Is Sodium Monocarboxylate Transporter (SMCT1) A Protein Involved in the Apical Iodide Transport?

Sunday, February 28, 2016 displays voltage-gated channels, enabling it to fire action potentials. Here we present evidences for the expression of Naþ-Hþ antiporters, as housekeepers maintaining the intracellular pH. Methods: Ganglia from newborn Wistar rats were digested with trypsin. The cells were maintained in DMEMþfetal bovine serum in CO2 5% atmosphere,  37 C. Intracellular pH was estimated with BCECF fluorescent indicator. The cells were perfused continuously with solutions, heated to 37 C. Ammonium pulses (NH4Cl, 20mM) or exposure to acidic extracellular solution (pH 6.5) leads to acidic load. On the recovery, [Hþ] decline could be well fitted by one exponential, whose rate constant is associated with the kinetics of the Naþ-Hþ antiporter and cytosolic buffering power. Naþ-Hþ antiporter was identified by the inhibitors amiloride (1 mM) or EIPA (5 mM) and by the effects of low Naþ extracellular solution (50 mM). Results and Conclusion: After acidic load, Hþ is pumped out at a rate of 0.013850.0061s1(n=63) (ammonium pulse) and 0.014650.0063s1 (n=43)(acidic extracellular pH), recovering intracellular pH to the previous levels. Amiloride completely blocked pH recovery in 17 cells and reduced the recovery rate in 10 (0.008650.0059s1). EIPA reduced rate to 0.006650.0045s1(n=23) in all cells. Further evidence for the Naþ-Hþ antiporter is reduced rate in low-Naþ (0.005550.0016s1, n=4). DRGneurons express Naþ-Hþ antiporter as housekeepers regulating the intracellular pH. Evidences for the Naþ-Hþ antiporter are provided by the effects, on the rates, of low-Naþ and classical inhibitors. Amiloride affects differentially cell populations, probably due to different isoforms. Financial support: Capes, CNPq and FAPESP. 701-Pos Board B481 Ribonucleotide Reductase Overexpression does not Alter Cardiomyocyte Mitochondrial Respiration Jason D. Murray1, Farid Moussavi-Harami2, David Marcinek3, Michael Regnier4. 1 Physiology and Biophysics, University of Washington, Seattle, WA, USA, 2 Cardiology, University of Washington, Seattle, WA, USA, 3Radiology, University of Washington, Seattle, WA, USA, 4Bioengineering, University of Washington, Seattle, WA, USA. Heart disease is already the leading cause of death in the United States. People over 65 years of age are the fastest growing age group, and the incidence of heart failure is expected to increase further in the coming decades. We have previously demonstrated that upregulation of ribonucleotide reductase (RNR) in cardiomyocytes results in an elevation in the cytosolic concentration of 2deoxy-ATP (dATP) and subsequent increase in contractility. For this approach to be an effective therapeutic option for treatment of heart failure we need to determine whether elevated cardiac dATP can be maintained long term and that it does not compromise cardiomyoctye metabolism. We found that dADP-dependent respiration of cardiac mitochondria was depressed when compared to ADP-dependent respiration, and higher concentrations of dADP are necessary to stimulate respiration. Despite this, physiological and even super-physiological (2% and 10% of adenonucleotides, respectively) dADP/ADP ratios elicited the same magnitude of respiration as 100% ADP. In addition, hearts from transgenic mice that overexpress RNR have the same maximum mitochondrial respiratory capacity as wild-type controls. We have demonstrated that the creatine phosphate/creatine phosphokinase system can phosphorylate dADP in solution, which we believe to be the predominant pathway by which dATP is produced in vivo. RNR-TG hearts showed no increase in the ratio of mitochondrial to nuclear DNA, but had slightly elevated mitochondrial volume as determined by citrate synthase activity. This may prove protective during ischemic events and prevent the shift towards a more glycolytic metabolic profile seen during cardiac stress. These data suggest that cardiomyocytes can generate dATP from dADP through the same metabolic pathways as ATP, levels of dADP associated with upregulation of RNR do not alter the cell’s ability to synthesize ATP, and that elevated RNR may impart a cardioprotective effect during ischemic events. 702-Pos Board B482 Slo2.1 Potassium Channel Knockout Mice have an Altered Metabolic Phenotype in Cardiac Mitochondria Charles O. Smith. Biochemistry, University of Rochester, Rochester, NY, USA. Mitochondrial Kþ channels are important mediators of cell protection against stress. Although the existence of mitochondrial Kþ channels can be demonstrated phenomenologically, intense debate surrounds their molecular identity, their role in patho-physiology, and their regulation by endogenous signals. The

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mammalian Slo family of Kþ channels consists of Slo1, Slo2.1, Slo2.2 and Slo3. While Slo3 expression is restricted to the germline, Slo1, Slo2.1 and Slo2.2 are widely expressed. Examination of these channels has been limited to pharmacologic profiling which is hampered by overlapping sensitivities to activators and inhibitors, and off-target effects of small molecules. Work from our lab using genetic knockout mice has demonstrated that Slo1 in intrinsic cardiac neurons is required for protection against ischemia/reperfusion injury elicited by ischemic preconditioning, while Slo2.1 in cardiomyocytes is required for protection via anesthetic preconditioning. Slo2.1 is expressed in the heart and has been detected in the cell membranes of cardiomyocytes. We predict that Slo2.1 may also be a mitochondrial potassium channel. Herein we use pharmacologic activators and inhibitors of Slo2 channels in wildtype and Slo2.1-/- mice to demonstrate the presence of Slo2.1 in cardiac mitochondria. This was also confirmed directly by electophysiology (patch clamp) of isolated mitochondrial inner membranes (mitoplasts). Additional analysis of hearts, cardiomyocytes, and cardiac mitochondria from these knockout mice has revealed a metabolic phenotype, indicating a functional relationship between mitochondrial potassium channels and regulation of mitochondrial oxidative phosphorylation. These data demonstrate a role for Slo2.1 in the regulation of cardiac mitochondrial function. 703-Pos Board B483 Is Sodium Monocarboxylate Transporter (SMCT1) A Protein Involved in the Apical Iodide Transport? Ariela Vergara-Jaque1, Peying Fong2, Jeffrey Comer1. 1 Institute of Computational Comparative Medicine, Nanotechnology Innovation Center of Kansas State, Kansas State University, Manhattan, KS, USA, 2Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA. SMCT1 is a sodium-coupled cotransporter of monocarboxylates and shortchain fatty acids, which has been found in the apical membrane of diverse epithelia such as colon, kidney, brain and thyroid. In thyroid follicular cells, SMCT1 originally was associated with passive apical iodide (I-) transport. However, this evidence was subsequently rejected, as Xenopus oocytes expression studies of SMCT1 demonstrated that this protein does not cotransport iodide or other inorganic anions. Interestingly, the role of SMCT1 in I- transport is now controversial, because an I- transport mode occurs at low extracellular sodium concentrations, such as that found in thyroid follicular lumen. Considering this discrepancy, here we have performed a comparative study between SMCT1 and the sodium iodide symporter (NIS) in order to find conserved sodium/iodide binding motifs between both proteins. Specifically, homology models of hSMCT1 and hNIS were built based on the crystal structure of vSGLT, a sodium/galactose transporter from Vibrio parahaemolyticus. The hNIS model predicts an iodide-binding pocket between the transmembrane TM3 and TM7 segments, where mutated residues previously were identified in patients with hypothyroidism. A similar iodide-biding pocket was identified in the hSMCT1 model involving some conserved residues in homologs of these proteins. Putative sodium binding sites were also identified in the two models, which comprise characteristic sequence motifs of sodium-dependent transporters. Finally, molecular dynamics simulations of the iodide-bound models were performed, demonstrating that conserved aromatic residues may be key in iodide coordination. Our results provide further evidence of homology between SMCT1 and NIS transporters and predict residues involved in binding sodium and iodide. Future work will be essential for determining the precise transport mechanism in these proteins. 704-Pos Board B484 A Biophysical Approach Towards Understanding the Transport Mechanism of an ABC L-Methionine Importer Qi W. Li. Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, CA, USA. ABC transporters are prevalent in many kingdoms of life. In E. coli, one third of the transporters are ABC transporters. Their proper functions are vital to cell survival. Multiple drug resistance (MDR) transporters are a type of ABC transporters. They pump the drug out of the cell before the drug can exert its effects. Multidrug resistance is a major challenge in cancer and infectious diseases. Such resistance interferes with treatments and leads to longer recovery times and higher mortality rates. In addition, ABC transporters are important in chloride conductance, cholesterol transport, and surface-antigen presentation. ABC transporters in general also play a role in the regulation of movements of small molecules, ions, and macromolecules across membranes. MetNI was discovered by Kadner in the 1970s to efficiently uptake L-methionine in E. coli. In