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Volumn 128, Issue 2, 2005, Pages 345-355

Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker

Author keywords

Acetylcholine receptor; Congenital myasthenic syndrome; M3 M4 loop; Mutagenesis; Single channel patch clamp recordings

Indexed keywords

ASPARAGINE; ASPARTIC ACID; CHOLINERGIC RECEPTOR; RECEPTOR SUBUNIT;

EID: 13544266220     PISSN: 00068950     EISSN: None     Source Type: Journal    
DOI: 10.1093/brain/awh364     Document Type: Article
Times cited : (29)

References (45)
  • 1
    • 0028600111 scopus 로고
    • Structural basis of different gating kinetics of fetal and adult acetylcholine receptors
    • Bouzat C, Bren N, Sine SM. Structural basis of different gating kinetics of fetal and adult acetylcholine receptors. Neuron 1994; 13: 1395-402.
    • (1994) Neuron , vol.13 , pp. 1395-1402
    • Bouzat, C.1    Bren, N.2    Sine, S.M.3
  • 2
    • 0000321435 scopus 로고    scopus 로고
    • Identification and functional characterization of eight novel acetylcholine receptor mutations in six congenital myasthenic syndrome kinships
    • Brengman JM, Ohno K, Milone M, Friedman RL, Feldman RG, Engel AG. Identification and functional characterization of eight novel acetylcholine receptor mutations in six congenital myasthenic syndrome kinships. Neurology 2000; 54 (Suppl. 3): A182-3.
    • (2000) Neurology , vol.54 , Issue.SUPPL. 3
    • Brengman, J.M.1    Ohno, K.2    Milone, M.3    Friedman, R.L.4    Feldman, R.G.5    Engel, A.G.6
  • 3
    • 1842475289 scopus 로고    scopus 로고
    • Nicotine and carbamycholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures
    • Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB, Sixma TK. Nicotine and carbamycholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 2004; 41: 907-14.
    • (2004) Neuron , vol.41 , pp. 907-914
    • Celie, P.H.1    Van Rossum-Fikkert, S.E.2    Van Dijk, W.J.3    Brejc, K.4    Smit, A.B.5    Sixma, T.K.6
  • 4
    • 0000410893 scopus 로고
    • A Q-matrix cookbook: How to write only one program to calculate the single-channel and macroscopic predictions for any kinetic mechanism
    • Sakmann B, Neher E, editors. New York: Plenum Press
    • Colquhoun D, Hawkes AG. A Q-matrix cookbook: how to write only one program to calculate the single-channel and macroscopic predictions for any kinetic mechanism. In: Sakmann B, Neher E, editors. Single-channel recording. New York: Plenum Press; 1995. p. 589-633.
    • (1995) Single-channel Recording , pp. 589-633
    • Colquhoun, D.1    Hawkes, A.G.2
  • 5
    • 0022272338 scopus 로고
    • Fast events in single channel currents activated by acetylcholine and its analogues at the frog muscle end-plate
    • Colquhoun D, Sakmann B. Fast events in single channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol (Lond) 1985; 369: 501-57.
    • (1985) J Physiol (Lond) , vol.369 , pp. 501-557
    • Colquhoun, D.1    Sakmann, B.2
  • 6
    • 0001774017 scopus 로고
    • Fitting and statistical analysis of single-channel records
    • Sakmann B, Neher E, editors. New York: Plenum Press
    • Colquhoun D, Sigworth FJ. Fitting and statistical analysis of single-channel records. In: Sakmann B, Neher E, editors. Single-channel recording. New York: Plenum Press; 1995. p. 483-587.
    • (1995) Single-channel Recording , pp. 483-587
    • Colquhoun, D.1    Sigworth, F.J.2
  • 7
    • 0034962933 scopus 로고    scopus 로고
    • Endplate γ- and ε subunit mRNA levels in AChR deficiency syndrome due to ε subunit null mutations
    • Croxen R, Young C, Slater C, Haslam S, Brydson M, Vincent A, et al. Endplate γ- and ε subunit mRNA levels in AChR deficiency syndrome due to ε subunit null mutations. Brain 2001; 124: 1362-72.
    • (2001) Brain , vol.124 , pp. 1362-1372
    • Croxen, R.1    Young, C.2    Slater, C.3    Haslam, S.4    Brydson, M.5    Vincent, A.6
  • 8
    • 0036623799 scopus 로고    scopus 로고
    • Desensitization of diliganded mouse muscle nicotinic acetylcholine receptor channels
    • Elenes S, Auerbach A. Desensitization of diliganded mouse muscle nicotinic acetylcholine receptor channels. J Physiol (Lond) 2002; 541: 367-83.
    • (2002) J Physiol (Lond) , vol.541 , pp. 367-383
    • Elenes, S.1    Auerbach, A.2
  • 9
    • 0000287048 scopus 로고
    • Quantitative morphological studies of muscle
    • Engel AG, Franzini-Armstrong C, editors. New York: McGraw-Hill
    • Engel AG. Quantitative morphological studies of muscle. In: Engel AG, Franzini-Armstrong C, editors. Myology. New York: McGraw-Hill; 1994a. p. 1018-45.
    • (1994) Myology , pp. 1018-1045
    • Engel, A.G.1
  • 10
    • 0000780544 scopus 로고
    • The muscle biopsy
    • Engel AG, Franzini-Armstrong C, editors. New York: McGraw-Hill
    • Engel AG. The muscle biopsy. In: Engel AG, Franzini-Armstrong C, editors. Myology. New York: McGraw-Hill; 1994b. p. 822-31.
    • (1994) Myology , pp. 822-831
    • Engel, A.G.1
  • 11
    • 0027501444 scopus 로고
    • Congenital myasthenic syndromes. I. Deficiency and short open-time of the acetylcholine receptor
    • Engel AG, Nagel A, Walls TJ, Harper CM, Waisburg HA. Congenital myasthenic syndromes. I. Deficiency and short open-time of the acetylcholine receptor. Muscle Nerve 1993; 16: 1284-92.
    • (1993) Muscle Nerve , vol.16 , pp. 1284-1292
    • Engel, A.G.1    Nagel, A.2    Walls, T.J.3    Harper, C.M.4    Waisburg, H.A.5
  • 12
    • 0017749807 scopus 로고
    • Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model
    • Engel AG, Lindstrom JM, Lambert EH, Lennon VA. Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model. Neurology 1977; 27: 307-15.
    • (1977) Neurology , vol.27 , pp. 307-315
    • Engel, A.G.1    Lindstrom, J.M.2    Lambert, E.H.3    Lennon, V.A.4
  • 13
    • 0037530441 scopus 로고    scopus 로고
    • Sleuthing molecular targets for neurological diseases at the neuromuscular junction
    • Engel AG, Ohno K, Sine SM. Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat Rev Neurosci 2003; 4: 339-52.
    • (2003) Nat Rev Neurosci , vol.4 , pp. 339-352
    • Engel, A.G.1    Ohno, K.2    Sine, S.M.3
  • 14
    • 0035025427 scopus 로고    scopus 로고
    • Assembly and clustering of acetylcholine receptors containing GFP-tagged ε or γ subunits. Selective targeting to the neuromuscular junction in vivo
    • Gensler S, Sander A, Korngreen A, Traina G, Giese G, Witzemann V. Assembly and clustering of acetylcholine receptors containing GFP-tagged ε or γ subunits. Selective targeting to the neuromuscular junction in vivo. Eur J Biochem 2001; 268: 2209-17.
    • (2001) Eur J Biochem , vol.268 , pp. 2209-2217
    • Gensler, S.1    Sander, A.2    Korngreen, A.3    Traina, G.4    Giese, G.5    Witzemann, V.6
  • 15
    • 0037444682 scopus 로고    scopus 로고
    • Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant εL221F, inferred from maximum likelihood fits
    • Hatton CJ, Shelley C, Brydson M, Beeson D, Colquhoun D. Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant εL221F, inferred from maximum likelihood fits. J Physiol (Lond) 2003; 547: 729-60.
    • (2003) J Physiol (Lond) , vol.547 , pp. 729-760
    • Hatton, C.J.1    Shelley, C.2    Brydson, M.3    Beeson, D.4    Colquhoun, D.5
  • 16
    • 0037441504 scopus 로고    scopus 로고
    • Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits
    • Huebsch KA, Maimone MM. Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits. J Neurobiol 2003; 54: 486-501.
    • (2003) J Neurobiol , vol.54 , pp. 486-501
    • Huebsch, K.A.1    Maimone, M.M.2
  • 18
    • 0033032332 scopus 로고    scopus 로고
    • Improved secondary structure predictions for a nicotinic receptor subunit: Incorporation of solvent accessibility and experimental data into a two-dimensional representation
    • Le Novère N, Corringer PJ, Changeux J-P. Improved secondary structure predictions for a nicotinic receptor subunit: incorporation of solvent accessibility and experimental data into a two-dimensional representation. Biophys J 1999; 76: 2329-45.
    • (1999) Biophys J , vol.76 , pp. 2329-2345
    • Le Novère, N.1    Corringer, P.J.2    Changeux, J.-P.3
  • 19
    • 0024596553 scopus 로고
    • A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671
    • Luther MA, Schoepfer R, Whiting P, Casey B, Blatt Y, Montal MS, et al. A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671. J Neurosci 1989; 9: 1082-96.
    • (1989) J Neurosci , vol.9 , pp. 1082-1096
    • Luther, M.A.1    Schoepfer, R.2    Whiting, P.3    Casey, B.4    Blatt, Y.5    Montal, M.S.6
  • 20
    • 0027328896 scopus 로고
    • Interaction of the 43 kd postsynaptic protein with all subunits of the muscle nicotinic acetylcholine receptor
    • Maimone MM, Merlie JP. Interaction of the 43 kd postsynaptic protein with all subunits of the muscle nicotinic acetylcholine receptor. Neuron 1993; 11: 53-66.
    • (1993) Neuron , vol.11 , pp. 53-66
    • Maimone, M.M.1    Merlie, J.P.2
  • 21
    • 0028019282 scopus 로고
    • Patch-clamp analysis of the properties of acetylcholine receptor channels at the normal human endplate
    • Milone M, Hutchinson DO, Engel AG. Patch-clamp analysis of the properties of acetylcholine receptor channels at the normal human endplate. Muscle Nerve 1994; 17: 1364-9.
    • (1994) Muscle Nerve , vol.17 , pp. 1364-1369
    • Milone, M.1    Hutchinson, D.O.2    Engel, A.G.3
  • 22
    • 0032031997 scopus 로고    scopus 로고
    • Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor ε subunit
    • Milone M, Wang H-L, Ohno K, Prince RJ, Shen X-M, Brengman JM, et al. Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor ε subunit. Neuron 1998; 20: 575-88.
    • (1998) Neuron , vol.20 , pp. 575-588
    • Milone, M.1    Wang, H.-L.2    Ohno, K.3    Prince, R.J.4    Shen, X.-M.5    Brengman, J.M.6
  • 23
    • 0000679188 scopus 로고    scopus 로고
    • Unusual congenital myasthenic syndrome with endplate AChR deficiency caused by alpha subunit mutations and a remitting-relapsing course
    • Milone M, Shen X-M, Ohno K, Harper CM, Fukudome T, Stilling G, et al. Unusual congenital myasthenic syndrome with endplate AChR deficiency caused by alpha subunit mutations and a remitting-relapsing course. Neurology 1999; 52 Suppl. 2: 185-6.
    • (1999) Neurology , vol.52 , Issue.SUPPL. 2 , pp. 185-186
    • Milone, M.1    Shen, X.-M.2    Ohno, K.3    Harper, C.M.4    Fukudome, T.5    Stilling, G.6
  • 24
    • 0032967642 scopus 로고    scopus 로고
    • Nicotinic acetylcholine receptor at 4.6 Å resolution: Transverse tunnels in the channel wall
    • Miyazawa A, Fujiyoshi Y, Unwin N. Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the channel wall. J Mol Biol 1999; 288: 765-86.
    • (1999) J Mol Biol , vol.288 , pp. 765-786
    • Miyazawa, A.1    Fujiyoshi, Y.2    Unwin, N.3
  • 25
    • 0038112088 scopus 로고    scopus 로고
    • Structure and gating mechanism of the acetylcholine receptor pore
    • Miyazawa A, Fujiyoshi Y, Unwin N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 2003; 423: 949-55.
    • (2003) Nature , vol.423 , pp. 949-955
    • Miyazawa, A.1    Fujiyoshi, Y.2    Unwin, N.3
  • 26
    • 15844429136 scopus 로고    scopus 로고
    • Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor ε subunit
    • Ohno K, Wang H-L, Milone M, Bren N, Brengman JM, Nakano S, et al. Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor ε subunit. Neuron 1996; 17: 157-70.
    • (1996) Neuron , vol.17 , pp. 157-170
    • Ohno, K.1    Wang, H.-L.2    Milone, M.3    Bren, N.4    Brengman, J.M.5    Nakano, S.6
  • 27
    • 8244225989 scopus 로고    scopus 로고
    • Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor ε subunit gene: Identification and functional characterization of six new mutations
    • Ohno K, Quiram P, Milone M, Wang H-L, Harper CM, Pruitt JN, et al. Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor ε subunit gene: identification and functional characterization of six new mutations. Hum Mol Genet 1997; 6: 753-66.
    • (1997) Hum Mol Genet , vol.6 , pp. 753-766
    • Ohno, K.1    Quiram, P.2    Milone, M.3    Wang, H.-L.4    Harper, C.M.5    Pruitt, J.N.6
  • 28
    • 0345530998 scopus 로고    scopus 로고
    • A frameshifting mutation in CHRNE unmasks skipping of the preceding exon
    • Ohno K, Milone M, Shen X-M, Engel AG. A frameshifting mutation in CHRNE unmasks skipping of the preceding exon. Hum Mol Genet 2003; 12: 3055-66.
    • (2003) Hum Mol Genet , vol.12 , pp. 3055-3066
    • Ohno, K.1    Milone, M.2    Shen, X.-M.3    Engel, A.G.4
  • 29
    • 0027048764 scopus 로고
    • Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in α-bungarotoxin treated rats
    • Plomp JJ, van Kempen GTH, Molenaar PC. Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in α-bungarotoxin treated rats. J Physiol (Lond) 1992; 458: 487-99.
    • (1992) J Physiol (Lond) , vol.458 , pp. 487-499
    • Plomp, J.J.1    Van Kempen, G.T.H.2    Molenaar, P.C.3
  • 31
    • 0021740393 scopus 로고
    • Nicotinic receptor of acetylcholine: Structure of an oligomeric integral membrane protein
    • Popot JL, Changeux J-P. Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein. Physiol Rev 1984; 64: 1162-239.
    • (1984) Physiol Rev , vol.64 , pp. 1162-1239
    • Popot, J.L.1    Changeux, J.-P.2
  • 32
    • 0030061670 scopus 로고    scopus 로고
    • Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events
    • Qin F, Auerbach A, Sachs F. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys J 1996; 70: 264-80.
    • (1996) Biophys J , vol.70 , pp. 264-280
    • Qin, F.1    Auerbach, A.2    Sachs, F.3
  • 33
    • 0032722151 scopus 로고    scopus 로고
    • Mutation causing congenital myasthenia reveals acetylcholine receptor β/δ subunit interaction essential for assembly
    • Quiram P, Ohno K, Milone M, Patterson MC, Pruitt NJ, Brengman JM, et al. Mutation causing congenital myasthenia reveals acetylcholine receptor β/δ subunit interaction essential for assembly. J Clin Invest 1999; 104: 1403-10.
    • (1999) J Clin Invest , vol.104 , pp. 1403-1410
    • Quiram, P.1    Ohno, K.2    Milone, M.3    Patterson, M.C.4    Pruitt, N.J.5    Brengman, J.M.6
  • 34
    • 0033561222 scopus 로고    scopus 로고
    • A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics
    • Salamone FN, Zhou M, Auerbach A. A re-examination of adult mouse nicotinic acetylcholine receptor channel activation kinetics. J Physiol (Lond) 1999; 516: 315-30.
    • (1999) J Physiol (Lond) , vol.516 , pp. 315-330
    • Salamone, F.N.1    Zhou, M.2    Auerbach, A.3
  • 35
    • 0023834181 scopus 로고
    • The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor. Cloning of the alpha-subunit cDNA
    • Schoepfer R, Luther MA, Lindstrom J. The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor. Cloning of the alpha-subunit cDNA. FEBS Lett 1988; 226: 235-40.
    • (1988) FEBS Lett , vol.226 , pp. 235-240
    • Schoepfer, R.1    Luther, M.A.2    Lindstrom, J.3
  • 36
    • 0037168777 scopus 로고    scopus 로고
    • Congenital myasthenic syndrome caused by low-expressor fast-channel AChR δ subunit mutation
    • Shen X-M, Ohno K, Fukudome T, Tsujino A, Brengman JM, De Vivo DC, et al. Congenital myasthenic syndrome caused by low-expressor fast-channel AChR δ subunit mutation. Neurology 2002; 59: 1881-8.
    • (2002) Neurology , vol.59 , pp. 1881-1888
    • Shen, X.-M.1    Ohno, K.2    Fukudome, T.3    Tsujino, A.4    Brengman, J.M.5    De Vivo, D.C.6
  • 37
    • 0037329589 scopus 로고    scopus 로고
    • Mutation causing severe myasthenia reveals functional asymmetry of AChR signature Cys-loops in agonist binding and gating
    • Shen X-M, Ohno K, Tsujino A, Brengman JM, Gingold M, Sine SM, et al. Mutation causing severe myasthenia reveals functional asymmetry of AChR signature Cys-loops in agonist binding and gating. J Clin Invest 2003; 111: 497-505.
    • (2003) J Clin Invest , vol.111 , pp. 497-505
    • Shen, X.-M.1    Ohno, K.2    Tsujino, A.3    Brengman, J.M.4    Gingold, M.5    Sine, S.M.6
  • 38
    • 0033817038 scopus 로고    scopus 로고
    • Immature end-plates and utrophin deficiency in congenital myasthenic syndrome caused by epsilon-AChR subunit truncating mutations
    • Sieb JP, Kraner S, Rauch M, Steinlein OK. Immature end-plates and utrophin deficiency in congenital myasthenic syndrome caused by epsilon-AChR subunit truncating mutations. Hum Genet 2000; 107: 160-4.
    • (2000) Hum Genet , vol.107 , pp. 160-164
    • Sieb, J.P.1    Kraner, S.2    Rauch, M.3    Steinlein, O.K.4
  • 39
    • 0023478292 scopus 로고
    • Data transformation for improved display and fitting of single-channel dwell time histograms
    • Sigworth FJ, Sine SM. Data transformation for improved display and fitting of single-channel dwell time histograms. Biophys J 1987; 52: 1047-54.
    • (1987) Biophys J , vol.52 , pp. 1047-1054
    • Sigworth, F.J.1    Sine, S.M.2
  • 40
    • 0027482551 scopus 로고
    • Molecular dissection of subunit interfaces in the acetylcholine receptor: Identification of residues that determine curare selectivity
    • Sine SM. Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity. Proc Natl Acad Sci USA 1993; 90: 9436-40.
    • (1993) Proc Natl Acad Sci USA , vol.90 , pp. 9436-9440
    • Sine, S.M.1
  • 41
    • 0018389601 scopus 로고
    • Functional consequences of agonist mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells
    • Sine SM, Taylor P. Functional consequences of agonist mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells. J Biol Chem 1979; 254: 3315-25.
    • (1979) J Biol Chem , vol.254 , pp. 3315-3325
    • Sine, S.M.1    Taylor, P.2
  • 42
    • 0027376417 scopus 로고
    • Congenital myasthenic syndromes. II. A syndrome attributed to abnormal interaction of acetylcholine with its receptor
    • Uchitel O, Engel AG, Walls TJ, Nagel A, Atassi ZM, Bril V. Congenital myasthenic syndromes. II. A syndrome attributed to abnormal interaction of acetylcholine with its receptor. Muscle Nerve 1993; 16: 1293-301.
    • (1993) Muscle Nerve , vol.16 , pp. 1293-1301
    • Uchitel, O.1    Engel, A.G.2    Walls, T.J.3    Nagel, A.4    Atassi, Z.M.5    Bril, V.6
  • 43
    • 0033363317 scopus 로고    scopus 로고
    • Acetylcholine receptor M3 domain: Stereochemical and volume contributions to channel gating
    • Wang H-L, Milone M, Ohno K, Shen X-M, Tsujino A, Batocchi AP, et al. Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating. Nat Neurosci 1999; 2: 226-33.
    • (1999) Nat Neurosci , vol.2 , pp. 226-233
    • Wang, H.-L.1    Milone, M.2    Ohno, K.3    Shen, X.-M.4    Tsujino, A.5    Batocchi, A.P.6
  • 44
    • 0033811495 scopus 로고    scopus 로고
    • Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome
    • Wang H-L, Ohno K, Milone M, Brengman JM, Evoli A, Batocchi AP, et al. Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome. J Gen Physiol 2000; 116: 449-60.
    • (2000) J Gen Physiol , vol.116 , pp. 449-460
    • Wang, H.-L.1    Ohno, K.2    Milone, M.3    Brengman, J.M.4    Evoli, A.5    Batocchi, A.P.6
  • 45
    • 0028118431 scopus 로고
    • The role of the cytoplasmic domains of individual subunits of the acetylcholine receptor in 43 kDa protein-induced clustering in COS cells
    • Yu X-M, Hall ZW. The role of the cytoplasmic domains of individual subunits of the acetylcholine receptor in 43 kDa protein-induced clustering in COS cells. J Neurosci 1994; 14: 785-95.
    • (1994) J Neurosci , vol.14 , pp. 785-795
    • Yu, X.-M.1    Hall, Z.W.2


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