microsomes and identified a novel Ca2+ pool in the central nervous system [5,6]. Ca2+ accumulates in the ER is via the ubiquitously expressed magnesium, ATP-dependent sarco-endoplasmic reticulum calcium ATPases (SERCAs) which rapidly transport excess Ca2+ from the cytosol into the ER lumen [7,8]. There are three genes that encode SERCAs in the mammalian genome and tissue specific alternative splicing of these gene products results in at least 11 known isoforms [9-11]. Of these, SERCA2b is expressed almost ubuiquitously whereas the others demonstrate temporal and tissue specific expression. In addition, all known isoforms are inhibited by the general P-type ATPase inhibitors such as La3+ and orthovanadate, as well as the more potent and specific inhibitor thapsigargin (TG) [12,13].Via conformational changes, SERCAs transfer two Ca2+ ions from the cytoplasm into the ER lumen per molecule of ATP hydrolyzed [9,14]. During this process, SERCAs transiently form a covalent bond with the gamma phosphate group of ATP [15]. Following the tra