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1.
New transport assay demonstrates vesicular acetylcholine transporter has many alternative substrates
The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [(3)H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12(A123.7) cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [(3)H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details. 相似文献
2.
This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily. 相似文献
3.
Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor 总被引:3,自引:3,他引:0
Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed. 相似文献
4.
J. Barbosa Jr. A. D. Clarizia M. V. Gomez M. A. Romano-Silva V. F. Prado M. A. M. Prado 《Journal of neurochemistry》1997,69(6):2608-2611
Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [3 H]-acetylcholine ([3 H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [3 H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [3 H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [3 H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by α-PMA, an inactive phorbol ester. PMA did not alter the release of [3 H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from 32 P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of 32 P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [3 H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process. 相似文献
5.
This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified. 相似文献
6.
The vesicular acetylcholine transporter (VAChT) resides in synaptic vesicles of cholinergic nerve terminals. It carries out vesicular storage of ACh. The amount of ACh stored determines, along with other factors, the amount of ACh released. Knowledge of the structure-function relationship in VAChT might enable pharmacological regulation of ACh storage and release at the level of VAChT. To this end, a quantitative model for the individual steps in the overall transport cycle of VAChT has been developed. Because of the particular values of the microscopic rate constants in the model, structure-function analysis of VAChT can be misleading. Attempts to devise a pro-storage strategy to increase ACh release from cholinergic nerve terminals should take into account the microscopic kinetics of this transporter. 相似文献
7.
Transport mechanisms in acetylcholine and monoamine storage. 总被引:10,自引:0,他引:10
S M Parsons 《FASEB journal》2000,14(15):2423-2434
Sequence-related vesicular acetylcholine transporter (VAChT) and vesicular monoamine transporter (VMAT) transport neurotransmitter substrates into secretory vesicles. This review seeks to identify shared and differentiated aspects of the transport mechanisms. VAChT and VMAT exchange two protons per substrate molecule with very similar initial velocity kinetics and pH dependencies. However, vesicular gradients of ACh in vivo are much smaller than the driving force for uptake and vesicular gradients of monoamines, suggesting the existence of a regulatory mechanism in ACh storage not found in monoamine storage. The importance of microscopic rather than macroscopic kinetics in structure-function analysis is described. Transporter regions affecting binding or translocation of substrates, inhibitors, and protons have been found with photoaffinity labeling, chimeras, and single-site mutations. VAChT and VMAT exhibit partial structural and mechanistic homology with lactose permease, which belongs to the same sequence-defined superfamily, despite opposite directions of substrate transport. The vesicular transporters translocate the first proton using homologous aspartates in putative transmembrane domain X (ten), but they translocate the second proton using unknown residues that might not be conserved between them. Comparative analysis of the VAChT and VMAT transport mechanisms will aid understanding of regulation in neurotransmitter storage. 相似文献
8.
Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding site was used to re-examine transport selectivity. Rat VAChT was expressed in PC12(A1237) cells, postnuclear supernatant-containing microvesicles was prepared, and the reorientation assay was conducted with unlabeled Ch and tetramethylammonium (TMA). Also, [(14)C]Ch and [(3)H]acetylcholine (ACh) were used in an optimized accumulation assay. The results demonstrate that Ch is transported at least as well as ACh is, but with sevenfold lower affinity. Even TMA is transported, but with 26-fold lower affinity. Ch transport by VAChT is of interest in view of the possibilities that Ch (i) occurs at higher concentration than ACh does in terminal cytoplasm under some conditions, and (ii) is an agonist for alpha 7 nicotinic receptors. 相似文献
9.
M F Diebler 《Neurochemistry international》1992,21(1):83-90
Vesamicol is a highly potent inhibitor of active acetylcholine transport into isolated cholinergic vesicles from Torpedo. On the basis of transport kinetics and vesamicol sensitivity, we have shown that the acetylcholine transporter could be in an activated state even in the absence of a stimulated ATPase. In this preparation, N,N'-dicyclohexylcarbodiimide (DCCD), an hydrophobic carbodiimide, inactivates both ACh transport and vesamicol binding. Inhibition of vesamicol binding by DCCD is time dependent, saturable and prevented by vesamicol. DCCD first affected the affinity constant for vesamicol. Ki-value for DCCD lies in the micromolar range. These results imply that there is a DCCD reactive site within the ACh transporter and that it is located in an hydrophobic environment near the vesamicol binding site. SDS-gel electrophoresis after labelling of the vesicle membrane proteins with [14C]DCCD shows that radioactivity is mainly incorporated in a 15 kDa subunit. Time-course and concentration dependence of [14C]DCCD labelling and vesamicol inhibition do not coincide. Hence, the two processes are probably unrelated and the result rather points to another inactivation mechanism which can be an intramolecular cross link. 相似文献
10.
T Kozaka I Uno Y Kitamura D Miwa K Ogawa K Shiba 《Bioorganic & medicinal chemistry》2012,20(16):4936-4941
A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and σ receptors (σ-1, σ-2) were evaluated by in vitro binding assays using rat cerebral or liver membranes. OIDV and OBDV showed greater binding affinity to VAChT (K(i)=20.5±5.6 and 13.8±1.2nM, respectively) than did vesamicol (K(i)=33.9±18.1nM) with low affinity to σ receptors. A saturation binding assay in rat cerebral membranes revealed that [(125)I]OIDV had a single high affinity binding site with a K(d) value of 1.73nM and a B(max) value of 164.4fmol/mg protein. [(125)I]OIDV revealed little competition with inhibitors, which possessed specific affinity to each σ (σ-1 and σ-2), serotonin (5-HT(1A) and 5-HT(2A)), noradrenaline, and muscarinic acetylcholine receptors. In addition, BBB penetration of [(125)I]OIDV was verified in in vivo. The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging probes with high affinity and selectivity. 相似文献
11.
Both the membrane-bound choline acetyltransferase (MChAT) and soluble ChAT (SChAT) were found to be activated by ATP-mediated protein phosphorylation. ATP activation of MChAT but not SChAT was found to depend on the integrity of proton gradient of synaptic vesicles because conditions disrupting the proton gradient also abolished the activation of MChAT by ATP. Among the kinases studied, Ca2+/calmodulin kinase II is most effective in activation of MChAT. Transport of ACh into synaptic vesicles by vesicular acetylcholine transporter (VAChT) is also proton gradient-dependent; therefore we proposed that there is a functional coupling between ACh synthesis and its packaging into synaptic vesicles. This notion is supported by the following findings: first, the newly synthesized [3H]-ACh from [3H]-choline was taken up much more efficiently than the pre-existing ACh; second, ATP-activation of MChAT was abolished when VAChT was inhibited by the specific inhibitor vesamicol; third, the activity of ChAT was found to be markedly increased when neurons are under depolarizing conditions. 相似文献
12.
The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT. 相似文献
13.
Cho GW Kim MH Chai YG Gilmor ML Levey AI Hersh LB 《The Journal of biological chemistry》2000,275(26):19942-19948
Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter. 相似文献
14.
Scheunemann M Sorger D Wenzel B Heinitz K Schliebs R Klingner M Sabri O Steinbach J 《Bioorganic & medicinal chemistry》2004,12(6):1459-1465
Detection of the central cholinergic deficits, a consistent feature of Alzheimer's disease, is essential to allow preventive measures and/or symptomatic treatment already at a very early stage of the disease. The vesicular acetylcholine transporter (VAChT) represents an appropriate target to establish PET radiotracer that are adequate for brain imaging the loss of cholinergic terminals. Here we describe the synthesis and binding characteristics of novel derivatives of vesamicol, known to represent a specific antagonist of VAChT sites. Novel benzyl ether derivatives of vesamicol either 4- or 5-substituted at the cyclohexylring have been synthesized by different regioselective ring opening reactions of a same epoxide precursor. The affinity and selectivity of the novel compounds to VAChT sites were analyzed by competitive radioligand binding studies in rat brain and liver membrane preparations using tritium labeled radioligands. The 4-substituted fluorobenzylether of vesamicol 10b was shown to exhibit a high affinity to VAChT sites (K(i)-value(10b)=10.7+/-1.7 nM), but demonstrated also binding capacities to sigma receptors (K(i-)value(10b)=18.5+/-6.9 nM, [(3)H]DTG; K(i)-value(10b)=30.6+/-9.6 nM, [(3)H]haloperidol). The data suggest the potential of vesamicol derivatives to design appropriate radiotracer for PET imaging of central cholinergic deficits. 相似文献
15.
Zea-Ponce Y Mavel S Assaad T Kruse SE Parsons SM Emond P Chalon S Giboureau N Kassiou M Guilloteau D 《Bioorganic & medicinal chemistry》2005,13(3):745-753
Our goal was to synthesize new stereospecific benzovesamicol analogues, which could potentially be used as SPECT or PET radioligands for the vesicular acetylcholine transporter (VAChT). This paper describes the chemical synthesis, resolution and determination of binding affinity for four enantiomeric pairs of derivatives. Their intrinsic affinities were determined by competition against binding of [3H]vesamicol to human VAChT. Of the eight enantiomers, (E)-(R,R)-5-AOIBV [(R,R)-3], and (R,R)-5-FPOBV [(R,R)-4] displayed the highest binding affinities for VAChT (Kd=0.45 and 0.77 nM, respectively), which indicated that an elongation of the chain from 5-idodo as in the case of 5-iodobenzovesamicol (5-IBVM), to a 5-(E)-3-iodoallyloxy or 5-fluoropropoxy substituent, as in 5-AOIBV and 5-FPOBV, respectively, was very well tolerated at the vesamicol binding site. The enantiomer (R,R)-4-MAIBV [(R,R)-16], which retains the basic structure of (-)-5-IBVM but possess an additional aminomethyl substituent in the 4-position of the piperidine ring, displayed lower binding affinity (Kd=8.8 nM). Nevertheless, the result suggests that substitution at this position may be an interesting alternative to investigate for development of new benzovesamicol analogues. As expected, the corresponding (S,S) enantiomers displayed lower Kd values, they were approximately 10-fold lower in the case of (S,S)-5-FPOBV (Kd=8.4 nM) and (E)-(S,S)-5-AOIBV (Kd=4.3 nM). (R,R)-3, and (R,R)-4 showed the same high affinity for VAChT as (-)-5-IBVM and may be suitable as imaging agents of cholinergic nerve terminals. 相似文献
16.
Hermann A. Rodrigues Matheus de C. Fonseca Wallace L. Camargo Patrícia M. A. Lima Patrícia M. Martinelli Lígia A. Naves Vania F. Prado Marco A. M. Prado Cristina Guatimosim 《PloS one》2013,8(11)
In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KDHOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KDHOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KDHOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KDHOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape. 相似文献
17.
Loureiro-dos-Santos NE Prado MA Reis RA Gardino PF de Mello MC de Mello FG 《Cellular and molecular neurobiology》2002,22(5-6):727-740
1. Previous studies have shown that phorbol esters induce protein kinase C (PKC) mediated phosphorylation of the vesicular acetylcholine transporter (VAChT) and change its interaction with vesamicol. However, it is not clear whether physiological activation of receptors coupled to PKC activation can alter VAChT behavior.2. Here we tested whether activation of kaianate (KA) receptors alters VAChT. Several studies suggest that the cholinergic amacrine cells display KA/AMPA receptors that mediate excitatory input to these neurons. In addition, KA in the chicken retina can generate intracellular messengers with the potential to regulate cellular functions.3. In cultured chicken retina (E8C11) KA reduced vesamicol binding to VAChT by 53%. This effect was potentiated by okadaic acid, a protein phosphatase inhibitor, and was totally prevented by BIM, a PKC inhibitor.4. Phorbol myristate acetate (PMA), but not -PMA, reduced in more than 85% the number of L-[3H]-vesamicol-specific binding sites in chicken retina, confirming that activation of PKC can influence vesamicol binding to chicken VAChT.5. The data show that activation of glutamatergic receptors reduces [3H]-vesamicol binding sites (VAChT) likely by activating PKC and increasing the phosphorylation of the ACh carrier. 相似文献
18.
Itakura M Misawa H Sekiguchi M Takahashi S Takahashi M 《Biochemical and biophysical research communications》1999,265(3):691-696
Classical neurotransmitters such as gamma-aminobutyric acid and glutamate are released from synaptic nerve terminals by exocytosis of synaptic vesicles. PC12 cells also have SSVs capable of storing acetylcholine (ACh). A novel method to examine the effect of transient transfection of any gene of interest on the exocytosis of SSVs was developed. The transfection of choline acetyltransferase (ChAT) into PC12 cells which have lost ACh synthesizing activity resulted in the accumulation of a substantial amount of ACh. Synthesized ACh was released in Ca(2+)-dependent manner. Release was thought to occur by an exocytosis of SSVs because: (1) release was abolished by treating the cells with vesamicol, a specific inhibitor of the vesicular ACh transporter (VAChT) localizing specifically in SSVs; and (2) the release was further increased by cotransfecting rat VAChT with the ChAT. By means of this method, we showed that overexpression of complexin I or II with ChAT markedly suppressed high-K(+)-dependent ACh release of SSVs. 相似文献
19.
Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed. 相似文献
20.
The vesicular acetylcholine transporter (VAChT) contains six conserved sequence motifs that are rich in proline and glycine. Because these residues can have special roles in the conformation of polypeptide backbone, the motifs might have special roles in conformational changes during transport. Using published bioinformatics insights, the amino acid sequences of the 12 putative, helical, transmembrane segments of wild-type and mutant VAChTs were analyzed for propensity to form non-alpha-helical conformations and molecular notches. Many instances were found. In particular, high propensity for kinks and notches are robustly predicted for motifs D2, C and C'. Mutations in these motifs either increase or decrease Vmax for transport, but they rarely affect the equilibrium dissociation constants for ACh and the allosteric inhibitor, vesamicol. The near absence of equilibrium effects implies that the mutations do not alter the backbone conformation. In contrast, the Vmax effects demonstrate that the mutations alter the difficulty of a major conformational change in transport. Interestingly, mutation of an alanine to a glycine residue in motif C significantly increases the rates for reorientation across the membrane. These latter rates are deduced from the kinetics model of the transport cycle. This mutation is also predicted to produce a more flexible kink and tighter tandem notches than are present in wild-type. For the full set of mutations, faster reorientation rates correlate with greater predicted propensity for kinks and notches. The results of the study argue that conserved motifs mediate conformational changes in the VAChT backbone during transport. 相似文献