Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchanger

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.     

Structural and functional characterization of transmembrane segment IX of the NHE1 isoform of the Na+/H+ exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339-363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu(346) and Ser(351) were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na(+) and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338-365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met(340)-Ser(344) and Ile(353)-Ser(359)) kinked with a large bend angle around a pivot point at amino acid Ser(351). The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser(351).     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na(+)/H(+) exchanger

The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.     

Solvent-induced beta-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central -Aib-Gly- segment capable of adopting beta-turn conformations compatible with both hairpin (beta(II') or beta(I')) and helical (beta(I)) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined beta-hairpin conformation in solvents capable of hydrogen bonding like (CD(3))(2)SO and CD(3)OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl(3) and CD(3)CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl(3) and (CD(3))(2)SO by 500 MHz (1)H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl(3). In (CD(3))(2)SO, peptide VII adopts a beta-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition.     

Structural and functional characterization of transmembrane segment VII of the Na+/H+ exchanger isoform 1

The Na(+)/H(+) exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H(+) for one extracellular Na(+). It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251-273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na(+)/H(+) exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly alpha-helical, with a break in the helix at the functionally critical residues Gly(261)-Glu(262). The relative positions and orientations of the N- and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition.     

Changes in Accessibility of Cytoplasmic Substances to the Pore Associated with Activation of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Opening of the cystic fibrosis transmembrane conductance regulator Cl⁻ channel is dependent both on phosphorylation and on ATP binding and hydrolysis. However, the mechanisms by which these cytoplasmic regulatory factors open the Cl⁻ channel pore are not known. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining sixth transmembrane region (TM6) of a cysteine-less variant of cystic fibrosis transmembrane conductance regulator. We find that methanethiosulfonate (MTS) reagents modify irreversibly cysteines substituted for TM6 residues Phe-337, Thr-338, Ser-341, Ile-344, Val-345, Met-348, Ala-349, Arg-352, and Gln-353 when applied to the cytoplasmic side of open channels. However, the apparent rate of modification by internal [2-sulfonatoethyl] methanethiosulfonate (MTSES), a negatively charged MTS reagent, is dependent on the activation state of the channels. In particular, cysteines introduced far along the axis of TM6 from the inside (T338C, S341C, I344C) showed no evidence of significant modification even after prolonged pretreatment of non-activated channels with internal MTSES. In contrast, cysteines introduced closer to the inside of TM6 (V345C, M348C) were readily modified in both activated and non-activated channels. Access of a permeant anion, Au(CN)₂⁻, to T338C was similarly dependent upon channel activation state. The pattern of MTS modification we observe allows us to designate different pore-lining amino acid side chains to distinct functional regions of the channel pore. One logical interpretation of these findings is that cytoplasmic access to residues at the narrowest region of the pore changes concomitant with activation.     

Solute carrier 11 cation symport requires distinct residues in transmembrane helices 1 and 6

Ubiquitous solute carriers 11 (SLC11) contribute to metal-ion homeostasis by importing Me(2+) and H(+) into the cytoplasm. To identify residues mediating cation symport, Escherichia coli proton-dependent manganese transporter (MntH) was mutated at five SLC11-specific transmembrane (TM) sites; each mutant activity was compared with wild-type MntH, and the biochemical results were tested by homology threading. Cd(2+) and H(+) uptake kinetics were analyzed in whole cells as a function of pH and temperature, and right-side out membrane vesicles were used to detail energy requirements and to probe site accessibility by Cys replacement and thiol modification. This approach revealed that TM segment 1 (TMS1) residue Asp(34) couples H(+) and Me(2+) symport and contributes to MntH forward transport electrogenicity, whereas the TMS6 His(211) residue mediates pH-dependent Me(2+) uptake; MntH Asn(37), Asn(250), and Asn(401) in TMS1, TMS7, and TMS11 participate in transporter cycling and/or helix packing interactions. These biochemical results fit the LeuT/SLC6 structural fold, which suggests that conserved peptide motifs Asp(34)-Pro-Gly (TMS1) and Met-Pro-His(211) (TMS6) form antiparallel "TM helix/extended peptide" boundaries, lining a "pore" cavity and enabling H(+)-dependent Me(2+) import.     

Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.     

Synthetic and conformational studies on dehydroalanine-containing model peptides

Three model dipeptides containing a dehydroalanine residue (delta Ala) at the C-terminal, Boc-X-delta Ala-NHCH3 [X = Ala, Val, and Phe,] have been synthesized and their solution conformations investigated by 1H-NMR, IR, and CD spectroscopy. NMR studies on these peptides in CDCl3 clearly indicate that the NH group of dehydroalanine is involved in an intramolecular hydrogen bond. This conclusion is supported by IR studies also. Nuclear Overhauser effect (NOE) studies are also accommodative of an inverse gamma-turn-type of conformation that is characterized by conformational angles of phi approximately -70 degrees and psi approximately +70 degrees around the X residue, and a C alpha i + 1 H-Ni + 2H interproton distance of 2.5 A. It appears that unlike dehydrophenylalanine or dehydroleucine, which tend to stabilize beta-turn type of structures occupying the i + 2 position of the turn, dehydroalanine favors the formation of an inverse gamma-turn, centered at the preceding L-residue in such solvents as CDCl3 and (CD3)2SO. A comparison of solution conformation of Boc Val-delta Ala-NHCH3 with the corresponding saturated analogue, Boc-Val-Ala-NHCH3, is also presented and shows that dehydroalanine is responsible for inducing the turn structure. It may be possible to design peptides with different preferred conformations using the suitable dehydroamino acid.     

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.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines

Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current–voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl⁻ permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.     

Mutational analysis of potential pore-lining amino acids in TM IV of the Na/H exchanger

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.     

A transmembrane site determines sensitivity of neuronal nicotinic acetylcholine receptors to general anesthetics

Neuronal nicotinic acetylcholine receptors (nAChRs) are potential targets for a wide variety of general anesthetics. We recently showed that alpha(4)beta(2) nAChRs are more sensitive than alpha(4)beta(4) receptors to the gaseous anesthetics nitrous oxide and xenon. The present study examines chimeric and point mutant rat nAChRs expressed in Xenopus oocytes and identifies a single amino acid residue (beta(2)-Val(253) or beta(4)-Phe(255)) near the middle of the second transmembrane segment (TM2) that determines gaseous anesthetic sensitivity. Mutations of this residue in beta subunits and the homologous residue of alpha(4) subunits (alpha(4)-Val(254)) showed that this position also determines sensitivities of nAChRs to acetylcholine, isoflurane, pentobarbital, and hexanol. In contrast, these mutations did not affect actions of ketamine. The positively charged sulfhydryl-specific reagent methanethiosulfonate ethylammonium reacted with a cysteine introduced at alpha(4)-Val(254) or beta(2)-Val(253), and irreversibly reduced anesthetic sensitivities of nAChRs. Propyl methanethiosulfonate is an anesthetic analog that covalently binds to a TM2 site of gamma-aminobutyric acid(A) and glycine receptors and irreversibly enhances receptor function. However, propyl methanethiosulfonate reversibly inhibited cysteine-substitution mutants at alpha(4)-Val(254) or beta(2)-Val(253) of nAChRs, and did not affect anesthetic sensitivity. Thus, residues alpha(4)-Val(254) and beta(2)-Val(253) alter channel gating and determine anesthetic sensitivity of nAChRs, but are not likely to be anesthetic-binding sites.     

Phe(303) in TMVI of the alpha(1B)-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation.

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M. (2000) EMBO J. 19, 4265-4271]. Here, we show that saturation mutagenesis of Phe(303) results in a series of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed increased basal activity but an attenuated maximal response to (-)-epinephrine-stimulation. F303L, on the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A > WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants result in TM helical movements that differ in accordance with their degree of constitutive activity. These findings, therefore, confirm and extend our previous data implicating Phe(303) as a key residue coupling TM helical movements to G-protein-activation.     

Functional response of the small multidrug resistance protein EmrE to mutations in transmembrane helix 2

Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity.     

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and alpha-methyl-D-glucopyranoside (alphaMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of alphaMG and phloridzin for T156C by approximately 5-fold and approximately 20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na(+) currents.     

Solid phase synthesis of a fully active analogue of cholecystokinin using the acid-stable Boc-Phe (p-CH2) SO3H as a substitute for Boc-Tyr(SO3H) in CCK8

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.