Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3

Abstract

Chandrasekera PC, Kargacin ME, Deans JP, Lytton J. Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3. Am J Physiol Cell Physiol 296: C1105-C1114, 2009. First published February 18, 2009 doi:10.1152/ajpcell.00650.2008.-The sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca 2ϩ concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca 2ϩ ; however, Ca 2ϩ affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca 2ϩ uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca 2ϩ activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/ IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca 2ϩ affinity for SERCA3 compared with SERCA2b (1.10 Ϯ 0.04 vs. 0.26 Ϯ 0.01 M), and using mixtures of recombinant protein isoforms, we validated the twocomponent uptake model. Then we determined apparent Ca 2ϩ affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca 2ϩ affinity in these two preparations was 1.04 Ϯ 0.07 and 1. ]), as well as the levels of Ca 2ϩ in the endoplasmic reticulum stores required for controlling complex dynamics of Ca 2ϩ signals. SERCA proteins are encoded by a family of structurally related and alternatively spliced transcripts from three distinct genes (SERCA1, SERCA2, and SERCA3) expressed in a tissue-dependent and/or developmentally regulated manner. Alternative splicing of the ATP2A1 gene generates transcripts encoding SERCA1a and SERCA1b isoforms, which are expressed exclusively in adult and neonatal fast-twitch skeletal muscle, respectively. The ATP2A2 gene encoding the SERCA2 protein is alternatively spliced and processed in a tissue-specific manner, giving rise to three isoforms: SERCA2a, which is expressed predominantly in cardiac/slow-twitch skeletal muscle; SERCA2b, which is expressed ubiquitously; and SERCA2c, which has been reported to have a broader expression pattern than that typically attributed to SERCA2a. The level of SERCA2c protein relative to coexpressed SERCA2a or SERCA2b is not known (13,19,35). SERCA2b has a higher apparent affinity for Ca 2ϩ and turns over at a rate twofold slower than SERCA2a. SERCA2c has a lower apparent Ca 2ϩ affinity and a slower turnover rate than SERCA2a. The functional differences are due to replacement of the COOH-terminal sequence AILE (amino acids 994 -997) in SERCA...

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