Water permeation through the sodium-dependent galactose cotransporter vSGLT

It is well accepted that cotransporters facilitate water movement by two independent mechanisms: osmotic flow through a water channel in the protein and flow driven by ion/substrate cotransport. However, the molecular mechanism of transport-linked water flow is controversial. Some researchers believe that it occurs via cotransport, in which water is pumped along with the transported cargo, while others believe that flow is osmotic in response to an increase in intracellular osmolarity. In this letter, we report the results of a 200-ns molecular dynamics simulation of the sodium-dependent galactose cotransporter vSGLT. Our simulation shows that a significant number of water molecules cross the protein through the sugar-binding site in the presence as well as the absence of galactose, and 70-80 water molecules accompany galactose as it moves from the binding site into the intracellular space. During this event, the majority of water molecules in the pathway are unable to diffuse around the galactose, resulting in water in the inner half of the transporter being pushed into the intracellular space and replaced by extracellular water. Thus, our simulation supports the notion that cotransporters act as both passive water channels and active water pumps with the transported substrate acting as a piston to rectify the motion of water.     

Ligand-induced differences in secondary structure of the Vibrio parahaemolyticus Na+/galactose cotransporter

A detailed structural study of the prokaryotic sodium/galactose transporter (vSGLT) from Vibrio parahaemolyticus using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy reveals stepwise increases in alpha-helicity upon binding of sodium and D-galactose. These increases in helicity correlate with decreases in beta-structural elements. The changes are accompanied by stepwise reductions in the degree of H/D exchange (HDX), suggesting reduced accessibility of water to the protein backbone. The data demonstrate discrete conformational changes from one intermediate to the next during the catalytic cycle of the protein and are interpreted in a model of the symport reaction mechanism.     

Functional expression of the Vibrio parahaemolyticus Na+/galactose (vSGLT) cotransporter in Xenopus laevis oocytes

We have successfully expressed a bacterial cotransporter in a functional form in the Xenopus laevis oocyte expression system. The goals were to compare the kinetics and selectivity of the cotransporter expressed in oocytes with those obtained in bacteria and in proteoliposomes, and to determine if it is possible to measure the electrical properties of the bacterial cotransporter expressed in oocytes. The Vibrio parahaemolyticus Na+/galactose cotransporter (vSGLT) expressed in oocytes has functional properties that are similar to those expressed in bacteria and those of the purified cotransporter reconstituted into liposomes. vSGLT is a Na+-dependent transporter that is saturable with Na+ (K(0.5)=17 mM) and D-galactose (K(0.5)=237 microM) and is sensitive to both D-fucose and phlorizin. In addition, vSGLT in oocytes shows a sugar specificity in the order of D-galactose >D-fucose > D-glucose, distinguishing it from the animal members of the Na+/glucose cotransporter family. The level of transport by vSGLT in oocytes is lower overall (V(max) approximately 10 pmol/oocyte/hour) compared to other plant and animal cotransporters (V(max) approximately 1000 pmol/oocyte/hour). The low level of expression does not permit us to carry out electrophysiological studies of the bacterial cotransporter. This study shows the potential and unique advantages of utilizing a eukaryotic oocyte expression system to study bacterial cotransporters.     

A proteomic study of sodium/D-glucose cotransporter 1 (SGLT1): topology of loop 13 and coverage of other functionally important domains

In order to obtain further information about the structure and function of human sodium/D-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate. Sequence coverage was provided from amino acids 559 to 644, suggesting that loop 13 is almost completely accessible at the extravesicular face of the proteoliposomes. These results support the notion that major parts of loop 13, essential for the interaction with transport inhibitors in vivo, are located extracellularly in intact cells. In-gel trypsin, chymotrypsin, and in particular trypsin/chymotrypsin digestion of recombinant SGLT1 in combination with LC-MS/MS provide extensive sequence coverage of the protein, including domains involved in sugar and inhibitor binding and potential phosphorylation sites. These studies demonstrate that proteomic analysis combined with mass spectrometry is a useful tool to characterize regions of SGLT1 that are important for its function and regulation.     

Reanalysis of structure/function correlations in the region of transmembrane segments 4 and 5 of the rabbit sodium/glucose cotransporter

The predicted topology of the mammalian high-affinity sodium/glucose cotransporter (SGLT1), in the region surrounding transmembrane segments 4 and 5, disagrees with the recent published crystal structure of bacterial SGLT from Vibrio parahaemolyticus (vSGLT). To investigate this issue further, 38 residues from I143 to A180 in the N-terminal half of rabbit SGLT1 were each replaced with cysteine and then expressed in COS-7 cells or Xenopus laevis oocytes. The membrane orientations of the substituted cysteines were determined by treatment with the thiol-specific reagent N-Biotinoylaminoethyl methanethiosulfonate (biotin-MTSEA), combined with the membrane impermeant thiol-specific reagent sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES). The present results combined with previous structure/function studies of SGLT1, suggest that transmembrane domain (TM) 4 of mammalian SGLT1 extends from residue 143-171 and support the topology observed in the crystal structure of vSGLT.     

Sodium-dependent extracellular accessibility of Lys-84 in the sodium/dicarboxylate cotransporter

The Na(+)/dicarboxylate cotransporter transports Na(+) with citric acid cycle intermediates such as succinate and citrate. The present study focuses on transmembrane helix 3, which is highly conserved among the members of the SLC13 family. Fifteen amino acids in the extracellular half of transmembrane helix (amino acids 98-112) as well as Lys-84, previously shown to affect substrate affinity, were mutated individually to cysteine and expressed in the human retinal pigment epithelial cell line. Transport specificity ratio analysis shows that determinants for distinguishing succinate and citrate are found at amino acids Lys-84, Glu-101, Trp-103, His-106, and Leu-111. All of the mutants were tested for sensitivity to the membrane-impermeant cysteine-specific reagent (2-sulfonatoethyl) methanethiosulfonate (MTSES), but only K84C was sensitive to MTSES inhibition. The sensitivity of K84C to MTSES was greatest in the presence of sodium, and the inhibition could be prevented by addition of substrate or replacement of sodium, indicating that the accessibility of Lys-84 changes with conformational state. The substrate protection of MTSES inhibition of K84C appears to occur early in the transport cycle, before the large-scale conformational change associated with translocation of substrate. The results point to a new location for Lys-84 within the substrate access pore of the Na(+)/dicarboxylate cotransporter, either in a transmembrane helix or a reentrant loop facing a water-filled pore.     

Cloning, expression and structure of the functionally active shortened lon gene in Escherichia coli

Lon gene of E. coli has been cloned into the plasmid pBR327. Full nucleotide sequence of the gene has been established. It was shown that the cloned gene does not possess the terminal codon and is somewhat shortened. Nevertheless it retains full phenotypic activity and expresses the C-end modified La proteinase which retains ATP-dependent proteolytic activity.     

A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1

Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.     

Oligomeric structure and minimal functional unit of the electrogenic sodium bicarbonate cotransporter NBCe1-A

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wild-type (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys(630) and Cys(642) in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys(992) construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A(T442C) mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.     

Organization of the membrane domain of the human liver sodium/bile acid cotransporter

Mammalian sodium/bile acid cotransporters (SBATs) are glycoproteins with an exoplasmic N-terminus, an odd number of transmembrane regions, and a cytoplasmic C-terminus. Various algorithms predict eight or nine membrane-embedded regions derived from nine hydrophobic stretches of the protein (H1-H9). Three methods were used to define which of these were transmembrane or membrane-associated segments in the liver bile acid transporter. The first was in vitro translation/insertion scanning using either single hydrophobic sequences between the N-terminal domain of the alpha-subunit of the gastric H,K-ATPase and the C-terminal domain of the beta-subunit that contains five N-linked glycosylation exoplasmic flags or using constructs beginning with the N-terminus of the transporter of various lengths and again ending in the C-terminus of the H,K-ATPase beta-subunit. Seven of the predicted segments, but not the amphipathic H3 and H8 sequences, insert as both individual signal anchor and stop transfer sequences in the reporter constructs. These sequences, H3 and H8, are contained within two postulated long exoplasmic loops in the classical seven-transmembrane segment model. The H3 segment acts as a partial stop transfer signal when expressed downstream of the endogenous H2. In a similar manner, the other amphipathic segment, H8, inserts as a signal anchor sequence when translated in the context with the upstream transporter sequence in two different glycosylation constructs. Alanine insertion scanning identified regions of the transporter requiring precise alignment of sequence to form competent secondary structures. The transport activity of these mutants was evaluated either in native protein or in a yellow fluorescent protein (YFP) fusion protein construct. All alanine insertions in H3 and H8 abolished taurocholate uptake, suggesting that both these regions have structures with critical intramolecular interactions. Moreover, these insertions also prevented trafficking to the plasma membrane as assessed by confocal microscopy with a polyclonal antibody against either the C-terminus of the transporter or the YFP signal of the YFP-transporter fusion protein. Two glycosylation signals inserted in the first postulated loop region and four of five such signals in the second postulated loop region were not recognized by the oligosaccharide transferase, and the L256N mutation exhibited 10% glycosylation and was inactive. These findings support a topography with nine membrane-spanning or membrane-associated segments.     

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.     

Expression of rat renal sodium/phosphate cotransporter in Xenopus laevis oocytes.

In an attempt to identify the renal Na+/Pi cotransporter, Xenopus laevis oocytes were used to express mRNA isolated from the renal cortex of rat kidney. Na(+)-dependent uptake of Pi in oocytes, injected with mRNA, resulted in an increase of 2-4-fold as compared to oocytes injected with water. Both the new expressed and endogenous Na(+)-dependent Pi uptake activity were inhibited with 2 mM phosphonoformic acid (PFA). Expression of Pi uptake into oocytes was dose-dependent with the amount of mRNA injected. When mRNA was fractionated on a sucrose gradient, a mRNA fraction of 2.5 kilobases expressed the Na+/Pi cotransport activity in oocytes. This fraction resulted in a 6-fold stimulation of Na(+)-dependent Pi transport when compared to oocytes injected with water. The Km and Vmax for Na(+)-dependent Pi uptake were 0.18 mM and 118 pmol/oocyte per 30 min, respectively.     

Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).     

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [¹⁴C]-α-methyl glucopyranoside ([¹⁴C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [¹⁴C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

Role of actin in the cAMP-dependent activation of sodium/glucose cotransporter in renal epithelial cells

We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [14C]-alpha-methyl glucopyranoside ([14C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [14C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments.     

A comparative study of galactose oxidase and active site analogs based on QM/MM Car-Parrinello simulations

A parallel study of the radical copper enzyme galactose oxidase (GOase) and a low molecular weight analog of the active site was performed with dynamical density functional and mixed quantum-classical calculations. This combined approach enables a direct comparison of the properties of the biomimetic and the natural systems throughout the course of the catalytic reaction. In both cases, five essential forms of the catalytic cycle have been investigated: the resting state in its semi-reduced (catalytically inactive) and its oxidized (catalytically active) form, A ^semi and A ^ox, respectively; a protonated intermediate B; the transition state for the rate-determining hydrogen abstraction step C, and its product D. For A and B the electronic properties of the biomimetic compound are qualitatively very similar to the ones of the natural target. However, in agreement with the experimentally observed difference in catalytic activity, the calculated activation energy for the hydrogen abstraction step is distinctly lower for GOase (16 kcal/mol) than for the mimetic compound (21 kcal/mol). The enzymatic transition state is stabilized by a delocalization of the unpaired spin density over the sulfur-modified equatorial tyrosine Tyr272, an effect that for geometric reasons is essentially absent in the biomimetic compound. Further differences between the mimic and its natural target concern the structure of the product of the abstraction step, which is characterized by a weakly coordinated aldehyde complex for the latter and a tightly bound linear complex for the former. Received 14 October 1999 · Accepted: 19 January 2000     

Characterizing the secondary structure and identifying functionally essential nucleotides of pH6DZ1, a fluorescence-signaling and RNA-cleaving deoxyribozyme

pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors.