A mutation in the C. elegans EXP-2 potassium channel that alters feeding behavior

Science

Volumn 286, Issue 5449, 1999, Pages 2501-2504

  Davis, M Wayne ^a,b   Fleischhauer, Richard ^a   Dent, Joseph A ^a,c   Joho, Rolf H ^a   Avery, Leon ^a  

^a UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER   (United States)

^b UNIVERSITY OF UTAH   (United States)

^c MCGILL UNIVERSITY   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

POTASSIUM CHANNEL;

ACTION POTENTIAL; ARTICLE; CAENORHABDITIS ELEGANS; ELECTROPHYSIOLOGY; FEEDING BEHAVIOR; GENE MUTATION; NONHUMAN; POTASSIUM CURRENT; PRIORITY JOURNAL;

ACTION POTENTIALS; ANIMALS; CAENORHABDITIS ELEGANS; FEEDING BEHAVIOR; GENES, HELMINTH; GENES, REPORTER; ION CHANNEL GATING; KINETICS; MEMBRANE POTENTIALS; MODELS, MOLECULAR; MUSCLES; MUTATION; NEURONS; OOCYTES; PHARYNGEAL MUSCLES; POTASSIUM CHANNELS; PROTEIN CONFORMATION; RECOMBINANT FUSION PROTEINS; RNA, COMPLEMENTARY; XENOPUS LAEVIS;

CAENORHABDITIS ELEGANS; INVERTEBRATA;

EID: 0033601287     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5449.2501     Document Type: Article

References (28)

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3. J. H. Thomas, Genetics 124, 855 (1990).
4. Worms were cultured and handled as described by J. Sulston and J. Hodgkin [in The Nematode Caenorhabditis elegans, W. B. Wood, Ed. (Cold Spring Harbor Laboratory Press, Plainview, NY, 1988), pp. 587-606] with minor modifications as described [L. Avery, Genetics 133, 897 (1993)]. Revertant alleles of exp-2(sa26) were isolated after ethyl methane sulfonate mutagenesis by looking for animals that no longer displayed exp-2(sa26) gain-of-function head movement or pharyngeal pumping phenotypes. We isolated exp-2(ad1201) and exp-2(ad1426) using DA1200 and DA1315 exp2(sa26sd)/unc-46(e177) sDf44 dpy-11(e224) V as the starting strain. All other revertant alleles were isolated using DA1090 exp-2(sa26sd) dpy-11 (e224)/unc-46(e177) sDf30 V; mnDp26 as the starting strain.
5. Worms were cultured and handled as described by J. Sulston and J. Hodgkin [in The Nematode Caenorhabditis elegans, W. B. Wood, Ed. (Cold Spring Harbor Laboratory Press, Plainview, NY, 1988), pp. 587-606] with minor modifications as described [L. Avery, Genetics 133, 897 (1993)]. Revertant alleles of exp-2(sa26) were isolated after ethyl methane sulfonate mutagenesis by looking for animals that no longer displayed exp-2(sa26) gain-of-function head movement or pharyngeal pumping phenotypes. We isolated exp-2(ad1201) and exp-2(ad1426) using DA1200 and DA1315 exp2(sa26sd)/unc-46(e177) sDf44 dpy-11(e224) V as the starting strain. All other revertant alleles were isolated using DA1090 exp-2(sa26sd) dpy-11 (e224)/unc-46(e177) sDf30 V; mnDp26 as the starting strain.
6. J. Thomas, personal communication.
7. M. W. Davis, J. A. Dent, L. Avery, data not shown.
8. Pharyngeal muscle voltage recordings were done and data analyzed as described (2). Pharynxes were dissected into Dent's saline. Cells were penetrated with 0.5 M or 3 M KOAc-filled electrodes (74 to 340 megohms) and potentials recorded with an Axoclamp amplifier. We analyzed 100-s sections of each record for action potential parameters and resting potential. The gain-of-function records were made from DA1090 animals, and loss-of-function records from DA1201, DA1426, and DA1482 unc-13(e51) eat-11(ad541) I; exp2(sa26sd ad1426) V animals. Average parameter values were (mean ± SEM): wild-type (n = 7) peak = +34 ± 3 mV (2), overshoot = -67 ± 2 mV (2), interpump potential = -45 ± 1 mV (2); gain-of-function heterozygote (n = 5) peak = +25 ± 4 mV, duration = 72 ± 10 ms, interpump potential -71 ± 3 mV; loss-of-function (n = 4) peak = +34 ± 4 mV, duration = 3.8 ± 1.3 s, interpump potential -40 ± 3 mV.
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11. The exp-2 loss-of-function worms were transformed [C. C. Mello, J. M. Kramer, D. Stinchcomb, V. Ambros, EMBO J. 10, 3959 (1991)] with a PCR product that began 3.4 kb 5′ to the initiating methionine and ended 854 bases 3′ to the stop codon of exp-2 (primers: 5′-CCGGATCCGAAAAATGATACGAGCAT-3′ and 5′-CTGCGGCCGGGCCCTTCAATGCCAGAATGCT-3′) using rol-6(d) as the co-transformation marker (2). Rescue of the exp-2 action potential defect was confirmed by electropharyngeogram [D. M. Raizen and L. Avery, Neuron 12, 483 (1994)], and rescue of the pharyngeal motion defect was confirmed by direct observation.
12. The exp-2 loss-of-function worms were transformed [C. C. Mello, J. M. Kramer, D. Stinchcomb, V. Ambros, EMBO J. 10, 3959 (1991)] with a PCR product that began 3.4 kb 5′ to the initiating methionine and ended 854 bases 3′ to the stop codon of exp-2 (primers: 5′-CCGGATCCGAAAAATGATACGAGCAT-3′ and 5′-CTGCGGCCGGGCCCTTCAATGCCAGAATGCT-3′) using rol-6(d) as the co-transformation marker (2). Rescue of the exp-2 action potential defect was confirmed by electropharyngeogram [D. M. Raizen and L. Avery, Neuron 12, 483 (1994)], and rescue of the pharyngeal motion defect was confirmed by direct observation.
13. PCR products that included the entire exp-2 coding region were generated using a single-worm PCR protocol [B. D. Williams, in Caenorhabditis elegans: Modern Biological Analysis of an Organism, H. F. Epstein and D. C. Shakes, Eds. (Academic Press, San Diego, CA, 1995), vol. 48, pp. 81-96] and were used as sequencing templates. The products from at least three independent PCR reactions were mixed before sequencing in order to limit the possibility of detecting PCR-induced mutations. Automated sequencing was done using ABI Prism Big-Dye Terminator cycle sequencing-ready reaction kits and an ABI Prism 377 machine.
14. A supplementary figure, which shows an alignment of the exp-2 sequence with Drosophila Kv channels, can be found at the Science Web site (www.sciencemag. org/feature/data/1044897.shl).
15. + selectivity filter on the correct side of the membrane, this mutation is a likely molecular null.
16. M. W. Davis, data not shown.
17. S. Owens, J. Pierce, S. Lockery, personal communication.
18. 2, 5 mM HEPES (pH 7.5) at 22°C ± 1°C].
19. 2, 5 mM HEPES (pH 7.5) at 22°C ± 1°C].
20. 2, 5 mM HEPES (pH 7.5) at 22°C ± 1°C].
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26. + channels near and in the pore region. The program Swiss-PDB Viewer was used to align the primary sequences and to prepare the files necessary for processing by the Swiss-MODEL server [N. Guex and M. C. Peitsch, Electrophoresis 18, 2714 (1997)] (www. expasy.ch/swissmod/SWISS-MODEL.html). The Swiss-MODEL server is an automated system that generates an alignment between the sequence of a known structure and the sequence to be modeled, generates a model structure for the new sequence based on the experimentally determined structure, and refines the model sequence using energy minimization and molecular dynamics. Swiss-PDB Viewer was used to generate the Cys-to-Tyr mutant structure, by replacement of the side chain and selection of the lowest energy rotamer. WebLab ViewerLite (www.msi.com/weblab/index.html) was used to manipulate the resulting models and prepare images for presentation.
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28. We would like to thank D. Hilgemann, R. Lin, and D. Smith for critical comments on the manuscript, A. Chiang for assistance in screening for exp-2(sa26) revertants, and A. Fire, J. Ahnn, G. Seydoux, and S. Xu for the gift of pPD95.77. Some strains were received from the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. J. Thomas kindly provided us with the exp-2(sa26) allele. Supported by NIH grants HL46154 to L.A. and NS28407 to R.H.J.