Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305126200
ISSN1083-351X
AutoresYasser A. Mahmmoud, Gordon Cramb, Arvid B. Maunsbach, Christopher P. Cutler, Lara Meischke, Flemming Cornelius,
Tópico(s)Hydrogen Storage and Materials
ResumoIn Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase α-subunit and PLMS showed the presence of α and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E 1∼P → E 2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E 2(K) → E 1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase α-subunit is important for the modulation of shark Na,K-ATPase activity. In Na,K-ATPase membrane preparations from shark rectal glands, we have previously identified an FXYD domain-containing protein, phospholemman-like protein from shark, PLMS. This protein was shown to associate and modulate shark Na,K-ATPase activity in vitro. Here we describe the complete coding sequence, expression, and cellular localization of PLMS in the rectal gland of the shark Squalus acanthias. The mature protein contained 74 amino acids, including the N-terminal FXYD motif and a C-terminal protein kinase multisite phosphorylation motif. The sequence is preceded by a 20 amino acid candidate cleavable signal sequence. Immunogold labeling of the Na,K-ATPase α-subunit and PLMS showed the presence of α and PLMS in the basolateral membranes of the rectal gland cells and suggested their partial colocalization. Furthermore, through controlled proteolysis, the C terminus of PLMS containing the protein kinase phosphorylation domain can be specifically cleaved. Removal of this domain resulted in stimulation of maximal Na,K-ATPase activity, as well as several partial reactions. Both the E 1∼P → E 2-P reaction, which is partially rate-limiting in shark, and the K+ deocclusion reaction, E 2(K) → E 1, are accelerated. The latter may explain the finding that the apparent Na+ affinity was increased by the specific C-terminal PLMS truncation. Thus, these data are consistent with a model where interaction of the phosphorylation domain of PLMS with the Na,K-ATPase α-subunit is important for the modulation of shark Na,K-ATPase activity. The Na,K-ATPase is the enzyme responsible for active transport of Na+ and K+ across the plasma membranes of animal cells (for recent review, see Ref. 1Kaplan J.H. Annu. Rev. Biochem. 2002; 71: 511-535Crossref PubMed Scopus (886) Google Scholar). It establishes and maintains the electrochemical gradients for Na+ and K+ responsible for generation of a resting membrane potential necessary for excitability of muscle and nerve cells, co- and counter-transport of ions and nutrient molecules across the cell membrane, as well as the regulation of cell volume. The enzyme is composed of two essential subunits; a catalytic α-subunit, which undergoes conformational changes that couple ATP hydrolysis to ion transport, and the heavily glycosylated β-subunit responsible for maturation, assembly, and membrane targeting of the enzyme. Different isoforms of the α- and β-subunit have been identified, and these have unique kinetic properties and tissue distributions. As a housekeeping enzyme the regulation of the Na,K-ATPase is very complex and occurs at many different levels, including both rapid (short term) and sustained (long term) hormonal control. Recently, considerable interest have been directed at studying the role of protein-protein interactions in the acute hormonal regulation of Na,K-ATPase activity. Indeed, regulation of transport ATPases by interaction with small regulatory proteins is a well known mechanism to achieve modulation of ATPase activity in vivo (for review see Ref. 2Cornelius F. Mahmmoud Y.A. Christensen H.R.Z. J. Bioenerg. Biomemb. 2001; 33: 415-423Crossref PubMed Scopus (28) Google Scholar). Such interactions are especially well described for the regulation of SERCA 1The abbreviations used are: SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase; PLN, phospholamban; PKA, protein kinase A; PKC, protein kinase C; DTT, dithiothreitol; RACE, rapid amplification of cDNA ends; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CDTA, 1,2-cyclo-hexylenedinitrilotetraacetic acid; EST, expressed sequence tag. by phospholamban (PLN) and sarcolipin (3Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 4Kimura Y. Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1998; 273: 14238-14241Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Reddy L.G. Autry J.M. Jones L.R. Thomas D.D. J. Biol. Chem. 1999; 274: 7649-7655Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 6Odermat A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 7Russell Tupling A. Asahi M. MacLennan D.H. J. Biol. Chem. 2002; 277: 44740-44746Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The small protein called the γ-subunit is the first example of a small single transmembrane protein interacting with and regulating Na,K-ATPase (8Béguin P. Wang X. Firsov D. Puoti A. Claeys D. Horisberger J.-D. Gerring K. EMBO J. 1997; 16: 4250-4260Crossref PubMed Scopus (214) Google Scholar, 9Therien A.G. Goldshleger R. Karlish S.J.D. Blostein R. J. Biol. Chem. 1997; 272: 32628-32634Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 10Therien A.G. Karlish S.J.D. Blostein R. J. Biol. Chem. 1999; 274: 12252-12256Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The γ-subunit has been shown to modulate Na,K-ATPase activity in the kidney by affecting the E 2/E 1 equilibrium toward E 1, thus regulating the affinity for ATP at its low affinity site and the cytoplasmic Na+ and K+ competition (11Pu H.X. Cluzeaud F. Goldshleger R. Karlish S.J.D. Farman N. Blostein R. J. Biol. Chem. 2001; 276: 20370-20378Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The γ-subunit has a highly distinct distribution along different parts of the nephron allowing differential regulation of ion transport along different nephron segments (12Arystarkhova E. Wetzel R.K. Asinovski N.K. Sweadner K.J. J. Biol. Chem. 1999; 274: 33183-33185Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The γ-subunit is a member of a family of small hydrophobic proteins, now termed the FXYD domain-containing protein family (13Sweadner K.J. Rael E. Genomics. 2000; 68: 41-56Crossref PubMed Scopus (353) Google Scholar). This family includes phospholemman (PLM or FXYD1) (14Palmer C.J. Scott D. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar), the γ-subunit (FXYD2) (15Minor N.T. Sha Q. Nichols C.G. Mercer R.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6521-6525Crossref PubMed Scopus (59) Google Scholar), mammary tumor protein of 8-kDa molecular mass (MAT-8 or FXYD3) (16Morrison B.W. Moorman J.R. Kowdley G.C. Kobayashi Y.M. Jones L.R. Leder P. J. Biol. Chem. 1995; 270: 2176-2182Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), channel-inducing factor (CHIF or FXYD4) (17Attali B. Latter H. Rachamim N. Garty H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6092-6096Crossref PubMed Scopus (176) Google Scholar), related to ion channel (RIC or FYXD5) (18Fu X. Kamps M.P. Mol. Cell. Biol. 1997; 17: 1503-1512Crossref PubMed Scopus (94) Google Scholar), as well as FXYD6 and FXYD7. Until recently the physiological functions of the FXYD proteins (except FXYD2) were unknown. However, it has now become evident that they are tissue-specific regulators of ion transporters (see recent reviews, Refs. 19Crambert, G., and Geering, K. (2003) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2003/166/re1.Google Scholar and 20Cornelius F. Mahmmoud Y.A. News. Physiol. Sci. 2003; 18: 119-124PubMed Google Scholar). Originally, this idea was substantiated by the identification of a PLM homologue (phospholemman-like protein from shark, PLMS) that was shown to specifically interact with and modulate Na,K-ATPase in the shark rectal gland (21Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 274: 35969-35977Abstract Full Text Full Text PDF Scopus (105) Google Scholar). Subsequent investigations by Crambert et al. (22Crambert G. Fuzesi M. Garty H. Karlish S. Geering K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11476-11481Crossref PubMed Scopus (223) Google Scholar), who used a co-expression system to study the effects of mammalian FXYD1 on Na,K-ATPase activity, indicated that PLM interacts with and regulates Na,K-ATPase isoforms. Indeed, both FXYD 4 (CHIF) and FXYD7 have been recently reported to be tissue-specific regulators of Na,K-ATPase (23Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.D. Gerring K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (133) Google Scholar, 24Béguin P. Crambert G. Monnet-Tschudi F. Uldry M. Horisberger J.D. Garty H. Geering K. EMBO J. 2002; 21: 3264-3273Crossref PubMed Scopus (119) Google Scholar). Therefore, it seems conceivable that most, if not all, members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase. The regulatory interaction of Na,K-ATPase with FXYD proteins seems to play an important role in cellular physiology and pathophysiology. For instance, the physiological relevance of the γ-subunit has recently been substantiated by identification of a point mutation of a glycine residue, which is highly conserved among all FXYD proteins, which correlates with a renal magnesium deficiency (25Meij I.C. Koenderink J.B. van Bokhoven H. Assink K.F. Groenestege W.T. de Pont J.J. Bindels R.J. Monnens L.A. van den Heuvel L.P. Knoers N.V. Nat. Genet. 2000; 26: 265-266Crossref PubMed Scopus (216) Google Scholar). In addition, phenotypic analysis of CHIF knockout mice indicated that CHIF plays a vital role in the tolerance to high K+ loading (26Aizman R. Asher C. Fuzesi M. Latter H. Lonai P. Karlish S.J.D. Garty H. Am. J. Physiol. 2002; 283: F569-F577Crossref Scopus (104) Google Scholar). Thus, characterization and localization of FXYD proteins in different tissues represents an important aim in identifying regulatory mechanisms of ion transport under physiological and pathophysiological states. Little is known about the three-dimensional functional interactions leading to regulation of the Na,K-ATPase by FXYD proteins. Spatial localization of the γ-subunit has been indirectly inferred from cryo-electron microscopy of two-dimensional crystals (27Hebert H. Purhonen P. Vorum H. Thomsen K. Maunsbach A.V. J. Mol. Biol. 2001; 314: 479-494Crossref PubMed Scopus (71) Google Scholar) or from thermal denaturation experiments (28Donnet C. Arystarkhova E. Sweadner K.J. J. Biol. Chem. 2001; 276: 7357-7365Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). They seem to indicate that the γ-subunit is associated with the C terminus of the α-subunit being located either between the M2/M9 or the M9/M10 transmembrane segments. Recently, the kinetic effects of γ on Na,K-ATPase were allocated to distinct domains within the γ-subunit (29Pu X.P. Scanzano R. Blostein R. J. Biol. Chem. 2002; 277: 20270-20276Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Also, mutagenesis studies of both the γ-subunit and CHIF indicated that the FXYD motif was important for long term and stable association with the α-subunit, whereas the basic residues located at the C terminus of CHIF are not necessary for association but are important determinants for the functional effects of CHIF on Na,K-ATPase (23Béguin P. Crambert G. Guennoun S. Garty H. Horisberger J.D. Gerring K. EMBO J. 2001; 20: 3993-4002Crossref PubMed Scopus (133) Google Scholar). Recently it was demonstrated that residues in the transmembrane segment of γ and CHIF are important for their association with and regulation of the Na,K-ATPase (30Lindzen M. Aizman R. Lifshitz Y. Lubarski I. Karlish S.J. Garty H. J. Biol. Chem. 2003; 278: 18738-18743Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). PLM and its homologue PLMS are the only members of the FXYD family known to be phosphorylated by protein kinases. The C terminus of PLMS is heavily phosphorylated by PKC (21Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 274: 35969-35977Abstract Full Text Full Text PDF Scopus (105) Google Scholar), as is the case for PLM (14Palmer C.J. Scott D. Jones L.R. J. Biol. Chem. 1991; 266: 11126-11130Abstract Full Text PDF PubMed Google Scholar). Co-immunoprecipitation experiments demonstrated that dephosphorylated PLMS associated more strongly with the α-subunit than PKC-phosphorylated PLMS (21Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 274: 35969-35977Abstract Full Text Full Text PDF Scopus (105) Google Scholar). This suggests that the interaction between PLMS and the shark α-subunit could be controlled by protein kinase-mediated phosphorylation reactions in a similar way to that proposed for the phospholamban (PLN) regulation of the Ca-ATPase in cardiac tissue in response to hormonal stimulation (3Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 4Kimura Y. Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1998; 273: 14238-14241Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Reddy L.G. Autry J.M. Jones L.R. Thomas D.D. J. Biol. Chem. 1999; 274: 7649-7655Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 6Odermat A. Becker S. Khanna V.K. Kurzydlowski K. Leisner E. Pette D. MacLennan D.H. J. Biol. Chem. 1998; 273: 12360-12369Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 7Russell Tupling A. Asahi M. MacLennan D.H. J. Biol. Chem. 2002; 277: 44740-44746Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Furthermore, PKC phosphorylation of the C-terminal cytoplasmic domain of PLMS, or disruption of interactions within the transmembrane domain by treatment with non-solubilizing concentrations of the non-ionic detergent C12E8 have been shown to result in activation of the shark Na,K-ATPase by relieving the inhibitory effect of PLMS (21Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 274: 35969-35977Abstract Full Text Full Text PDF Scopus (105) Google Scholar). This again emphasizes the implication of multiple domain interaction between FXYD regulatory proteins and Na,K-ATPase, as is the case for PLN regulation of Ca-ATPase. In the present study we aim to further characterize the molecular interactions that result in the regulation of shark Na,K-ATPase by PLMS. To begin this, we have first cloned PLMS and determined its primary amino acid sequence from cDNA. In addition, we have characterized the cellular distribution of both PLMS and the α-subunit in rectal gland cells using immunocytochemical methods. Finally, through controlled proteolysis we have been able to preferentially cleave a 5-kDa fragment from the C terminus of PLMS, which contains the protein kinase phosphorylation sites. Using this approach, we have characterized the functional effects of the interaction between the C-terminal domain of PLMS and shark Na,K-ATPase. Some results of this study have been previously reported (31Cornelius F. Mahmmoud Y.A. Ann. N. Y. Acad. Sci. U. S. A. 2003; 986: 579-586Crossref PubMed Scopus (12) Google Scholar). Total RNA Extraction—Total RNA was extracted using a modification of the Chomczynski and Sacchi method (32Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar) as described previously (33Cutler C.P. Brezillon S. Bekir S. Sanders I.L. Hazon N. Cramb G. Am. J. Physiol. 2000; 279: R222-R229Google Scholar). In brief, tissues were collected and rapidly frozen in liquid nitrogen before transfer and storage at –80 °C. The tissue was pulverized using a mortar and pestle and then homogenized in 10 volumes (w/v) of 4 m guanidinium isothiocyanate, 25 mm sodium citrate, 0.5% (v/v) Sarkosyl, and 90 mm 2-mercaptoethanol using a Polytron PT 10 homogenizer (Kinematica Ltd.) set at position 5, for 2 × 20–30 s. Following homogenization, total RNA was extracted by the sequential addition of 0.1 volume of 2 m sodium acetate, pH 4.0, 0.5 volume of water-saturated phenol, and finally 0.2 volume of 1-bromo-3-chloropropane. Tubes were vortexed briefly between the additions of each solution and then centrifuged at 3900 × g for 30 min at 4 °C in a Beckman J6-MC centrifuge (Beckman Instruments Inc.). The upper aqueous phase was carefully transferred to a fresh tube, and then 2.5 volumes of 2-propanol and 0.2 volume of 1.2 m NaCl, 0.8 m sodium citrate, pH 7.0, was added sequentially with vortexing. The resulting solution was incubated at room temperature for 10 min, before centrifugation at 3900 × g for 30 min. The supernatant was aspirated, and the pellet was washed twice in 80% ethanol before drying under vacuum at room temperature for 5 min. After resuspension of the pellet in diethylpyrocarbonate-treated Milli-Q water, diluted samples (1:100) were prepared and the absorbance measured at 260 and 280 nm (Philips PU 8620 spectrophotometer) to estimate both the concentration and purity of the RNA samples. RNA samples from each extract were also run on denaturing formaldehyde gels and stained with ethidium bromide (as detailed below) to ensure that no degradation of the RNA had occurred. Cloning and Sequencing—First strand cDNA synthesis was carried out in a total reaction volume of 20 μl containing 5 μg of total rectal gland RNA, 75 mm Tris-HCl, pH 8.3, 3 mm MgCl2, 10 mm DTT, 10 μm oligo(dT)12–18, 1 mm each of deoxyribonucleotide triphosphates (dNTPs; dATP, dGTP, dCTP, and dTTP), and 200 units of Superscript II (Invitrogen, Paisley, UK). The reaction was incubated at 45 °C for 2 h and then stored frozen at –20 °C for use in PCR. This single strand cDNA template was used for the amplification and isolation of the initial 203-bp fragment with the sequences further 5′ or 3′ to this subsequently obtained by rapid amplification of cDNA ends using the Marathon RACE kit (Clontech, Basingstoke, UK) as described previously (33Cutler C.P. Brezillon S. Bekir S. Sanders I.L. Hazon N. Cramb G. Am. J. Physiol. 2000; 279: R222-R229Google Scholar). All PCR reactions were carried out using 0.5 μl of cDNA template in a total volume of 20 μl, comprising 10 mm Tris-HCl, pH 9.0, 50 mm KCl, 1.5 mm MgCl2, 0.2 mm dNTPs, 4 pmol each of sense and antisense primers, and 1.25 unit of Taq DNA polymerase (BioGene Ltd., Cambs, UK). Primers used were as follows. Initial amplifications were carried out using Squ-1 sense (CGXTTCACTTACGACTACTAC) and Squ-1 antisense (CCGCCTGCGGGTGGACAGACGGCG) primer pairs (X = any base). Subsequent nested 5′ and 3′-RACE reactions employed 5′-RACE-1 (CACACAGCACTGCGGCCAC), 5′-RACE-2 (CCACAATCAGTCCGACAACACGC), 3′-RACE-1 (GCGTGTTGTCGGACTGATTGTGG), and 3′-RACE-2 (GTGGCCGCAGTGCTGTGTG) primers in amplifications with the Marathon kit primers AP-1 (CCATCCTAATACGACTCACTATAGGGC) and AP-2 (ACTCACTATAGGGCTCGAGCGGC). PCR was performed using a hot start technique with an initial 2-min incubation at 92 °C, followed by 40 cycles of 94 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s, with a final incubation of 72 °C for 10 min. DNA fragments within PCR reactions were either purified directly using an Edge Biosystems Quick-Step PCR purification kit (VH Bio Ltd., Gosforth, UK) or separated by Tris acetate-EDTA-agarose gel electrophoresis (34Sambrook J. Gething M.J. Nature. 1989; 342: 224-225Crossref PubMed Scopus (62) Google Scholar) and bands of interest were purified using a Gene-clean II DNA purification kit (Anachem Ltd., Luton, UK). 3′- and 5′-RACE products were produced in nested PCR reactions using Squalus PLMS-specific sense and antisense primers in conjunction with the Marathon kit nested primers AP1 and AP2. PCR fragments generated using the initial primers or by RACE amplification were blunt-ended by incubation for 15 min at 72 °C with 0.025 unit/μl Pfu DNA polymerase in 1× Pfu buffer (Stratagene) containing 0.2 mm dNTPs and then cloned into TOP10 cells using a Zero Blunt TOPO PCR cloning kit (Invitrogen, Leek, The Netherlands). Positive colonies were identified by colony PCR, and cDNA fragments from at least four different clones were sequenced in both directions using a Big Dye Terminator sequencing kit (PerkinElmer Life Sciences Biosystems, Warrington, UK) as described previously (33Cutler C.P. Brezillon S. Bekir S. Sanders I.L. Hazon N. Cramb G. Am. J. Physiol. 2000; 279: R222-R229Google Scholar). Sequences were combined and analyzed using the GeneJockey II software package (Biosoft, Cambridge, UK). Northern Blotting—Northern blotting was performed as described previously (33Cutler C.P. Brezillon S. Bekir S. Sanders I.L. Hazon N. Cramb G. Am. J. Physiol. 2000; 279: R222-R229Google Scholar). The probe used for Northern analysis was a full-length cDNA containing the complete sequence shown in Fig. 1. Total RNA (5 μg, as measured by absorbance at 260 nm) extracted from various Squalus tissues was resuspended in MOPS buffer (20 mm MOPS, 8 mm sodium acetate, 1 mm EDTA, pH 7.8) containing 12.5 m formamide and 2.2 m formaldehyde and then denatured at 65 °C for 15 min and snap-cooled on ice before adding 0.1 volume of 5% "Loading Dyes" (0.025% bromphenol blue, 0.025% xylene cyanol, and 50% glycerol; all w/v). Samples (30–100 μl) were then loaded onto the agarose gel (1.2% agarose w/v (Biogene Ltd.), MOPS buffer containing 6.7% (v/v) formaldehyde) and electrophoresed at 135 V (5 V/cm) for 1.5–2 h in MOPS buffer. After electrophoresis, gels were stained for 30 min in 0.1 m ammonium acetate, 5 μg/ml ethidium bromide before destaining for 1–2 h in several changes of 0.1 m ammonium acetate before viewing on a UV transilluminator. The integrity and relative amounts of RNA loaded onto each lane were qualitatively assessed by viewing the sharpness and intensity levels of ethidium bromide-stained 18 S and 28 S ribosomal RNA bands as quantified using a gel documentation and analysis system (Syngene, Cambridge, UK). The staining intensities of the tissue rRNA bands were compared with a known standard, and the amount of total RNA loaded on each lane was re-determined. The separated RNAs were electroblotted overnight using TAE (40 mm Tris base, 0.35% (v/v) glacial acetic acid, 10 mm EDTA, pH 8.0) as blotting buffer (25 V, 0.75 amps) onto a Zeta Probe nylon membrane (Bio-Rad, Hemel Hempstead, UK). RNA blots were prehybridized in 10 ml of UltraHyb (Ambion, Huntingdon, UK) for 6 h at 47 °C and then hybridized overnight in the same solution with the 32P-labeled Squalus PLMS probe (Megaprime DNA labeling system, Amersham Biosciences, Little Chalfont, UK). Membranes were washed sequentially at 47 °C in 0.5× SSC, 1% SDS, then 0.2× SSC, 0.1% SDS and finally 0.1× SSC, 0.1% SDS for 15 min before analysis of radioactive intensity by electronic autoradiography (Instant Imager, Canberra Packard Instruments, Meriden, CT). The blots were finally incubated at –80 °C with x-ray film (Kodak BioMax MS) for autoradiography (1× SSC = 0.15 m NaCl, 15 mm sodium citrate, pH 7.0). Cellular Localization of PLMS and Na,K-ATPase α-Subunit—Salt glands from two sharks were immersion-fixed for 2 h with a solution containing 4% paraformaldehyde, 150 mm NaCl, and 100 mm sodium cacodylate buffer (pH 7.2). Small tissue blocks from the middle of the glands were cryo-protected with 2.3 m sucrose and frozen in liquid nitrogen. Immunoelectron microscopy was performed on thin (60–80 nm) cryosections, which were cut at –120 °C from the frozen tissue on a Reichert Ultracut S cryo-ultramicrotome (Leica, Vienna, Austria). Immunolabeling and staining was performed as previously described (35Manusbach A.B. Celis J.E. Cell Biology: A Laboratory Handbook. 2nd Ed. 3. Academic Press, San Diego1998: 268-275Google Scholar). Briefly, the sections were first preincubated in PBS containing 0.1% skimmed milk powder and 50 mm glycine. The sections were then incubated for 1 h at room temperature with rabbit anti-PLMS antibodies, rabbit anti-Na,K-ATPase α-subunit antibodies, or pre-immune sera diluted 1:100–1:1600 in PBS containing 0.1% skimmed milk powder. The primary antibodies were visualized using goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM1O, Bio-Cell Research Laboratories, Cardiff, UK) diluted 1:50 in PBS with 0.1% skimmed milk powder and polyethylene glycol (5 mg/ml). The Ultrathin cryosections were stained with uranyl acetate in methylcellulose before examination in a Zeiss 912 or a Philips 208 electron microscope. Immunolabeling controls consisted of substitution of the primary antibody with rabbit pre-immune IgG or incubation without primary antibody. All controls showed absence of specific labeling. Na,K-ATPase Preparation and Hydrolytic Activity—In this study, purified Na,K-ATPase-containing membranes from the rectal gland of Squalus acanthias were used. Purification of membrane fragments was as previously described (36Skou J.C. Esmann M. Biochim. Biophys. Acta. 1979; 567: 436-444Crossref PubMed Scopus (130) Google Scholar). Protein concentrations, ranging from 3 to 5 mg/ml, were determined using Peterson's modification of the Lowry method (37Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7134) Google Scholar), using bovine serum albumin as a standard. The specific activity was ∼ 30 units/mg at 37 °C and 10.5 units/mg at 24 °C (1 unit = 1 μmol of Pi/min). The ATPase activity was measured in a reaction mixture containing 30 mm histidine, pH 7.4, 3 mm MgCl2, 0.06% bovine serum albumin, 10% glycerol, 10 μm ATP (containing 0.03 μCi of γ-[32P]ATP), and variable concentrations of NaCl, KCl, and ATP as indicated in separate figure legends. The concentration of Pi hydrolyzed from ATP was measured as previously described (38Lindberg O. Ernster L. Methods Biochem. Anal. 1956; 3: 1-12Crossref PubMed Google Scholar). PKA and PKC Phosphorylation of Na,K-ATPase—PKA phosphorylation was performed in a reaction mixture containing 50 mm Hepes, 10 mm MgCl2, 1 mm EGTA, 0.1 mm ATP (Tris salt) containing γ-[32P]ATP (3 μCi/pmol), 0.1% Triton X-100, 4 μg of protein, and 2 milliunits of PKA. The catalytic subunit of PKA was purchased from Sigma. PKC phosphorylation was performed in a typical assay mixture containing 50 mm Hepes, 10 mm MgCl2, 0.5 mm CaCl2, 20 μm l-α-phosphatidylserine (Avanti Polar Lipids, Alabaster, AL), 10 μm dioleoyl 1, 2-sn-glycerol (Sigma, St. Louis, MO), 100 μm ATP (Tris-salt) containing 3 μCi/pmol γ-[32P]ATP, 4 μg of protein, and 0.13 μg of purified PKC. PKC was from Calbiochem (La Jolla, CA), and contained the Ca2+-dependent (conventional) isoforms (α, βI, βII, and γ). The phosphorylation reaction for both kinases was initiated by the addition of ATP, allowed to proceed for 30 min at 24 °C, and terminated by the addition of 16 μl of electrophoresis sample buffer (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207200) Google Scholar). Gel Electrophoresis and Immunoblotting—The phosphorylated proteins were separated using Tricine-based SDS-PAGE (3% resolution gel, 9% intermediate, and 16% resolving gels, unless indicated elsewhere). Molecular weight standards were from Bio-Rad (Hercules, CA). For the detection of 32P-assisted kinase phosphorylation, the gels were stained with Coomassie Blue, destained, dried, and then analyzed by autoradiography overnight at –80 °C. For immunoblotting, proteins were transferred to polyvinylidene difluoride membranes, then washed for 1 h with PBS buffer containing 5% Tween 20, and incubated overnight at room temperature with primary antibody. The membranes were washed again with PBS and incubated with goat anti-rabbit antibody for 2 h. After washing, the proteins were detected using ECL reagents (Amersham Biosciences). For the detection of the α-subunit from shark rectal gland and pig kidney, the antibody NKA1002–1016 was used (kindly provided by Jesper V. Møller, Department of Biophysics, University of Aarhus). Preparation of Trypsinized PLMS—To obtain cleavage of the C terminus of PLMS, membrane-bound enzyme was incubated with trypsin (w/w trypsin to protein 1:1000) for 0–10 min on ice in the presence of 130 mm NaCl or 20 mm KCl, plus 1 mm EDTA. The trypsinization reaction was started by the addition of trypsin and stopped by adding a 10-fold excess of soybean trypsin inhibitor. The mixtures was diluted 10-fold with imidazole buffer (25 mm) and centrifuged at 170,000 × g for 1 h. The membranes were washed with imidazole and centrifuged again, then finally suspended in a 30 mm histidine buffer, pH 7.4, containing 25% glycerol, and stored at –20 °C. All proce
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