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Na+-, Ouabain-, Ca2 -, and Thapsigargin-Sensitive Atpase

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Na+-, Ouabain-, Ca2 -, and Thapsigargin-Sensitive Atpase Proc. Nadl. Acad. Sci. USA Vol. 91, pp. 6103-6107, June 1994 Biochemnstry Na+-, ouabain-, Ca2 -, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump a subunits (Na+,K+-ATPase/Ca2+-ATPase/arac glycoside/ /chimeric moecule) TOSHIAKI ISHII, M. VICTOR LEMAS*, AND KUNIO TAKEYASUt The Department of Medical Biochemistry and Biotechnology Center, The Ohio State University, Columbus, OH 43210 Communicated by Joseph F. Hoffman, February 28, 1994 ABSTRACT Using the chicken sarcoplasmic/endoplasmic tion of their amino acid sequences (14) and the similar reticulum Ca2+ (SERCA)-ATPase as a parental molecule and distribution of hydrophobic domains throughout the mole- replacing various portions with the corresponding portions of cules (15), a series of chimeric ATPases were constructed the chicken Na+,K+-ATPase a, subunit, Ca+/th argin- (15-17). Functional comparison of these chimeric molecules and Na+/ouabin-sensitive domains critical for these P-type should provide critical information about the structure- ATPase activities were identified. In the chimera, [n/c]CC, the function relationships of the SERCA ATPase and the amno-ternminal amino acids Met-i to Asp-162 of the SERCA Na+,K+-ATPase. In this report, we examined the effects of (isoform 1) (SERCA1) ATPase were replaced with the corre- Na+, Ca2+, ouabain, and thapsigargin on a series of chimeric sponding portion (Met-l-Asp-200) of the Na+,K+-ATPase a, and wild-type molecules side by side in transfected mouse L subunit. In the chimera CC[c/n], the carboxyl-terminal amino cells. acids (Ser-830 to COOH) of the SERCA1 ATPase were re- placed with the corresponding segment (Leu-861 to COOH) of MATERIALS AND METHODS the Na+,K+-ATPase a, subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was Expression of Chicken Chimeric cDNAs in Mouse L Cells. exchanged with the Na+,K+-ATPase a, subunit (Gly-378-Lys- Fig. 1 schematically represents the wild-type and chimeric 724). None of the chimeric molecules exhibited any detectable ATPases expressed in this study. The wild-type and chimeric ouabain-sentive Na+,K+-ATPase activity, but they did ex- chicken cDNA constructs encoding NNN (19), CCC (20), hibit thsigargin-sensitive Ca2+-ATPase activity. Therefore, [n/c]CC (16), CNC (17), CC[c/n] (15), and [An/c]CC (C and the segments e-i163-Gly-354 and Lys-712-Ser-830 of the c stand for parts of SERCA1 ATPase, N and n stand for parts SERCA1 ATPase are sufcent for Ca2+ and of Na+,K+-ATPase, and An represents a deleted portion of sensiivity. The SERCAi-ATPase activity of [n./cICC, but not n) were cloned intomammalian expression vector pRc/CMV ofCCC, CNC, or CC[c/n], was further stimulated by addition (Invitrogen, no. V750-20), introduced into mouse Ltk+,B cells of Na+ in the assay medium contining Ca2+. This additional that had been transfected with a cDNA encoding the chicken timulation of SERCAl-ATPase activity by Na+ was abolished Na+,K+-ATPase 81 subunit (21), and G418-resistant colonies when the amino-terminal region (Met-l-Leu-69) of [n/c]CC were isolated. At least 10 different cell lines per cDNA was deleted ([An/c]CC). In the absence of Na+, the SERCA1- construct were selected on the basis of high levels of immu- ATIase activity of [n/c]CC was inhibited by ouain, and, in nofluorescence staining (15, 21) and subjected to immuno- the presence ofNa+, its activity was stimulated by this drug. On precipitation to identify individual cell lines that expressed the other hand, the ATPase activity of [An/c]CC was not chicken chimeric molecules at a similar level. In immuno- affeted byouabain, al [A/cJCC can still bind [3HJoua- logical detection, monoclonal antibody (mAb) IgG 5 specific bain. These results suggest that a distinct Na+-sensltive domain to chicken Na+/K+-ATPase a subunit (19) was used for (Na+ sensor) located within the restred amino-terminal NNN and CNC, and mAb IgG 5D2 specific to chicken region (Met-i-Leu-69) of the Na+,K+-ATPase a, subunit SERCA1 ATPase (22) was used for CCC, [n/c]CC, [An/c]- regulates ATPase activity. The Na+ sensor also controls oua- CC, and CC[c/n]. The encoding cDNA for [An/c]CC was bain action in concert with the major ouabain-binding region constructed by replacing the first 207 bp encoding Met-1- between Ala-70 and Asp-200 of a, subunit. Leu-69 of the Na+,K+-ATPase portion in the [n/c]CC chi- mera with an artificial start codon, ATG. For this replace- The plasma membrane Na+,K+-ATPase and the sarcoplas- ment, the fragment between the artificial start codon and the mic/endoplasmic reticulum Ca2+-(SERCA) ATPase belong unique recognition site for endonuclease EcoRV at the chi- to a family of P-type ATPases that undergo a cycle of meric junction was amplified by oligonucleotide-directed conformational changes between phosphorylated and de- mutagenesis using PCR and inserted into the EcoRV site in phosphorylated stages (1, 2). Major research effort for the [n/c]CC. For PCR, 50 ng each of the synthetic primers past years has been directed toward identification of specific 5'-GCACCAIGGCTCGTGATGGCCCAAATCCCT-3' (ar- ion- and inhibitor-binding domains (3). Point mutations and tificial start codon is underlined) and 5'-CCGQ&IAICAGC- chemical modification in transmembrane segments have pro- TGGAATTCT-3' (EcoRV site is underlined) and 50 ng ofthe vided initial evidence for functional domains responsible for chicken Na+,K+-ATPase al subunit cDNA were used, and inhibitor (4-9) and ion binding (10-12) by these ATPases. More recently, the use of chimeric ATPases with character- Abbreviations: SERCA, sarcoplasmic/endoplasmic reticulum Ca2+; istics ofdistinct ion pumps for functional studies has become SERCA1, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (iso- possible (13-18). Taking advantage of the limited conserva- form 1); mAb, monoclonal antibody. *Present address: The Johns Hopkins Oncology Center, The Johns Hopkins University Medical Institution, Baltimore, MD 21203. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed at: The Biotechnol- payment. This article must therefore be hereby marked "advertisement" ogy Center, Rightmire Hall, 1060 Carmack Road, The Ohio State in accordance with 18 U.S.C. §1734 solely to indicate this fact. University, Columbus, OH 43210. 6103 Downloaded by guest on September 25, 2021 6104 Biochemistry: Ishii et al. Proc. Nati. Acad. Sci. USA 91 (1994) with -260 pM NaOH). The SERCA-ATPase activity was defined as the thapsigargin- (500 nM) inhibitable Ca2+- dependent ATP hydrolysis in 100 mM KCI/50 mM Tris-HCl, pH 7.4/2 mM ATP (containing 100 juM KOH and [3VP]ATP), 3 mM MgCl2, 2 pM ionophore A23187 (Boehringer Mann- heim), 1 mM NaN3, and appropriate amounts of CaCl2 and Cytoplasm EGTA (25). Crude membrane preparations [('1 mg ofprotein per ml in TME buffer (75 mM Tris HCI, pH 7.5/12.5 mM MgCl2/1.5 mM EDTA)] were obtained from monolayers of cells and used for these ATPase assays (60 ng protein per assay) as described (16). Specific ouabain binding to the crude membrane preparation was measured as described (16). CCC ASPI162 (GI.Y 354 ILE163 T1HR355 LYS712 SER830) RESULTS 6 - -i - 11 -X-1C - -4-62 AN_ 192 .04 348 1 II8 -16.4 _Om Expression of Chimeric Molecdes In MouseL Cels. All chicken cDNA constructs were stably transfected into G EY 54 ncCC asp2Ol} mouse L cells that had been expressing the chicken subunit ln/cICC ltEIL163 THR355 LYS712 SER830 8i a_ f] (21), because one of the chimera, CC[c/nJ, was known to assemble with the .8 subunit (15). The unmunoprecipitation asp20f) G profiles of the wild-type (NNN and CCC) and chimeric IA.nlcl(( ,J IS 1LE163 TlHR355.354 LIS712 SERS83f Is ryl o------L rL X molecules ([n/c]CC, [An/c]CC, CNC, and CC[c/n]) in six EJ selected cell lines indicated that these chicken molecules are CNC ASPl 62 (IlYA354 expressed at a similar level (Fig. 2), and, therefore, these cell ILE163 thr378 Ivs724 SER83O lines were used for further functional studies. __I I I _ |___ I El AN Cheric ATases Eibitd SERCA-ATPase Actity ASI" Y 354 SER830 But No Na+,K+-ATPase None of the chimeric mol- C Cc/n 11LF:6l62 (;1 THR355 Activity. I163 LYS7 12 ieu8bl ecules ([n/c]CC, CNC, and CC[c/n]) exhibited any detect- . T LL able ouabain-sensitive Na+,K+-ATPase activity over the NNN asp200 g1y377 background of endogenous mouse enzyme (data not shown) leu2Ol thr378 Ivs724 leu861 but did exhibit SERCA-ATPase activity, defined as the 200 177 - 4- -346 - 4 137 -*4161_ thapsigargin-inhibitable Ca2+-dependent ATP hydrolysis (27-30), at least 300-500% higher than that of mouse endog- FiG. 1. Schematic representation of wild-type and chimeric pro- enous enzyme at optimum free Ca2+ concentrations of 1-5 teins. (Upper) Working model for structure and function of the pM (Fig. 3A). The background level of mouse endogenous Na+,K+-ATPase a subunit that accommodates all results with SERCA-ATPase activity does not change in NNN- (wild- SERCA ATPase as a parental molecule. Thec]model includes Na+ type Na,K-ATPase) transfected mouse L cells (16). These sensor, ouabain-binding, and subunit-assembly domains. The topol- ogy model proposed for the wild-type sarcoplasmic reticulum Ca2+- differences in activities in transfected and control mouse cells ATPase (10) is adapted to all constructs on the basis of reported can be easily and repeatedly identified due to the advantage results (13-17).
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