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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

Departments of ¹Biochemistry and Molecular Biology and ²Physiology and Biophysics, ³Libin Cardiovascular Institute of Alberta, ⁴Hotchkiss Brain Institute, and ⁵Snyder Institute of Infection, Immunity, and Inflammation, University of Calgary, Calgary, Alberta, Canada

Submitted 22 December 2008 ; accepted in final form 10 February 2009

The sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca²⁺ concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca²⁺; however, Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ affinity for SERCA3 compared with SERCA2b (1.10 ± 0.04 vs. 0.26 ± 0.01 µM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca²⁺ affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca²⁺ affinity in these two preparations was 1.04 ± 0.07 and 1.1 ± 0.2 µM for SERCA3 and 0.27 ± 0.02 and 0.26 ± 0.01 µM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca²⁺ is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms.

calcium pump; calcium uptake; continuous fura 4F fluorescence assay