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Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1
M Panayotova-Heiermann DD Loo JT Lam EM Wright 《Canadian Metallurgical Quarterly》1998,37(29):10522-10528
Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode. 相似文献
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Site-directed mutagenesis was used to assess the role of transmembrane (TM)-charged amino acids in the expression and function of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP). Charged residues that are conserved in the TM regions of most or all members of the PTH/secretin receptor subfamily were targeted. Four mutants (E296A, R337A, H414A, and E459K) displayed properties similar to the wild type PTH/PTHrP receptor with respect to agonist binding and stimulation of adenylyl cyclase when expressed in COS-7 cells. Several mutations, all in TM II, produced receptors that signaled extremely poorly. Mutation of three residues (227S, 230R, and 233S), predicted to be aligned on one helical face of TM II, displayed a similar phenotype: markedly blunted adenylyl cyclase activity in response to PTH (20-30% of the wild type response) and a lower binding affinity for agonist, with no reduction in cell surface receptor expression. These results suggest that TM II contains a polar face that is involved in TM signaling by the PTH/PTHrP receptor. Two of these mutations were made at the corresponding sites in the secretin receptor, and a similar reduction in secretin-stimulated adenylyl cyclase activity was observed. Thus this region of TM II may participate in a mechanism of TM signal transduction that is shared by the PTH/secretin sub-family of G protein-coupled receptors. 相似文献
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Glucagon is a peptide hormone that plays a central role in the maintenance of normal circulating glucose levels. Structure-activity studies have previously demonstrated the importance of histidine at position 1 and the absolute requirement for aspartic acid at position 9 for transduction of the hormonal signal. Site-directed mutagenesis of the receptor protein identified Asp64 on the extracellular N-terminal tail to be crucial for the recognition function of the receptor. In addition, antibodies generated against aspartic acid-rich epitopes from the extracellular region competed effectively with glucagon for receptor sites, which suggested that negative charges may line the putative glucagon binding pocket in the receptor. These observations led to the idea that positively charged residues on the hormone may act as counterions to these sites. Based on these initial findings, we synthesized glucagon analogs in which basic residues at positions 12, 17, and 18 were replaced with neutral or acidic residues to examine the effect of altering the positive charge on those sites on binding and adenylyl cyclase activity. The results indicate that unlike N-terminal histidine, Lys12, Arg17, and Arg18 of glucagon have very large effects on receptor binding and transduction of the hormonal signal, although they are not absolutely critical. They contribute strongly to the stabilization of the binding interaction with the glucagon receptor that leads to maximum biological potency. 相似文献
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Irritable bowel syndrome is frequently encountered in clinical practice, and it has been repeatedly suggested that abnormal colonic motor activity is one of the major pathophysiological mechanisms responsible for the origin of symptoms in such disorder. If this statement is true, then high-amplitude propagated colonic contractions (HAPCs), i.e. the mass movements, may play an important role. To test this hypothesis, we conducted an investigation by recording colonic motility for a prolonged (24 h) period in 25 patients with irritable bowel syndrome and in 18 healthy volunteers, to compare the number of mass movements over 24 h in patients (constipation-predominant, alternating bowel habits) and controls. The overall amount of motility was also assessed in twelve patients and 13 controls. We also looked for the possible changes in mass movements and motility which may occur with defecation and after a meal. The results showed that 1) with respect to HAPCs and motility index, neither group was significantly different from controls; 2) HAPCs and the motility index were significantly reduced during sleep in all groups tested; 3) HAPCs were significantly more common before as compared to after defecation and after as compared to before meals; 4) HAPCs are not independent from the segmental contractile activity; 5) the motility index/24 h was lower in the constipation-predominant group of patients with respect to controls. We conclude that in patients with irritable bowel syndrome colonic motility per se may play a pathophysiological role in the genesis of the symptoms, although other mechanisms are likely to concur, or to be responsible for the complaints of these patients. However, colonic prolonged recordings are very useful for studying physiological and pathophysiological correlates of sleep, eating, and defecation. 相似文献
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The stability of a coiled coil or leucine zipper is controlled by hydrophobic interactions and electrostatic forces between the constituent helices. We have designed a 30-residue peptide with the repeating seven-residue pattern of a coiled coil, (abcdefg)n, and with Glu in positions e and g of each heptad. The glutamate side chains prevented folding at pH values above 6 because of electrostatic repulsion across the helix dimer interface as well as within the individual helices. Protonation of the carboxylates changed the conformation from a random coil monomer to a coiled coil dimer. Folding at alkaline pH where the peptide had a net charge of -7e was promoted by the addition of salts. The nature of the charge screening cation was less important than that of the anion. The high salt concentrations (>1 M) necessary to induce folding indicated that the salt-induced folding resulted from alterations in the protein-water interaction. Folding was promoted by the kosmotropic anions sulfate and fluoride and to a lesser extent by the weak kosmotrope formate, whereas chloride and the strong chaotrope perchlorate were ineffective. Kosmotropes are excluded from the protein surface, which is preferentially hydrated, and this promotes folding by strengthening hydrophobic interactions at the coiled coil interface. Although charge neutralization also contributed to folding, it was effective only when the screening cation was partnered by a good kosmotropic anion. Folding conformed to a two-state transition from random coil monomer to coiled coil dimer and was enthalpy driven and characterized by a change in the heat capacity of unfolding of 3.9 +/- 1.2 kJ mol-1 K-1. The rate of folding was analyzed by fluorescence stopped-flow measurements. Folding occurred in a biphasic reaction in which the rapid formation of an initial dimer (kf = 2 x 10(7) M-1 s-1) was followed by an equally rapid concentration-independent rearrangement to the folded dimer (k > 100 s-1). 相似文献
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Phospholamban is a small membrane protein which can form cation selective ion channels in lipid bilayers. Each subunit contains a single, largely hydrophobic transmembrane helix. The helices are thought to assemble as a pentameric and approximately parallel bundle surrounding a central pore. A model of this assembly (PDB code IPSL) has been used as the starting point for molecular dynamics (MD) simulations of a system consisting of the pentameric helix bundle, plus 217 water molecules located within and at either mouth of the pore. Interhelix distance restraints were employed to maintain the integrity of the helix bundle during a 500 ps MD simulation. Water molecules within the pore exhibited reduced diffusional and rotational mobility. Interactions between the alpha-helix dipoles and the water dipoles, the latter aligned anti-parallel to the former, contribute to the stability of the system. Analysis of the potential energy of interaction of a K+ ion as it was moved through the pore suggested that unfavourable interactions of the cation with the aligned helix dipoles at the N-terminal mouth were overcome by favourable ion-water interactions. Comparable analysis for a Cl ion revealed that the ion-(pore + water) interactions were unfavourable along the whole of the pore, increasingly so from the N- to the C-terminal mouth. Overall, the interaction energy profiles were consistent with a pore selective for cations over anions. Pore radius profiles were used to predict a channel conductance of 50 to 70 ps in 0.2 M KCl, which compares well with an experimental value of 100 ps. 相似文献
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MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an alpha-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating. 相似文献
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F Polticelli G Bottaro A Battistoni MT Carrì K Djinovic-Carugo M Bolognesi P O'Neill G Rotilio A Desideri 《Canadian Metallurgical Quarterly》1995,34(18):6043-6049
The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Glu131, and Lys134, has been determined by pulse radiolysis as a function of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutralization of Asp130, while neutralization of either of the two lysines drastically decreases the enzyme activity. The Lys120Leu-Lys134Thr and Lys134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives rise to a faster enzyme at any ionic strength value. The effects observed in the single Asp130Gln and Lys120Leu mutants differ from those reported on human or bovine enzymes [Getzoff et al. (1992) Nature (London) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indicating that some residues occupying the same position in the linear sequence of different Cu,Zn superoxide dismutases have a different functional weight. Our results also suggest that the strategy of multiple charge mutation may be a promising approach in order to increase the catalytic rate of Cu,Zn SODs independently of ionic strength. 相似文献
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Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues 总被引:1,自引:0,他引:1
P Van Gelder N Saint P Phale EF Eppens A Prilipov R van Boxtel JP Rosenbusch J Tommassen 《Canadian Metallurgical Quarterly》1997,269(4):468-472
The porins PhoE and OmpF form anion and cation-selective pores, respectively, in the outer membrane of Escherichia coli. Each monomer of these trimeric proteins consists of a 16-stranded beta-barrel, which contains a constriction at half the height of the channel. The functional significance of a transverse electrical field that is formed by charged amino acid residues within the constriction zone was investigated. For this purpose, the PhoE residues R37, R75, K18 and E110 were substituted by neutral amino acids. The mutant pores allowed an increased permeation of beta-lactam antibiotics across the outer membrane in vivo, although the single channel conductance, measured in planar lipid bilayers, was not increased or even slightly decreased. Replacement of the positively charged residues resulted in a decreased voltage sensitivity, whereas the substitution of a negatively charged residue resulted in an increased voltage sensitivity. Similar substitutions in OmpF caused the opposite effects, i.e. the substitution of positive and negative charges resulted in increased and decreased voltage sensitivity, respectively. Together, the results suggest that opposite charges, i.e. positive charges in anion-selective and negative charges in cation-selective porins, act as sensors for voltage gating. 相似文献
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X Gallet F Festy P Ducarme R Brasseur A Thomas-Soumarmon 《Canadian Metallurgical Quarterly》1998,16(2):72-82, 97-8
The cystic fibrosis transmembrane conductance regulator is a cAMP-regulated chloride channel. We used molecular modelling to predict 3-D models for the CFTR membrane domain. Hydropathy and residue conservation in all CFTRs as well as in other proteins suggested that the membrane domain is a 12-helix bundle. If the domain is enclosing a channel for chloride, it could be made of five helices. We propose two structural models in which both lumenal and cytoplasmic entrances to the chloride pore have a ring of positively charged residues. The inner surface of the channel is covered with neutral polar plus one or two charged residues. Helices that are not directly involved in the chloride channel could organise to form a second channel; a dimeric symmetrical structure is proposed. Analysis raised interest for helix 5: this hydrophobic fragment is conserved in all CFTRs and aligns with segments present in several different ion channels and transporters. The existence of an FFXXFFXXF motif is proposed. Helix 5 could be an important domain of CFTRs. The models agree with available data from pathological mutations but does not account for the membrane insertion of a hydrophilic fragment of NBDI. 相似文献
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Three experiments showed that constraints imposed early in learning have different effects on variability when they are in effect and after they are removed. Task constraints, which determine how something can be done, limited the number of possible responses in a computer game. Variability constraints, which specify how differently something must be done, required that each response differ from some number of prior responses. Less restrictive constraints (Experiments 1 and 2) produced higher variability during the constraints. More restrictive constraints (Experiments 2 and 3) led to higher variability after the constraints were relaxed. The authors discuss how these differences reflect strategies acquired during the constraints (default rules) and modified in closely related ways (exception rules) afterward. (PsycINFO Database Record (c) 2010 APA, all rights reserved) 相似文献
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The liver-type (GLUT2) and brain-type (GLUT3) human facilitative glucose transporters exhibit distinct kinetics (Km values for deoxyglucose transport of approximately 11 mM and approximately 1.5 mM, respectively) and patterns of substrate transport (GLUT2 is capable of D-fructose transport, while GLUT3 is not). Using a range of chimeric glucose transporters comprised of regions of GLUT2 and GLUT3 studied by expression in Xenopus oocytes after microinjection of cRNA, we have proposed that the seventh putative transmembrane helix is intimately involved in the selection of transported substrate and that this region plays an important role in determining the Km for 2-deoxyglucose [Arbuckle, M. I., Kane, S., Porter, L. M., Seatter, M. J., and Gould, G. W. (1996) Biochemistry 35, 16519-16527]. Inspection of the predicted amino acid sequence of this region reveals that GLUTs 1, 3, and 4 (high-affinity glucose transporters) contain a conserved QLS motif in this helix (residues 277-279 in human GLUT3). In the glucose/fructose transporter (GLUT2) this motif is replaced by HVA. To study the role of the QLS motif in substrate selection, we have engineered substitutions in this region between GLUT2 and GLUT3. GLUT3 (QLS > HVA) exhibits a Km for deoxyglucose transport identical to that of native GLUT3 but increased sensitivity for inhibition of deoxyglucose transport by D-fructose. However, unlike native GLUT3, this species is capable of transporting D-fructose. Compared to wild-type GLUT2, GLUT2 (HVA > QLS) exhibits a lower Km for deoxyglucose transport (approximately 3 mM vs approximately 11 mM), the ability to transport D-fructose is reduced, and D-fructose is a less efficient inhibitor of deoxyglucose transport. Analysis of the ability of a range of glucose epimers and analogues to inhibit transport by these species suggests that the QLS motif interacts with the incoming D-glucose at the C-1 position; this may be a key interaction in the high-affinity recognition of the transported substrate. We further argue that this interaction acts as a molecular filter that is involved in the selection of the transported substrate. 相似文献
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ME Kuipers PJ Swart M Schutten C Smit JH Proost AD Osterhaus DK Meijer 《Canadian Metallurgical Quarterly》1997,33(2):99-108
Changes of blood metabolites and hormones were studied in female breeding calves before, during and after weaning from 4 to 18 weeks of age. Calves were initially fed increasing amounts of whole milk (up to 7 kg/day in week 8 of life). Milk intake was then gradually decreased up to the age of 16 weeks, when calves were completely weaned and only fed hay and concentrates. Average daily gain was 0.85 kg. Postprandial concentrations of glucose, insulin, insulin-like growth factor-I and 3.5.3'-triiodothyronine concentrations gradually decreased (P < 0.05) with age, while those of beta-hydroxybutyrate, protein, albumin, haemoglobin and iron increased (P < 0.05). Concentrations of cholesterol transiently increased, whereas those of urea reversibly decreased. Non-esterified fatty acids, triglycerides and growth hormone did not consistently change during the duration of the study. In conclusion, changes of glucose, beta-hydroxybutyrate, haemoglobin, iron, insulin, insulin-like growth factor-I and 3.5.3'-triiodothyronine were markedly different from those usually seen in veal calves of the same age. 相似文献
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Role of aromatic transmembrane residues of the delta-opioid receptor in ligand recognition 总被引:1,自引:0,他引:1
K Befort L Tabbara D Kling B Maigret BL Kieffer 《Canadian Metallurgical Quarterly》1996,271(17):10161-10168
In the present study we examine the role of transmembrane aromatic residues of the delta-opioid receptor in ligand recognition. Three-dimensional computer modeling of the receptor allowed to identify an aromatic pocket within the helices bundle which spans transmembrane domains (Tms) III to VII and consists of tyrosine, phenylalanine, and tryptophan residues. Their contribution to opioid binding was assessed by single amino acid replacement: Y129F and Y129A (Tm III), W173A (Tm IV), F218A and F222A (Tm V), W274A (Tm VI), and Y308F (Tm VII). Scatchard analysis shows that mutant receptors, transfected into COS cells, are expressed at levels comparable with that of the wild-type receptor. Binding properties of a set of representative opioids were examined. Mutations at position 129 most dramatically affected the binding of all tested ligands (up to 430-fold decrease of deltorphin II binding at Y129A), with distinct implication of the hydroxyl group and the aromatic ring, depending on the ligand under study. Affinity of most ligands was also reduced at Y308F mutant (up to 10-fold). Tryptophan residues seemed implicated in the recognition of specific ligand classes, with reduced binding for endogenous peptides at W173A mutant (up to 40-fold) and for nonselective alkaloids at W274A mutant (up to 65-fold). Phenylalanine residues in Tm V appeared poorly involved in opioid binding as compared with other aromatic amino acids examined. Generally, the binding of highly selective delta ligands (TIPPpsi, naltrindole, and BW373U86) was weakly modified by these mutations. Noticeably, TIPPpsi binding was enhanced at W274A receptor by 5-fold. Conclusions from our study are: (i) aromatic amino acid residues identified by the model contribute to ligand recognition, with a preponderant role of Y129; (ii) these residues, which are conserved across opioid receptor subtypes, may be part of a general opioid binding domain; (iii) each ligand-receptor interaction is unique, as demonstrated by the specific binding pattern observed for each tested opioid compound. 相似文献
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Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca(2+)-ATPase 总被引:1,自引:0,他引:1
AO Adebayo A Enyedi AK Verma AG Filoteo JT Penniston 《Canadian Metallurgical Quarterly》1995,270(46):27812-27816
In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882. 相似文献