A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorga
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Biological Procedures Online
METHODOLOGY
Open Access
A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorganic phosphate David C McMullen1*, William S Kean2, Ajay Verma3, Jeffrey T Cole3 and William D Watson3
Abstract Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format. Keywords: Calcium, SERCA activity, Microsomes, Inorganic phosphate, Malachite green
Introduction 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 45 Ca 2+ accumulation in ER microsomes and identified a novel Ca 2+ pool in the central nervous system [5,6]. Ca 2+ accumulates in the ER is via the ubiquitously expressed magnesium, ATP-dependent sarco-endoplasmic reticulum calcium ATPases (SERCAs) which rapidly transport * Correspondence: [email protected] 1 Department of Neurology, Uniformed Services University of the Health Sciences, B-3059, 4301 Jones Bridge Road, Bethesda, MD 20814, USA Full list of author information is available at the end of the article
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 molecul
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