Dissection of the Functional Differences between Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) 1 and 3 Isoforms by Steady-state and Transient Kinetic Analyses

Abstract

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca 2؉ -transport cycle mediated by the human sarco(endo)plasmic reticulum Ca 2؉ -ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca 2؉ in activation of the overall reaction due to a decreased E 2 to E 1 Ca 2 transition rate and an increased rate of Ca 2؉ dissociation from E 1 Ca 2 . At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca 2؉ level (either an enhanced efflux of lumenal Ca 2؉ through the pump in E 2 form or insensitivity to inhibition by lumenal Ca 2؉ ). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca 2؉ and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E 2 P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.Sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs) 1 are single-subunit membrane-spanning P-type ATPases that mediate the uphill transport of cytoplasmic Ca 2ϩ into the lumen of intracellular stores with a stoichiometry of two Ca 2ϩ per ATP hydrolyzed (1-3). Studies of the SERCA1a enzyme have shown that Ca 2ϩ transport and ATP utilization are coupled through long-range intramolecular interactions between the 10-helix transmembrane domain containing the Ca 2ϩ binding sites and the cytoplasmic actuator, phosphorylation, and nucleotidebinding domains (1-4). In the reversible catalytic cycle (5), the binding of the two calcium ions with high affinity from the cytoplasmic side of the membrane triggers the phosphorylation of Asp 351 by ATP, whereas the dephosphorylation occurs when the translocated calcium ions have been released from lowaffinity lumenally facing sites in exchange with protons being countertransported (Scheme 1).The SERCAs have long been known as key enzymes in the cytoplasmic Ca 2ϩ signaling events, and more recent studies have also drawn attention to the importance of the luminal Ca 2ϩ content for Ca 2ϩ signaling, folding and processing of newly synthesized proteins, and control of cell growth and apoptosis (6 -9). In addition to the functionally well characterized SERCA1a isoform, the 3 human SERCA genes encode at least 8 other SERCA proteins by alternative splicing (10 -15). Whereas SERCA2b seems to be a ubiquitously expressed housekeeping enzyme, the other isoforms are limited to specific c...

Effect of Hailey-Hailey Disease Mutations on the Function of a New Variant of Human Secretory Pathway Ca2+/Mn2+-ATPase (hSPCA1)

“…The rate of dephosphorylation of the phosphoenzyme intermediate formed from ATP was analyzed after treatment with EGTA and ADP for serial time intervals prior to acid quenching (23,24). Phosphorylation was performed in 100 l of a solution containing 160 mM KCl, 17 mM potassium Hepes (pH 7), 5 mM NaN 3 , 1 mM dithiothreitol, and 2 M thapsigargin.…”

“…Quantification of the separated phosphoenzyme band was performed by imaging using the Packard Cyclone TM storage phosphor system (Packard Bioscience, Berkshire, UK). Appropriate background phosphorylation levels were subtracted before data analysis as described (24).…”

Identification, Expression, Function, and Localization of a Novel (Sixth) Isoform of the Human Sarco/Endoplasmic Reticulum Ca2+ATPase 3 Gene

“…Phosphorylation from inorganic phosphate was performed for 10 min at 25°C in the presence of 0.5 mM 32 P i , 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 10 mM MgCl 2 , and 30% (v/v) Me 2 SO. Dephosphorylation was studied at 25°C by a 19-fold dilution of the phosphorylated sample into 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 10 mM EDTA (removing Mg 2ϩ and thereby terminating phosphorylation), 15% (v/v) Me 2 SO, and 0.5 mM non-radioactive P i (25,27). In all cases, the phosphoenzyme was quenched with 0.5 volume of 25% (w/v) trichloroacetic acid containing 100 mM H 3 PO 4 .…”

“…Appropriate background phosphorylation levels (obtained in the presence of excess Ca 2ϩ for phosphorylation from 32 P i ) were subtracted, and data analysis was performed as in Ref. 25.…”

“…Finally, we found that the insertion of an additional exon 22 in human SERCA3b and SERCA3c mRNAs led to SERCA3d and SERCA3e isoforms (24). Although the rationale for such a diversity of novel SERCA3 isoforms is still not understood, functional differences in the human SERCA3a, -3b, and -3c isoforms were recently pointed out, particularly one step of their catalytic cycle, the dephosphorylation of the E 2 P intermediate, varying with the length of the alternatively spliced C terminus (25).…”