Zebrafish Slc5a12 Encodes an Electroneutral Sodium Monocarboxylate Transporter (SMCTn)
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m609313200
ISSN1083-351X
AutoresConsuelo Plata, Caroline R. Sussman, Aleksandra Sinđić, Jennifer O. Liang, David B. Mount, Zara M. Josephs, Min‐Hwang Chang, Michael F. Romero,
Tópico(s)Ion channel regulation and function
ResumoWe have identified and characterized two different sodium-coupled monocarboxylate cotransporters (SMCT) from zebrafish (Danio rerio), electrogenic (zSMCTe) and electroneutral (zSMCTn). zSMCTn is the 12th member of the zebrafish Slc5 gene family (zSlc5a12). Both zSMCT sequences have ∼50% homology to human SLC5A8 (hSMCT). Transport function and kinetics were measured in Xenopus oocytes injected with zSMCT cRNAs by measurement of intracellular Na+ concentration ([Na+]i) and membrane potential. Both zSMCTs oocytes increased [Na+]i with addition of monocarboxylates (MC) such as lactate, pyruvate, nicotinate, and butyrate. By using two electrode voltage clamp experiments, we measured currents elicited from zSMCTe after MC addition. MC-elicited currents from zSMCTe were similar to hSMCT currents. In contrast, we found no significant MC-elicited current in either zSMCTn or control oocytes. Kinetic data show that zSMCTe has a higher affinity for lactate, nicotinate, and pyruvate (KmL-lactate = 0.17 ± 0.02 mm, Kmnicotinate = 0.54 ± 0.12 mm at -150 mV) than zSMCTn (KmL-lactate = 1.81 ± 0.19 mm, Kmnicotinate = 23.68 ± 4.88 mm). In situ hybridization showed that 1-, 3-, and 5-day-old zebrafish embryos abundantly express both zSMCTs in the brain, eyes, intestine, and kidney. Within the kidney, zSMCTn mRNA is expressed in pronephric tubules, whereas zSMCTe mRNA is more distal in pronephric ducts. zSMCTn is expressed in exocrine pancreas, but zSMCTe is not. Roles for Na+-coupled monocarboxylate cotransporters have not been described for the brain or eye. In summary, zSMCTe is the zebrafish SLC5A8 ortholog, and zSMCTn is a novel, electroneutral SMCT (zSlc5a12). Slc5a12 in higher vertebrates is likely responsible for the electroneutral Na+/lactate cotransport reported in mammalian and amphibian kidneys. We have identified and characterized two different sodium-coupled monocarboxylate cotransporters (SMCT) from zebrafish (Danio rerio), electrogenic (zSMCTe) and electroneutral (zSMCTn). zSMCTn is the 12th member of the zebrafish Slc5 gene family (zSlc5a12). Both zSMCT sequences have ∼50% homology to human SLC5A8 (hSMCT). Transport function and kinetics were measured in Xenopus oocytes injected with zSMCT cRNAs by measurement of intracellular Na+ concentration ([Na+]i) and membrane potential. Both zSMCTs oocytes increased [Na+]i with addition of monocarboxylates (MC) such as lactate, pyruvate, nicotinate, and butyrate. By using two electrode voltage clamp experiments, we measured currents elicited from zSMCTe after MC addition. MC-elicited currents from zSMCTe were similar to hSMCT currents. In contrast, we found no significant MC-elicited current in either zSMCTn or control oocytes. Kinetic data show that zSMCTe has a higher affinity for lactate, nicotinate, and pyruvate (KmL-lactate = 0.17 ± 0.02 mm, Kmnicotinate = 0.54 ± 0.12 mm at -150 mV) than zSMCTn (KmL-lactate = 1.81 ± 0.19 mm, Kmnicotinate = 23.68 ± 4.88 mm). In situ hybridization showed that 1-, 3-, and 5-day-old zebrafish embryos abundantly express both zSMCTs in the brain, eyes, intestine, and kidney. Within the kidney, zSMCTn mRNA is expressed in pronephric tubules, whereas zSMCTe mRNA is more distal in pronephric ducts. zSMCTn is expressed in exocrine pancreas, but zSMCTe is not. Roles for Na+-coupled monocarboxylate cotransporters have not been described for the brain or eye. In summary, zSMCTe is the zebrafish SLC5A8 ortholog, and zSMCTn is a novel, electroneutral SMCT (zSlc5a12). Slc5a12 in higher vertebrates is likely responsible for the electroneutral Na+/lactate cotransport reported in mammalian and amphibian kidneys. The liver and the kidneys are the major sites of lactate uptake and metabolism. Normally the blood lactate concentration is ∼1.2 mm; however, lactate levels can increase to 10 mm during maximal exercise. Lactate is reabsorbed in kidney such that only trace amounts of lactate are normally excreted in the urine. Lactate in the glomerular ultrafiltrate is almost completely reabsorbed by the proximal nephron (1Geibisch G. Windhager E. Boron W.F. Boulpaep E.L. Medical Physiology. 1st Ed. W. B. Saunders Co., Philadelphia2003: 790-813Google Scholar). The movement of lactate and other monocarboxylates across plasma membranes is thought to occur by proton-linked monocarboxylate transporters (MCT) 4The abbreviations used are: MCT, monocarboxylate transporter; SMCT, sodium monocarboxylate cotransporter; MCs, monocarboxylates; [Na+]i, intracellular Na+ concentration; AcAc, acetoacetate; SMCTe, electrogenic; SMCTn, electroneutral; EST, expressed sequence tag; AP, alkaline phosphatase; βOH-buty, β-hydroxybutyrate; hpf, hours post-fertilization; dpf, days post-fertilization. (2Juel C. Halestrap A.P. J. Physiol. (Lond.). 1999; 517: 633-642Crossref Scopus (332) Google Scholar, 3Halestrap A.P. Meredith D. Pfluegers Arch. 2004; 447: 619-628Crossref PubMed Scopus (855) Google Scholar). The MCT gene family (Slc16) now includes 14 members, of which only the first 4 members (MCT1–MCT4) have been demonstrated experimentally to mediate the proton-linked transport of metabolically important monocarboxylates, such as lactate, pyruvate, and short chain fatty acids. MCTs are expressed in a number of tissues, including kidney (2Juel C. Halestrap A.P. J. Physiol. (Lond.). 1999; 517: 633-642Crossref Scopus (332) Google Scholar, 3Halestrap A.P. Meredith D. Pfluegers Arch. 2004; 447: 619-628Crossref PubMed Scopus (855) Google Scholar). However, a significant body of data indicates an important role for luminal Na+/monocarboxylate cotransport in the reabsorption of filtered lactate by the proximal tubule. Some studies have concluded that luminal Na+/lactate transport is electroneutral (4Barbarat B. Podevin R.A. J. Biol. Chem. 1988; 263: 12190-12193Abstract Full Text PDF PubMed Google Scholar, 5Siebens A.W. Boron W.F. J. Gen. 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Turk E. Pfluegers Arch. 2004; 447: 510-518Crossref PubMed Scopus (30) Google Scholar). In humans, 11 SLC5 genes have been identified (15Wright E.M. Turk E. Pfluegers Arch. 2004; 447: 510-518Crossref PubMed Scopus (30) Google Scholar). SLC5 members function as Na+ and solute (e.g. glucose, myoinositol, iodide and multivitamins) cotransporters (15Wright E.M. Turk E. Pfluegers Arch. 2004; 447: 510-518Crossref PubMed Scopus (30) Google Scholar). Although the functions of some of the family members are well understood, e.g. Slc5a1 (SGLT1; the Na+/glucose cotransporter) and Slc5a5 (NIS; the Na+/I- cotransporter), others family members (SLC5A9-A12) have not been well characterized (15Wright E.M. Turk E. Pfluegers Arch. 2004; 447: 510-518Crossref PubMed Scopus (30) Google Scholar, 16Wright E.M. Loo D.D. Hirayama B.A. Turk E. Physiology (Bethesda). 2004; 19: 370-376Crossref PubMed Scopus (152) Google Scholar). SLC5A8 was originally cloned by Rodriguez et al. (17Rodriguez A.M. Perron B. 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Recently, the SLC5A8 gene was shown to play an important role in controlling the development of colon cancers (19Li H. Myeroff L. Smiraglia D. Romero M.F. Pretlow T. Kasturi L. Lutterbaugh J. Casey G. Issa J.-P. Willis J. Willson J.K.V. Plass C. Markowitz S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8412-8417Crossref PubMed Scopus (256) Google Scholar) and classical papillary thyroid carcinomas (PTC-cf) (20Porra V. Ferraro-Peyret C. Durand C. Selmi-Ruby S. Giroud H. Berger-Dutrieux N. Decaussin M. Peix J.L. Bournaud C. Orgiazzi J. Borson-Chazot F. Dante R. Rousset B. J. Clin. Endocrinol Metab. 2005; 90: 3028-3035Crossref PubMed Scopus (96) Google Scholar). Silencing of SLC5A8 is a common and early event in both tumors. Promoter methylation that inactivates SLC5A8 is found in ∼60% of colon cancer cell lines and primary colon cancers and >80% adenomas (19Li H. Myeroff L. Smiraglia D. Romero M.F. Pretlow T. Kasturi L. Lutterbaugh J. Casey G. Issa J.-P. Willis J. Willson J.K.V. Plass C. Markowitz S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8412-8417Crossref PubMed Scopus (256) Google Scholar). In the case of PTC-cf, SLC5A8 silencing by methylation was found in 90% of thyroid carcinomas (follicular, the most common type) and occurred in about 20% of other papillary thyroid carcinomas (20Porra V. Ferraro-Peyret C. Durand C. Selmi-Ruby S. Giroud H. Berger-Dutrieux N. Decaussin M. Peix J.L. Bournaud C. Orgiazzi J. Borson-Chazot F. Dante R. Rousset B. J. Clin. Endocrinol Metab. 2005; 90: 3028-3035Crossref PubMed Scopus (96) Google Scholar). SLC5A8 is thus also hypothesized to be a tumor suppressor gene. We reasoned that such important functions of SLC5A8 should be evolutionarily preserved and could be crucial in development and differentiation, particularly of epithelial tissues. In fact, Costa and co-workers (21Costa R.M. Mason J. Lee M. Amaya E. Zorn A.M. Gene Expr. Patterns. 2003; 3: 509-519Crossref PubMed Scopus (26) Google Scholar) have found that Slc5a8 (Vito) is first expressed at the blastopore lip (the beginning of neurulation and gastrulation) in Xenopus tadpoles. Human SLC5A8 and mouse Slc5a8 protein functions have been studied previously in Xenopus laevis oocytes. As a transporter, SLC5A8 is an electrogenic Na+/monocarboxylate cotransporter (SMCT) moving substrates such as short chain fatty acids (propionate, butyrate, and valerate) as well as monocarboxylates such as lactate, pyruvate, nicotinate, and pyrazinoate (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar, 14Miyauchi S. Gopal E. Fei Y.J. Ganapathy V. J. Biol. Chem. 2004; 279: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 22Gopal E. Fei Y.J. Miyauchi S. Zhuang L. Prasad P.D. Ganapathy V. Biochem. J. 2005; 388: 309-316Crossref PubMed Scopus (79) Google Scholar). Yet, these substrates are apparently moved with varied affinity and coupling to Na+ (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar, 14Miyauchi S. Gopal E. Fei Y.J. Ganapathy V. J. Biol. Chem. 2004; 279: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 22Gopal E. Fei Y.J. Miyauchi S. Zhuang L. Prasad P.D. Ganapathy V. Biochem. J. 2005; 388: 309-316Crossref PubMed Scopus (79) Google Scholar). Slc5a8 was also cloned from mouse kidney (23Gopal E. Fei Y.J. Sugawara M. Miyauchi S. Zhuang L. Martin P. Smith S.B. Prasad P.D. Ganapathy V. J. Biol. Chem. 2004; 279: 44522-44532Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Not surprisingly, mouse Slc5a8 expressed in Xenopus oocytes is electrogenic, transporting the same substrates as human SMCT (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar, 14Miyauchi S. Gopal E. Fei Y.J. Ganapathy V. J. Biol. Chem. 2004; 279: 13293-13296Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Thus, Slc5a8 is the first transporter known to be expressed in mammalian colon and kidney that has the ability to mediate the electrogenic absorption of lactate and other monocarboxylates with Na+ (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar, 23Gopal E. Fei Y.J. Sugawara M. Miyauchi S. Zhuang L. Martin P. Smith S.B. Prasad P.D. Ganapathy V. J. Biol. Chem. 2004; 279: 44522-44532Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 24Ganapathy V. Gopal E. Miyauchi S. Prasad P.D. Biochem. Soc. Trans. 2005; 33: 237-240Crossref PubMed Scopus (89) Google Scholar). The Km and Vmax values for MC substrates of SLC5A8 indicate that it is a high affinity and low capacity transporter that could easily be saturated by MCs in the renal ultrafiltrate or in the colonic lumen. Thus, an electroneutral Na+/MC cotransporter, particularly with high transport capacity, would allow MC absorption without excess loss in the urine or stool. Yet the molecular identity of such electroneutral Na+/lactate transport proteins in kidney remains unknown. In this study we identified, localized, and functionally characterized two zebrafish SMCTs. One is the electrogenic SMCT (Slc5a8), and the other is a novel electroneutral form of the Na+/monocarboxylate cotransporter (SMCTn) with high transport capacity. SMCTn is the zebrafish ortholog of Slc5a12 recently reported as a second electrogenic Na+/monocarboxylate cotransporter (32Srinivas S.R. Gopal E. Zhuang L. Itagaki S. Martin P.M. Fei Y.J. Ganapathy V. Prasad P.D. Biochem. J. 2005; 392: 655-664Crossref PubMed Scopus (107) Google Scholar). Our data demonstrate that both SMCTn and the electrogenic SMCT (SMCTe/Slc5A8) are present in kidney and brain of the teleost Danio rerio (zebrafish) and have different kinetic properties. Both proteins increase intracellular Na+ in response to a variety of MCs as follows: short chain fatty acids, lactate, pyruvate, nicotinate, acetoacetate, and 3-β-hydroxybutyrate. Slc5a8 mRNA is also found in the gut consistent with its original description in human. cDNA Cloning of Zebrafish SMCT, zSlc5a8 and zSlc5a12—A BLAST search of the EST data base revealed two D. reiro cDNAs that were homologous to human SLC5A8, yet clearly distinct from one another. The cDNAs corresponding these ESTs (IMAGE clones zSMCTe (zSlc5a8, 7212813) and zSMCTn (Slc5a12, 6793401)) were obtained from the IMAGE consortium (Research Genetics, Genome Systems) and sequenced (GenBank™ accession numbers AY727859 for zSMCTe and AY727860 for zSMCTn). The complete EST inserts were sequenced (W. M. Keck Facility, New Haven, CT) and then subcloned in pGEMHE Xenopus oocyte expression vector between EcoRI (5′) and XbaI (3′). Oocyte Isolation and Injection—Oocytes were removed from female X. laevis (Xenopus Express, Beverly Hills, FL) as described previously (25Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar). Excised lobes of oocytes were placed into a Ca2+-free buffered saline solution (200 mosm) and defolliculated by collagenase digestion as described previously (26Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar). Capped zSlc5a8 and zSlc5a12 cRNAs were synthesized using a linearized cDNA template and the T7 mMessage mMachine (Ambion, Austin, TX). Oocytes were injected with 50 nl of 0.5 ng/nl (25 ng/oocyte) cRNA of zSMCTe, zSMCTn, hSMCT, or water. The oocytes were maintained at 18 °C in filtered ND96 medium (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1.8 mm CaCl2, 5 mm HEPES-Tris (pH 7.5)) (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar). Oocytes were studied 3–10 days after injection. All experiments were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of Case Western Reserve University. Electrophysiology—All monocarboxylate solutions (1 mm lactate, pyruvate, propionate, and butyrate) were prepared in ND96 (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5 mm HEPES (pH 7.5)) as described previously (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar). Unless otherwise stated, MCs were all used at 1 mm. To examine the kinetics of lactate and nicotinate transport of zSMCTn, a low chloride ND96 was used to maintain the same tonicity for all solutions (∼200 mosmol). For lactate curves, we used (in mm) 66 NaCl base solution with 30 to 0 sodium gluconate and 0 to 30 sodium lactate. For nicotinate curves, we used 56 NaCl base with 50 to 0 sodium gluconate and 0–50 sodium nicotinate. All solutions also contained 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES and were pH 7.5. Ion-selective Microelectrode—Ion-selective microelectrodes were used to monitor intracellular Na+ concentration ([Na+]i) of zSMCTs and water-injected oocytes as described previously (25Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar, 26Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar, 27Romero M.F. Henry D. Nelson S. Harte P.J. Dillon A.K. Sciortino C.M. J. Biol. Chem. 2000; 275: 24552-24559Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Intracellular [Na+] was measured as the difference between the Na+ electrode and a KCl voltage electrode impaled into the oocyte; and membrane potential (Vm) was the potential difference between the KCl microelectrode and an extracellular calomel (26Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar, 28Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (392) Google Scholar). Na+ microelectrodes were calibrated using 10 mm NaCl and 100 mm NaCl solutions followed by point calibration in ND96 (96 mm Na+) as performed previously (26Romero M.F. Fong P. Berger U.V. Hediger M.A. Boron W.F. Am. J. Physiol. 1998; 274: F425-F432Crossref PubMed Google Scholar, 28Romero M.F. Hediger M.A. Boulpaep E.L. Boron W.F. Nature. 1997; 387: 409-413Crossref PubMed Scopus (392) Google Scholar). Intracellular Na+ microelectrodes had slopes of at least -50 mV/p(Na+). Two Electrode Voltage Clamp—Oocyte membrane currents were recorded using an OC-720C voltage clamp (Warner Instruments, Hamden, CT), filtered at 2–5 kHz, digitized at 10 kHz, and recorded with the Pulse software, and data were analyzed using the PulseFit program (HEKA, Germany) as described previously (25Sciortino C.M. Romero M.F. Am. J. Physiol. 1999; 277: F611-F623Crossref PubMed Google Scholar, 29Dinour D. Chang M.-H. Satoh J.-I. Smith B.L. Angle N. Knecht A. Serban I. Holtzman E.J. Romero M.F. J. Biol. Chem. 2004; 279: 52238-52246Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). For periods when I-V protocols were not being run, oocytes were clamped at a holding potential (Vh) of -50 mV; and the current was constantly monitored and recorded at 1 Hz. I-V protocols consisted of 400-ms steps from Vh to -150 mV and +50 mV in 20-mV steps as described previously (13Coady M.J. Chang M.-H. Charron F.M. Plata C. Wallendorff B. Sah J.F. Markowitz S.D. Romero M.F. Lapointe J.-Y. J. Physiol. (Lond.). 2004; 557: 719-731Crossref Scopus (141) Google Scholar).The I-V protocols were run in the absence and in the presence of a particular substrate. The substrate-specific current is determined by subtraction of the pre-substrate current from the substrate current. All of the experiments were performed at room temperature. Test substrates bathed oocytes for 1–3 min. For kinetic analysis, oocytes were exposed to only one monocarboxylate at one concentration and then discarded. Calculations—Oocytes were perfused with ND96 for 5 min at which time "initial [Na+]i" is measured. The solution was switched to different monocarboxylates for 8–10 min (i.e. [Na+]i and Vm or I plateau), and the "final [Na+]i" is measured. Oocytes came from at least four separate donor animals. 22Na+ Uptakes—Functions of zSMCTe and zSMCTn were assessed by measuring 22Na+ uptake in groups of 15–20 oocytes 4 days after water or cRNA injection. 22Na+ uptake was performed as described previously (30Plata C. Meade P. Vazquez N. Hebert S.C. Gamba G. J. Biol. Chem. 2002; 277: 11004-11012Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Briefly, 22Na+ uptake was measured with the following protocol: a 30-min incubation period ND96 with 1 mm ouabain, 100 μm amiloride, 100 μm bumetanide, followed by a 60-min uptake period in ND96. To analyze the pyruvate transport kinetics, we varied pyruvate concentration from 0 to 5 mm in ND96 for zSMCTe and from 0 to 20 mm for zSMCTn. These uptake solutions also contained 1 μCi/ml of 22Na+ (PerkinElmer Life Sciences) and the same inhibitors used during the incubation period. Uptakes were performed at 30 °C. At the end of the uptake period, oocytes were washed five times in ice-cold ND96 solution without isotope to remove extracellular tracer. Next, individual oocytes were dissolved in 10% SDS, and tracer activity was determined by β scintillation counting. RNA-injected oocytes were compared with water controls subjected to identical conditions, using oocytes from the same donor. Kinetic analysis was performed by estimating the Km values for pyruvate of each SMCT. The Km values were calculated from log [ion] versus V/Vmax plots using GraphPad Prism (GraphPad Software, Inc., San Diego). RNA in Situ Hybridization—Single label in situ hybridization was carried out as described (31Thisse C. Thisse B. Schilling T.F. Postlethwait J.H. Development (Camb.). 1993; 119: 1203-1215Crossref PubMed Google Scholar). Reagents were obtained from Roche Applied Science. Briefly, zebrafish embryo groups were incubated at 70 °C with digoxigenin-UTP-labeled antisense RNA probe for zSMCTe or zSMCTn in hybridization solution containing 50% formamide, detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase (AP), and visualized with a combination of 4-nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (blue precipitate). We alkali-hydrolyzed cRNA probes with 0.2 m Na2CO3 (pH 10.2) for 14 min at 60 °C to obtain 200–300-bp fragments (size verified by RNA gel and [cRNA] determined by UV spectrometry). AP buffer is 100 mm Tris-HCl (pH 9.5), 50 mm MgCl2, 100 mm NaCl, 0.1% Tween 20. The developing solution was 225 μg/ml 4-nitro blue tetrazolium and 175 μg/ml 5-bromo-4-chloro-3-indolyl phosphate in AP buffer. Endogenous AP activity was inhibited by adding 2 mm levamisole (Sigma) to all solutions after antibody incubation. Following in situ hybridization, embryos were postfixed in 4% paraformaldehyde, dehydrated through a graded ethanol series, destained with undiluted methyl salicylate (M6752, Sigma), digitally photographed (27Romero M.F. Henry D. Nelson S. Harte P.J. Dillon A.K. Sciortino C.M. J. Biol. Chem. 2000; 275: 24552-24559Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), and stored in mineral oil. Fish Embryo Sections—After in situ hybridization, 5 dpf embryos were postfixed in 4% paraformaldehyde for 1 h at room temperature. Fixed embryos were incubated overnight in 30% sucrose in phosphate-buffered saline and then embedded in OCT. 15-μm cryosections were cut with a Leica cryostat and mounted on gelatin-coated slides. Embryo sections were observed using a Zeiss Axiovert 25 microscope and acquired with an AxioCam digital camera and AxioVision software (Carl Zeiss, Germany) as described previously (27Romero M.F. Henry D. Nelson S. Harte P.J. Dillon A.K. Sciortino C.M. J. Biol. Chem. 2000; 275: 24552-24559Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Statistical Analysis—The results are presented as means ± S.E. The significance of the differences between groups was tested by one-way analysis of variance with multiple comparison using Bonferroni correction or by the Kruskal-Wallis one-way analysis of variance on ranks with the Dunn method for multiple comparison procedure, as needed. Molecular Cloning of the zSMCTs—We found two cDNAs for the zebrafish (D. rerio) homologs of the human electrogenic sodium/monocarboxylate cotransporter (hSMCTe/SLC5A8). One cDNA (zSMCTe) encodes a 610-amino acid protein (GenBank™ accession number AY727859), whereas the other cDNA (zSMCTn) encodes a 623 amino acid protein (GenBank™ accession number AY727860). zSMCTe and zSMCTn are 46% identical. Both zSMCT sequences are roughly equally divergent from mammalian Slc5a8. zSMCTe has 55 and 56% amino acid identity, and zSMCTn has 51 and 53% amino acid identity to human and mouse Slc5a8, respectively. zSMCTn shows 64% identity to mouse Slc5a12, and zSMCTe is 48% identical to mammalian Slc5a12 (Fig. 1). Secondary structure of zSMCT proteins was predicted using TMHMM. This analysis predicts that zSMCTe has 13 transmembrane spans, as indicated for hSMCT (19Li H. Myeroff L. Smiraglia D. Romero M.F. Pretlow T. Kasturi L. Lutterbaugh J. Casey G. Issa J.-P. Willis J. Willson J.K.V. Plass C. Markowitz S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8412-8417Crossref PubMed Scopus (256) Google Scholar). The zSMCTe protein contains three potential N-linked glycosylation sites in extracellular loops (Asn-112, Asn-439, and Asn-485), 19 Ser, 8 Thr, and 4 Tyr-like potential phosphorylation sites but only 7 Ser (Ser-477, Ser-523, Ser-541, Ser-556, Ser-568, Ser-570, and Ser-588), 2 Thr (Thr-75 and Thr-84), and one Tyr (Tyr-80) are outside of the membrane, increasing the probability these are actual phosphorylation sites. A similar sequence analysis of zSMCTn also predicted 13 transmembrane spans. There are two potential N-linked glycosylation sites in extracellular loops (Asn-478 and Asn-614). Several potential phosphorylation sites are predicted, but only a few are not in the membrane making them accessible to kinases: 9 Ser (Ser-111, Ser-216, Ser-232, Ser-266, Ser-303, Ser-314, Ser-379, Ser-547, and Ser-597), 3 Thr (Thr-217, Thr-495, and Thr-587), and two Tyr (Tyr-12 and Tyr-82). BLAST analysis of the zSMCTn sequence against the human genome data base resulted in a hypothetical protein MGC52019 (SLC5A12; NT_086780.1 and NT_009237.17) with ∼65% identity to zSMCTn. Recently, Srinivas et al. (32Srinivas S.R. Gopal E. Zhuang L. Itagaki S. Martin P.M. Fei Y.J. Ganapathy V. Prasad P.D. Biochem. J. 2005; 392: 655-664Crossref PubMed Scopus (107) Google Scholar) identified and cloned mouse Slc5a12 indicating that mammalian homologs are not just putative. Thus, zSMCTn is the zebrafish ortholog of mouse Slc5a12, and zSMCTe is the ortholog of human SLC5A8 (Fig. 1). Functional Expression of zSMCT Proteins in Xenopus Oocytes— We analyzed the function of both zSMCT clones by simultaneous measurement of membrane potential (Vm) and intracellular Na+ concentration ([Na+]i) with ion-selective microelectrodes in Xenopus oocytes injected with zSMCTn or zSMCTe cRNA. Using two electrode voltage clamp experiments, we also examined currents elicited by monocarboxylates for both SMCT cotransporters. All MCs tested induced a significant increase of intracellular [Na+] in both zSMCT oocytes (Δ[Na+] zSMCTei = 2.76 ± 0.29 mm, n = 8; Δ[Na+] zSMCTni = 2.28 ± 0.33 mm, n = 9). In contrast, control oocytes displayed only minor increments of intracellular Na+ (Δ[Na+] controli = 0.31 ± 0.10 mm; n = 6). Comparison to the human transporter indicates that hSMCTe transports slightly more Na+ Δ[Na+]hSMCTi = 3.47 ± 0.77 mm) than both zSMCT clones expressed in oocytes. Fig. 2, A–D, shows individual experiments of simultaneous measurement of [Na+]i (top traces) and Vm (bottom traces) in oocytes. These experiments show that addition of 1 mm MC 5Several
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