Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
14
pubmed:dateCreated
2010-4-6
pubmed:abstractText
Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC 2.A.1.2.13) [Vardy, E., et al. (2004) Protein Sci. 13, 1832-1840]. In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12(A123.7) cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with a pK of 6.5 for binding of vesamicol and with a pK of approximately 5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [(3)H]vesamicol binding centered at pH 7.4, mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH >or=9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far from D398 and the ACh binding site in three-dimensional space. Also, a probable network of interactions involving E309 and an unknown residue having a pK of 10 has been revealed.
pubmed:grant
pubmed:commentsCorrections
http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-10692409, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-11099460, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-12893935, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-12893936, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-15215526, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-15924414, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-1649250, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-16675700, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-19685929, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-2724295, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-3950625, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-7798315, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-8177877, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-8342028, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-8662678, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-9038139, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-9182805, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-942051, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-9721736, http://linkedlifedata.com/resource/pubmed/commentcorrection/20225888-9873001
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1520-4995
pubmed:author
pubmed:issnType
Electronic
pubmed:day
13
pubmed:volume
49
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3049-59
pubmed:dateRevised
2011-9-26
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Possible important pair of acidic residues in vesicular acetylcholine transporter.
pubmed:affiliation
Department of Chemistry and Biochemistry, Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural