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• W2064371172 abstract "Cholinergic neurotransmission depends upon the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of the VAChT gene of Caenorhabditis elegans(CeVAChT) (unc-17) and exhibit behavioral phenotypes consistent with a reduction in vesicular transport activity and neurosecretion. Several of these mutants express normal amounts of VAChT protein and exhibit appropriate targeting of VAChT to synaptic vesicles. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stably expressed these cDNAs in PC-12 cells. These mutants display selective defects in initial acetylcholine transport velocity (Km), with values ranging from 2- to 8-fold lower than that of the wild-type. One of these mutants has lost its specific interaction with vesamicol, a selective inhibitor of VAChT, and displays vesamicol-insensitive uptake of acetylcholine. The relative order of behavioral severity of the CeVAChT point mutants is identical to the order of reduced affinity of VAChT for acetylcholine in vitro. This indicates that specific structural changes in VAChT translate into specific alterations in the intrinsic parameters of transport and in the storage and synaptic release of acetylcholine in vivo. Cholinergic neurotransmission depends upon the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of the VAChT gene of Caenorhabditis elegans(CeVAChT) (unc-17) and exhibit behavioral phenotypes consistent with a reduction in vesicular transport activity and neurosecretion. Several of these mutants express normal amounts of VAChT protein and exhibit appropriate targeting of VAChT to synaptic vesicles. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stably expressed these cDNAs in PC-12 cells. These mutants display selective defects in initial acetylcholine transport velocity (Km), with values ranging from 2- to 8-fold lower than that of the wild-type. One of these mutants has lost its specific interaction with vesamicol, a selective inhibitor of VAChT, and displays vesamicol-insensitive uptake of acetylcholine. The relative order of behavioral severity of the CeVAChT point mutants is identical to the order of reduced affinity of VAChT for acetylcholine in vitro. This indicates that specific structural changes in VAChT translate into specific alterations in the intrinsic parameters of transport and in the storage and synaptic release of acetylcholine in vivo. vesicular acetylcholine transporter transmembrane domain vesicular monoamine transporter VAChT gene of Caenorhabditis elegans Acetylcholine is synthesized by choline acetyltransferase in the cytoplasm of cholinergic nerve terminals and transported into synaptic vesicles by a vesicular acetylcholine transporter (VAChT).1 Genetic and behavioral analyses of various point mutants in Caenorhabditis elegans have revealed that the genes encoding both cholinergic proteins map to the same genetic locus and that their co-expression is required for acetylcholine release (1Rand J.B. Russell R.L. Genetics. 1984; 106: 227-248Crossref PubMed Google Scholar, 2Rand J.B. Genetics. 1989; 122: 73-80Crossref PubMed Google Scholar, 3Alfonso A. Grundahl K. Duerr J.S. Han H.-P. Rand J.B. Science. 1993; 261: 617-619Crossref PubMed Scopus (312) Google Scholar, 4Alfonso A. Grundahl K. McManus J.R. Asbury J.M. Rand J.B. J. Mol. Biol. 1994; 241: 627-630Crossref PubMed Scopus (85) Google Scholar). Mutations in the gene encoding choline acetyltransferase (cha-1) and in the gene encoding VAChT (unc-17) lead to similar behavioral defects, and both mutations protect against the toxicity of organophosphorus compounds such as aldicarb. Aldicarb inhibits the degradative enzyme acetylcholinesterase, allowing the toxic accumulation of acetylcholine in the synaptic cleft. These mutations confer resistance to esterase inhibition by reducing the efficiency of acetylcholine neurosecretion. One difference in phenotype between mutants in the two genes is that choline acetyltransferase mutants have decreased levels of acetylcholine within cholinergic nerve terminals, whereas VAChT mutants have increased levels of acetylcholine within cholinergic nerve terminals (5Hosono R. Sassa T. Kuno S. Zool. Sci. 1989; 6: 697-702Google Scholar, 6Nguyen M. Alfonso A. Johnson C.D. Rand J.B. Genetics. 1995; 140: 527-535Crossref PubMed Google Scholar). However, the increased acetylcholine level in CeVAChT mutants is not available for synaptic release due to specific defects in the level of expression or in the intrinsic activity of VAChT. Vesicular acetylcholine transport is essential for cholinergic neurotransmission because homozygous VAChT knockout mutants in C. elegans and in Drosophila melanogaster do not live for more than a few days (3Alfonso A. Grundahl K. Duerr J.S. Han H.-P. Rand J.B. Science. 1993; 261: 617-619Crossref PubMed Scopus (312) Google Scholar, 7Kitamoto R. Xie X. Wu C.F. Salvaterra P.M. J. Neurobiol. 2000; 42: 161-171Crossref PubMed Scopus (30) Google Scholar). Genetic manipulation of the level of VAChT expression can influence acetylcholine synaptic release. In Drosophila, VAChT heterozygotes show decreased cholinergic transmission with high frequency stimulation (7Kitamoto R. Xie X. Wu C.F. Salvaterra P.M. J. Neurobiol. 2000; 42: 161-171Crossref PubMed Scopus (30) Google Scholar). Conversely, overexpression of VAChT in Xenopus embryos increases the magnitude of postsynaptic currents at developing neuromuscular junctions by 2- to 3-fold (8Song H. Ming G. Fon E. Bellocchio E. Edwards R.H. Poo M. Neuron. 1997; 18: 815-826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This is consistent with the increase in vesicular packaging of acetylcholine observed in vitro after overexpression of VAChT in cholinergic cells (9Varoqui H. Erickson J.D. J. Biol. Chem. 1996; 271: 27229-27232Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (80) Google Scholar). Thus, the level of VAChT expression may alter the rate and extent of synaptic vesicle accumulation of acetylcholine. Relatively little is known regarding the role of the intrinsic activity of VAChT on the vesicular accumulation and synaptic release of acetylcholine. Inhibition of acetylcholine transport into synaptic vesicles by vesamicol, which selectively binds to VAChT, blocks quantal acetylcholine secretion in vivo (9Varoqui H. Erickson J.D. J. Biol. Chem. 1996; 271: 27229-27232Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (80) Google Scholar, 11Varoqui H. Diebler M.-F. Meunier F.-M. Rand J.B. Usdin T.B. Bonner T.I. Eiden L.E. Erickson J.D. FEBS Lett. 1994; 342: 97-102Crossref PubMed Scopus (95) Google Scholar, 12Erickson J.D. Varoqui H. Schäfer M.K.-H. Diebler M.-F. Weihe E. Modi W. Rand J.B. Eiden L.E. Bonner T.I. Usdin T. J. Biol. Chem. 1994; 269: 21929-21932Abstract Full Text PDF PubMed Google Scholar, 13Brittain R.T. Levy G.P. Tyers M.B. Eur. J. Pharmacol. 1969; 8: 93-99Crossref PubMed Scopus (51) Google Scholar, 14Marshall I.G. Br. J. Pharmacol. 1970; 38: 503-516Crossref PubMed Scopus (73) Google Scholar, 15Vizi E.S. Br. J. Pharmacol. 1989; 98: 898-902Crossref PubMed Scopus (20) Google Scholar). Sequence conservation of the aspartic acid residues within the putative hydrophobic transmembrane domains (TMDs) of VAChT from several species suggests functional importance in intrinsic activity. Some of these aspartate residues have been shown to be critical for the activity of VAChT (16Kim M.-H. Lu M. Kelly M. Hersh L.B. J. Biol. Chem. 2000; 275: 6175-6180Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Kim M.-H. Lu M. Lim E.-H. Chai Y.-H. Hersh L.B. J. Biol. Chem. 1999; 274: 673-680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). When an aspartate residue is changed in TMD IV (D193N) or in TMD X (D398N), overexpression of rat VAChT in Xenopusneuromuscular junctions no longer leads to an increase in quantal acetylcholine secretion (8Song H. Ming G. Fon E. Bellocchio E. Edwards R.H. Poo M. Neuron. 1997; 18: 815-826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Interestingly, overexpression of D398N appears to result in a dominant negative effect that reduces endogenous quantal acetylcholine secretion (8Song H. Ming G. Fon E. Bellocchio E. Edwards R.H. Poo M. Neuron. 1997; 18: 815-826Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In vesicles isolated from PC-12 cells, the D398N mutation completely abolishes acetylcholine transport and vesamicol binding, whereas the D193N mutant transports acetylcholine at the same rate as wild-type transporter (16Kim M.-H. Lu M. Kelly M. Hersh L.B. J. Biol. Chem. 2000; 275: 6175-6180Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). VAChT is part of a gene family that includes the vesicular monoamine transporters VMAT1 and VMAT2 (18Eiden L.E. J. Neurochem. 1998; 70: 2227-2240Crossref PubMed Scopus (172) Google Scholar). The functional activity of vacht relies upon the proton electrochemical gradient established by a vacuolar-type H+-ATPase (9Varoqui H. Erickson J.D. J. Biol. Chem. 1996; 271: 27229-27232Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 10Liu Y. Edwards R.H. J. Cell Biol. 1997; 139: 907-916Crossref PubMed Scopus (80) Google Scholar, 19Nguyen M.L. Cox G.D. Parson S.M. Biochemistry. 1998; 37: 13400-13410Crossref PubMed Scopus (53) Google Scholar). A chimeric VAChT molecule in which TMD I is replaced by TMD I of VMAT2 shows a reduced affinity for acetylcholine but retains vesamicol binding (20Varoqui H. Erickson J.D. J. Physiol. (Paris). 1998; 92: 141-144Crossref PubMed Scopus (17) Google Scholar). This suggests that TMD I of VAChT, like the TMD I of VMAT, is involved in substrate translocation (21Merickel A. Rosandich P. Peter D. Edwards R.H. J. Biol. Chem. 1995; 270: 25798-25804Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However, the ammonium headgroup of acetylcholine may interact with Asp-425 in TMD XI instead of Asp-46 in TMD I, which is hypothesized to interact with the primary amino group of monoamines (16Kim M.-H. Lu M. Kelly M. Hersh L.B. J. Biol. Chem. 2000; 275: 6175-6180Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 21Merickel A. Rosandich P. Peter D. Edwards R.H. J. Biol. Chem. 1995; 270: 25798-25804Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The roles of conserved basic amino acid residues within the TMDs of VAChT have also been examined by mutagenesis. A putative charge-pair interaction between His-338 in TMD VIII and Asp-398 in TMD X has been proposed to anchor the TMD helices together (17Kim M.-H. Lu M. Lim E.-H. Chai Y.-H. Hersh L.B. J. Biol. Chem. 1999; 274: 673-680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The presence of a salt bridge in membrane-spanning regions of VAChT may be important in facilitating the conformational changes associated with H+/acetylcholine antiport. In VMATs, the negative charge in TMD X is hypothesized to play a direct role in H+ translocation (22Steiner-Mordoch S. Shirvan A. Schuldiner S. J. Biol. Chem. 1996; 271: 13048-13054Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). A conserved lysine residue in TMD II (Lys-131) is also critical for VMAT and VAChT function (17Kim M.-H. Lu M. Lim E.-H. Chai Y.-H. Hersh L.B. J. Biol. Chem. 1999; 274: 673-680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,23Merickel A. Kaback H.R. Edwards R.H. J. Biol. Chem. 1997; 272: 5403-5408Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Thus, Lys-131, His-338, and Asp-398 play important roles in the mechanism of action of VAChT, possibly as components of a H+ relay system, as described for VMATs and the lactose permease, a H+-symporter that belongs to the same sequence-defined superfamily (22Steiner-Mordoch S. Shirvan A. Schuldiner S. J. Biol. Chem. 1996; 271: 13048-13054Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 23Merickel A. Kaback H.R. Edwards R.H. J. Biol. Chem. 1997; 272: 5403-5408Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 24Lee J.A. Püttner I.B. Kaback H.R. Biochemistry. 1989; 28: 2540-2544Crossref PubMed Scopus (22) Google Scholar, 25He M.M. Kaback H.R. Biochemistry. 1997; 36: 13688-13692Crossref PubMed Scopus (40) Google Scholar, 26Sahin-Toth M. Karlin A. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10729-10732Crossref PubMed Scopus (64) Google Scholar, 27Parsons S.M. FASEB J. 2000; 14: 2423-2434Crossref PubMed Scopus (132) Google Scholar). In the present report, we have analyzed 12 different nonlethal point mutations in the CeVAChT in an effort to identify additional domains of VAChT involved in acetylcholine translocation. Several mutations are at residues that are conserved in vertebrate VAChTs and are associated with normal levels of VAChT protein in cholinergic nerve endings. These mutant proteins also exhibit appropriate targeting to synaptic vesicles. The analysis of the acetylcholine transport and vesamicol binding properties of hVAChT constructs containing these mutations expressed in a heterologous system in vitroprovides new information regarding the location of the acetylcholine transport recognition site in VAChT. Furthermore, the combination of a genetic approach in C. elegans and biochemistry in vitro establishes a direct relationship between the intrinsic parameters of vesicular acetylcholine transport in vitro and the storage and synaptic release of acetylcholine in vivo. C. elegansstrains were grown on solid medium as described previously (3Alfonso A. Grundahl K. Duerr J.S. Han H.-P. Rand J.B. Science. 1993; 261: 617-619Crossref PubMed Scopus (312) Google Scholar, 28Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). The unc-17 alleles md65 and md69 were ethyl methane sulfonate-induced and identified by noncomplementation with pre-existing unc-17 mutations (3Alfonso A. Grundahl K. Duerr J.S. Han H.-P. Rand J.B. Science. 1993; 261: 617-619Crossref PubMed Scopus (312) Google Scholar); md334,md414, and md1107 were isolated as spontaneous mutants resistant to the acetylcholinesterase inhibitor aldicarb (6Nguyen M. Alfonso A. Johnson C.D. Rand J.B. Genetics. 1995; 140: 527-535Crossref PubMed Google Scholar,29Miller K.G. Alfonso A. Nguyen M. Crowell J.A. Johnson C.D. Rand J.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12593-12598Crossref PubMed Scopus (338) Google Scholar). Alleles with p designations were ethyl methane sulfonate-induced (in Richard Russell's laboratory) and isolated by resistance to aldicarb (30Rand J.B. Russell R.L. Psychopharmacol. Bull. 1985; 21: 623-630PubMed Google Scholar). The unc-17 alleles with e allele designations were isolated at the Medical Research Council (Cambridge, United Kingdom) and generously provided by Jonathan Hodgkin. All mutants were outcrossed to wild-type (N2 Bristol) at least four times. The sequence alterations associated with the e245 and p1160 alleles have been published previously (3Alfonso A. Grundahl K. Duerr J.S. Han H.-P. Rand J.B. Science. 1993; 261: 617-619Crossref PubMed Scopus (312) Google Scholar). Sequence analysis of the remaining unc-17 mutations involved amplification of specific 1–2-kilobase unc-17 genomic regions using direct “single-worm polymerase chain reaction” from individual mutant animals (31Williams B.D. Schrank B. Huynh C. Shownkeen R. Waterston R.H. Genetics. 1992; 131: 609-624Crossref PubMed Google Scholar) followed by sequencing of the amplified genomic DNA with internal primers. Oligonucleotide primers were synthesized by the Molecular Biology Resource Facility of the University of Oklahoma Health Sciences Center. Nematodes were prepared with a variation of the “freeze-crack” method and stained with an anti-CeVAChT mouse monoclonal antibody (Mab 1403) as described previously (32Albertson D.G. Dev. Biol. 1984; 101: 61-72Crossref PubMed Scopus (231) Google Scholar, 33Duerr J.S. Gaskin J. Rand J.B. Am. J. Physiol. Cell Physiol. 2001; 280: C1616-C1622Crossref PubMed Google Scholar, 34Lickteig K.M. Duerr J.S. Frisby D.L. Hall D.H. Rand J.B. Miller III., D.M. J. Neurosci. 2001; 21: 2001-2014Crossref PubMed Google Scholar). All behavioral measurements were performed at 20 °C. The procedures for observing and quantifying pharyngeal pumping and body thrashing in liquid have been described previously (29Miller K.G. Alfonso A. Nguyen M. Crowell J.A. Johnson C.D. Rand J.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12593-12598Crossref PubMed Scopus (338) Google Scholar, 35Duerr J.S. Frisby D.L. Gaskin J. Duke A. Asermely K. Huddleston D. Eiden L.E. Rand J.B. J. Neurosci. 1999; 19: 72-84Crossref PubMed Google Scholar). Values are normalized to N2 = 100% and are presented ± S.D. (10–50 individuals were counted for each allele). An approximate measure of growth rate at 20 °C was determined by a “plate-clearing” method (36Rand J.B. Johnson C.D. Epstein H.F. Shakes D.C. C. elegans: Modern Biological Analysis of an Organism. Academic Press, San Diego1995: 187-204Google Scholar). Twenty L1 animals were placed on a streak plate and examined once or twice a day, until all the food was consumed. A total of one to four plates were analyzed per allele. The growth rate for each strain is defined as the inverse of the number of days to consume all the food, and the “relative growth rate” is calculated as ratio of the mutant growth rate to the wild-type rate. Site-directed mutagenesis was performed using a 640-base pair Sse8387I/SalI fragment of hVAChT (contains coding sequences that include TMD IV through TMD X) subcloned into pUC18 using the method of Deng and Nickoloff (37Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-85Crossref PubMed Scopus (1079) Google Scholar). The Escherichia coliBMH 71-18 mutS bacterial strain and buffers were provided with the Transformer Site-directed Mutagenesis Kit (CLONTECH). Transformants were screened by cDNA double-stranded sequencing using the Sequenase kit (United States Biochemical). The mutated hVAChT fragment was then reintroduced into the wild-type hVAChT cDNA and subcloned into pRc/CMV at HindIII/NotI for transfection into PC-12 cells. Rat PC-12 cells were maintained at 37 °C in an atmosphere of 95% air, 5% CO2 in Dulbecco’s modified Eagle’s medium containing 7% fetal bovine serum, 7% heat-inactivated horse serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 4 mm glutamine. PC-12 cells were transfected with the hVAChT constructs using Lipofectin (10 μg/ml; Life Technologies, Inc.), and stable transformants were selected with 0.5 mg/ml Geneticin (G418; Life Technologies, Inc.). Geneticin-resistant colonies were picked and screened by immunocytochemistry as described previously (38Varoqui H. Erickson J.D. J. Biol. Chem. 1998; 273: 9094-9098Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Cells were fixed for 2 h in 10% formalin/phosphate-buffered saline and incubated with species-specific hVAChT antipeptide rabbit polyclonal antisera (39Weihe E. Tao-Chemg J. Schäfer M.K.-H. Erickson J.D. Eiden L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3547-3552Crossref PubMed Scopus (268) Google Scholar) at a final dilution of 1:2000 in phosphate-buffered saline, 0.1% Triton, and 3% normal goat serum. Primary antibodies were visualized using VectaStain Kit (Vector Laboratories Inc.). Control PC-12 cells and the lines expressing hVAChT and the hVAChT mutants were grown to confluence in a T175 flask, treated with 6 mm sodium butyrate, and incubated for an additional 36 h. This treatment increases expression from viral promoters (40Grote E. Hao J. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (144) Google Scholar) and does not affect the biochemical parameters of acetylcholine transport or targeting of VAChT to cholinergic vesicles in PC-12 cells. Transient transfections were also performed in CV-1 fibroblasts by the T7-vaccinia hybrid system as described previously (11Varoqui H. Diebler M.-F. Meunier F.-M. Rand J.B. Usdin T.B. Bonner T.I. Eiden L.E. Erickson J.D. FEBS Lett. 1994; 342: 97-102Crossref PubMed Scopus (95) Google Scholar). The cells were rinsed with phosphate-buffered saline, collected in phosphate-buffered saline containing 10 mm EDTA (pH 7.4), and centrifuged at 800 × g for 5 min. To prepare postnuclear supernatants, the cell pellets were resuspended and homogenized in a ball-bearing device (20 strokes, 11 μm clearance) in ice-cold buffer containing 80 mm potassium tartrate, 20 mmHEPES, 0.5 mm EGTA, and 1 mm ascorbic acid, adjusted to pH 7.0 with KOH, and supplemented with a protease inhibitor mixture (0.2 mm phenylmethylsulfonyl fluoride, 2 mg/ml leupeptin, 2 mg/ml aprotinin, and 2 mg/ml pepstatin) and 50 μm esterase inhibitor echothiophate (Wyeth Ayerst Laboratories). The nuclei and unbroken cells were removed by centrifugation at 2000 × g for 10 min. The protein content in the supernatant was measured using the Bradford assay (41Bradford M.M. Anal. Biochem. 1976; 73: 248-254Crossref Scopus (214455) Google Scholar). The levels of expression of the human VAChT proteins were assessed on Western blot and, when possible, by [3H]vesamicol (l-[piperidinyl-3,4-3H]vesamicol; Amersham Pharmacia Biotech) binding measurements. Fresh preparations were used to perform [3H]acetylcholine transport assays. For [3H]acetylcholine uptake assay, aliquots (100 μl) of postnuclear homogenates containing 200 μg of protein were preincubated at 37 °C for 5 min in the presence and absence ofl-vesamicol (Research Biochemicals Inc.) as described previously (42Varoqui H. Erickson J.D. Amara S.G. Methods in Enzymology: Neurotransmitter Transporters. Academic Press, New York1998: 84-98Google Scholar). After preincubation, 100 μl of uptake buffer containing 110 mm potassium tartrate, 20 mmHEPES, 1 mm ascorbic acid, 50 μmechothiophate, and 10 mm Mg2+-ATP (neutralized with KOH to pH 7.4) and various concentrations of tritiated (0.4 mm; 55.2 mCi/mmol; PerkinElmer Life Sciences) and unlabeled acetylcholine were added. The final concentration of Mg2+-ATP was 5 mm. Uptake was terminated after 10 min by placing tubes in an ice water bath, filtering through GF/C glass fiber filters (Whatman), and washing with 5 ml of ice-cold uptake buffer. Homogenates from control PC-12 cells and human VAChT-expressing PC-12 cells were always analyzed in parallel to assess the specific acetylcholine uptake derived from endogenous PC-12 VAChT, which is generally less than 2-fold more than that seen at 4 °C or in the presence of 2 μm vesamicol. Aliquots of postnuclear supernatant (30 μg of protein in 50 μl) were mixed with binding buffer containing 110 mm potassium tartrate and 20 mmHEPES at pH 7.4 (50 μl) and various concentrations of [3H]vesamicol (31 Ci/mmol; PerkinElmer Life Sciences). For acetylcholine displacement of [3H]vesamicol binding, the organelles in the postnuclear homogenates were first lysed by dilution (1:10) in ice-cold 20 mm HEPES containing peptidase inhibitors and centrifugation at 100,000 × gfor 1 h. The pellets were resuspended in binding buffer, and [3H]vesamicol (final concentration, 20 nm) and various amounts of unlabeled acetylcholine were added. After a 30-min incubation at 20 °C, the reactions were stopped by vacuum filtration through GF/C glass fiber filters (Whatman) presoaked in 0.5% polyethyleneimine, followed by a 5-ml wash with ice-cold buffer containing 110 mm potassium tartrate and 20 mmHEPES, pH 7.4. Radioactivity bound to the filters was solubilized in 1 ml of 1% SDS and measured by scintillation counting in 10 ml of EcoScint (National Diagnostics). Nonspecific binding was determined in the presence of 30 μml-vesamicol and subtracted from the total binding. For equilibrium density fractionation, postnuclear supernatants (about 1 mg of protein) were loaded onto a continuous 4.4 ml of 0.6–1.6 m (20–46%) sucrose gradient buffered with 20 mm HEPES, pH 7, and 5 mm EGTA for sedimentation at 46,000 rpm at 4 °C for 3 h in a SW55 rotor. For glycerol velocity fractionation, we used a modification of the method described by Kelly and co-workers (40Grote E. Hao J. Bennett M.K. Kelly R.B. Cell. 1995; 81: 581-589Abstract Full Text PDF PubMed Scopus (144) Google Scholar). Briefly, the postnuclear supernatant was centrifuged at 10,000 ×g for 10 min, and the resulting supernatant (about 1 mg of protein) was loaded onto a 4.4-ml 5–25% glycerol gradient prepared in homogenization buffer for a 45-min centrifugation at 55,000 rpm in a SW55 rotor. For both gradients, a first fraction equivalent to the volume loaded and successive 350-μl fractions were collected from the top. The linearity of the gradients was verified by refractometry. Equivalent amounts of protein from the postnuclear supernatants of the stable PC-12 lines expressing hVAChT and the hVAChT mutants or equal volumes of each gradient fraction were processed for Western blot analysis. High-speed pellets were prepared from diluted gradient fractions. Sucrose gradient fractions were brought to 320 mm sucrose with 20 mm HEPES, pH 7, whereas glycerol gradient fractions were diluted 5-fold in the same buffer. Both were spun for 1 h at 45,000 rpm at 4 °C in a Ti50.4 rotor. Pellets were solubilized in sample buffer containing 62 mm Tris-HCl, pH 6.8, 1 mm EDTA, 10% glycerol, 5% SDS, and 50 mmdithiothreitol. Membrane proteins were separated on 8% polyacrylamide gels and electrotransferred onto nitrocellulose (Hybond-ECL; Amersham Pharmacia Biotech). Chromogranin B and synaptophysin (p38) were detected in aliquots taken directly from gradient fractions and dissolved in sample buffer. After a 1-h preincubation in TBS (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk, the blots were incubated for 2 h at room temperature with affinity-purified anti-hVAChT at 1 mg/ml (38Varoqui H. Erickson J.D. J. Biol. Chem. 1998; 273: 9094-9098Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), anti-chromogranin B at 1:1000 (a gift from R. Fischer-Colbrie, Innsbruck, Austria), or anti-p38 at 1:5000 (Sigma) in TBS-1% bovine serum albumin. Bound primary antibodies were visualized using a monoclonal anti-rabbit antibody or a polyclonal anti-mouse antibody, both of which were coupled to peroxidase (Sigma), and an enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech). The sequence alterations of 19 independent unc-17 point mutants, corresponding to 12 distinct amino acid substitutions, are presented in Fig.1. These fall predominantly into three categories: (a) those at amino acids that are conserved among VMATs and VAChT, (b) those found only in VAChTs, and (c) those unique to CeVAChT. Only one mutation (CeR60W) was found at a residue unique to CeVAChT; it was localized in the first intralumenal loop. Six mutants (CeE98K, CeG103R, CeG103E,CeG342E, CeG347R, and CeY400N) had sequence alterations at amino acid residues that are conserved in VMATs and VAChT. Five of these mutations lie in predicted TMDs (TMDs II, IX, and XI), whereas one (CeE98K) is in the luminal loop between TMD I and TMD II. The CeG103R/E, CeG342E, and CeG347R mutations introduce a charged residue into the putative TMDs. Four CeVAChT mutations (CeA206V,CeC230F, CeT292del, and CeC370Y) were at amino acids that are relatively conserved in Torpedo, rat, and human VAChT but are not conserved in VMATs. Three of these VAChT-specific residues introduce a larger and more hydrophobic amino acid residue in predicted TMDs (TMDs V, VI, and X), and one (CeT292del) was found in the luminal loop between TMD VII and VIII. One mutation (CeS211F) is at an amino acid conserved in VMATs, whereas this residue in vertebrate VAChTs is glycine (G233). C. elegans mutants were screened for normal levels of CeVAChT immunoreactivity. Mutations at two locations (CeG342E and CeG347R) led to lower levels of CeVAChT protein. These mutations altered residues conserved in VAChTs and VMATs, and they probably destabilized the VAChT protein. All of the VAChT-specific mutations as well as CeS211F express relatively normal amounts of protein that is appropriately targeted to cholinergic endings (Fig.2). We measured three phenotypes of the unc-17 mutants (Fig.3). The rates of pharyngeal pumping and thrashing in liquid are measures of neuromuscular function in the pharynx and the body muscles, respectively. The “plate clearing” assay is affected by both the generation time and the fecundity of the mutants. By each of these criteria, e327(CeS211F), e795 (CeA206V), and p1160 (CeC230F) form a graded series of increasing severity, whereas md414 (CeC370Y) homozygotes have approximately normal growth and only moderately impaired neuromuscular function. For comparison, the properties of the unc-17 reference allele e245 (CeG347R) are also presented in Fig. 3. However, because e245 has lower levels of VAChT immunoreactivity in vivo (Fig. 2), it is difficult to compare the e245 phenotypic data directly to the other mutants. We have further examined the effect of these mutations on VAChT function in a heterologous system that has been used to study the vesicular trafficking and transport function of mammalian VAChT (9Varoqui H. Erickson J.D." @default.
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