Functional Comparison between Secretory Pathway Ca2+/Mn2+-ATPase (SPCA) 1 and Sarcoplasmic Reticulum Ca2+-ATPase (SERCA) 1 Isoforms by Steady-state and Transient Kinetic Analyses

Abstract

Steady-state and transient kinetic studies were performed to functionally analyze the overall and partial reactions of the Ca 2؉ transport cycle of the human secretory pathway Ca 2؉ /Mn 2؉ -ATPase 1 (SPCA1) isoforms: SPCA1a, SPCA1b, SPCA1c, and SPCA1d (encoded by ATP2C1, the gene defective in Hailey-Hailey disease) upon heterologous expression in mammalian cells. The expression levels of SPCA1 isoforms were 200 -350-fold higher than in control cells except for SPCA1c, whose low expression level appears to be the effect of rapid degradation because of protein misfolding. Relative to SERCA1a, the active SPCA1a, SPCA1b, and SPCA1d enzymes displayed extremely high apparent affinities for cytosolic Ca 2؉ in activation of the overall ATPase and phosphorylation activities. The maximal turnover rates of the ATPase activity for SPCA1 isoforms were 4.7-6.4-fold lower than that of SERCA1a (lowest for the shortest SPCA1a isoform). The kinetic analysis traced these differences to a decreased rate of the E 1 ϳP(Ca) to E 2 -P transition. The apparent affinity for inorganic phosphate was reduced in the SPCA1 enzymes. This could be accounted for by an enhanced rate of the E 2 -P hydrolysis, which showed constitutive activation, lacking the SERCA1a-specific dependence on pH and K ؉ .Secretory pathway Ca 2ϩ /Mn 2ϩ -ATPases (SPCAs) 3 represent a new group of ion-motive ATPases consisting of single subunit integral membrane enzymes specifically mediating the ATP-powered uphill transport of either Ca 2ϩ or Mn 2ϩ from cytosol into the Golgi lumen (1-4).SPCAs (encoded by ATP2C1-2 genes) as well as sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (SERCAs; encoded by ATP2A1-3 genes) and plasma membrane Ca 2ϩ -ATPases (PMCAs; encoded by ATP2B1-4 genes) are characterized by the obligatory autophosphorylation during their reaction cycle (5, 6). Analysis of the deduced amino acid sequences of human (7), rat (8), worm (9), insect (9), and yeast (2) SPCA proteins indicates that despite the relatively low amino acid sequence homology (30 -35%) with SERCAs, the SPCAs retain the key architectural features revealed in the recently elucidated high resolution crystal structures of the rabbit SERCA1a (994 amino acids) in Ca 2ϩ -free thapsigargin-bound E 2 and Ca 2ϩ -bound E 1 forms either with or without bound nucleotide (10 -15). Relative to SERCA1a, SPCAs appear to be similarly composed of 10 hydrophobic helices (M1-M10) displaying strong membranespanning propensity. Other SERCA1a-specific structural determinants conserved in SPCAs are the phosphorylation (P, containing the phosphorylatable Asp 350 in human SPCA1 and Asp 351 in SERCA1a), nucleotide-binding (N) and actuator (A) domains present in the large "headpiece" emerging into the cytosol. The direct structural information obtained from SERCA1a crystals (10) has confirmed that two Ca 2ϩ ions are bound at two high affinity binding sites (site I and site II) located in the transmembrane region, as previously proposed on the basis of extensive mutational studies (16 -18). The sequence alignment of SPCA...

Dissection of the Functional Differences between Human Secretory Pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 Isoenzymes by Steady-state and Transient Kinetic Analyses

“…Relative to SERCA1a, the SPCA1 enzymes display remarkably high apparent affinities for cytosolic Ca 2ϩ (K 0.5 ϭ 0.01-0.04 M) in activation of the ATPase and phosphorylation activities and relatively low maximal turnover rates (3). Further investigations showed that these differences are mainly due to a reduction in the rate of the energytransducing E 1 ϳP(Ca) to E 2 -P conformational transition (3).…”

“…SPCA2 colocalizes with Golgispecific markers in human colon epithelial cells (10). For SPCA1, further isoform diversity is brought about by alternative splicing of the ATP2C1 pre-mRNA at its 3Ј end giving rise to three functionally active splice variants (SPCA1a of 919 amino acids, SPCA1b of 939 amino acids, and SPCA1d of 949 amino acids) and one inactive truncated isoform (SPCA1c of 888 amino acids) (3,12). So far, no splice variants have been reported for ATP2C2.…”

Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn 2+ efflux and protects against toxicity

“…P-type pumps share a high degree of sequence, structural and functional similarity, and their varied substrate specificities arise from minor changes in their ionbinding residues. During ion transport, autophosphorylation at an invariant aspartic acid residue in the cytoplasmic domain induces a characteristic conformation change from the E1 state, which has high affinity for the ion, to the E2 state, which has low affinity for the ion (1)(2)(3)(4). Transport through these pumps is limited by the rate of this conformational change (2).…”

“…During ion transport, autophosphorylation at an invariant aspartic acid residue in the cytoplasmic domain induces a characteristic conformation change from the E1 state, which has high affinity for the ion, to the E2 state, which has low affinity for the ion (1)(2)(3)(4). Transport through these pumps is limited by the rate of this conformational change (2). Devising molecular techniques that can increase ion transport through P-type ATPases will significantly improve our understanding of cellular processes regulated by these pumps and may also contribute to new treatments of diseases related to these processes.…”

Regulation of Ca2+release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells

“…Typically, a new steady-state level of fluorescence was established after 2-3 min of superfusion. The catalytic transport cycle of SERCA involves a phosphor-enzyme intermediate, and the phosphorylation and dephosphorylation of the enzyme during Ca 2ϩ transport are markedly enhanced by Mg 2ϩ (16,25,51). Therefore, 1 min before addition of InsP3 and throughout application of InsP3 in the test period, SERCA pump activity was effectively disabled by removal of Mg 2ϩ from the superfusate.…”