{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,4]],"date-time":"2026-10-04T09:53:00Z","timestamp":1791107580295,"version":"4.1.0"},"reference-count":21,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5360","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1998,4,3]]},"abstract":"<jats:p>\n            Toxins from scorpion venom interact with potassium channels. Resin-attached, mutant K\n            <jats:sup>+<\/jats:sup>\n            channels from\n            <jats:italic>Streptomyces lividans<\/jats:italic>\n            were used to screen venom from\n            <jats:italic>Leiurus quinquestriatus hebraeus<\/jats:italic>\n            , and the toxins that interacted with the channel were rapidly identified by mass spectrometry. One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding. The results show that a prokaryotic K\n            <jats:sup>+<\/jats:sup>\n            channel has the same pore structure as eukaryotic K\n            <jats:sup>+<\/jats:sup>\n            channels. This structural conservation, through application of techniques presented here, offers a new approach for K\n            <jats:sup>+<\/jats:sup>\n            channel pharmacology.\n          <\/jats:p>","DOI":"10.1126\/science.280.5360.106","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T09:42:47Z","timestamp":1027762967000},"page":"106-109","source":"Crossref","is-referenced-by-count":362,"title":["Structural Conservation in Prokaryotic and Eukaryotic Potassium Channels"],"prefix":"10.1126","volume":"280","author":[{"given":"Roderick","family":"MacKinnon","sequence":"first","affiliation":[{"name":"R. MacKinnon and A. Kuo, Laboratory of Molecular Neurobiology and Biophysics and the Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"S. L. Cohen and B. T. Chait, Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"A. Lee, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven L.","family":"Cohen","sequence":"additional","affiliation":[{"name":"R. MacKinnon and A. Kuo, Laboratory of Molecular Neurobiology and Biophysics and the Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"S. L. Cohen and B. T. Chait, Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"A. Lee, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anling","family":"Kuo","sequence":"additional","affiliation":[{"name":"R. MacKinnon and A. Kuo, Laboratory of Molecular Neurobiology and Biophysics and the Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"S. L. Cohen and B. T. Chait, Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"A. Lee, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alice","family":"Lee","sequence":"additional","affiliation":[{"name":"R. MacKinnon and A. Kuo, Laboratory of Molecular Neurobiology and Biophysics and the Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"S. L. Cohen and B. T. Chait, Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"A. Lee, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian T.","family":"Chait","sequence":"additional","affiliation":[{"name":"R. MacKinnon and A. Kuo, Laboratory of Molecular Neurobiology and Biophysics and the Howard Hughes Medical Institute, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"S. L. Cohen and B. T. Chait, Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."},{"name":"A. Lee, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/0896-6273(95)90057-8","volume":"15","author":"Miller C.","year":"1995","unstructured":"M. L. Garcia, et al., J. Bioenerg. Biomembr. 23, 615 (1991); Miller C., Neuron 15, 5 (1995).","journal-title":"Neuron"},{"key":"e_1_3_1_3_2","doi-asserted-by":"crossref","first-page":"6719","DOI":"10.1021\/bi00144a011","volume":"31","author":"Giangiacomo K.M.","year":"1992","unstructured":"R. MacKinnon, C. Miller, J. Gen. Physiol. 91, 335 (1988); Giangiacomo K.M., Garcia M. L., McManus O. B., Biochemistry 31, 6719 (1992).","journal-title":"Biochemistry"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/0896-6273(92)90169-E","volume":"9","author":"Park C. S.","year":"1992","unstructured":"Park C. S., Miller C., Neuron9, 307 (1992).","journal-title":"Neuron"},{"key":"e_1_3_1_5_2","first-page":"131","volume":"16","author":"Ranganathan R.","year":"1996","unstructured":"Ranganathan R., Lewis J. H., MacKinnon R., ibid16, 131 (1996).","journal-title":"ibid"},{"key":"e_1_3_1_6_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.280.5360.69"},{"key":"e_1_3_1_7_2","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/S0896-6273(00)80029-X","volume":"16","author":"Naranjo D.","year":"1996","unstructured":"R. MacKinnon, C. Miller, ibid 245, 1382 (1989); R. MacKinnon, L. Heginbotham, T. Abramson, Neuron 5, 767 (1990); M. Stocker, C. Miller, Proc. Natl. Acad. Sci. U.S.A. 91, 9509 (1994); Naranjo D., Miller C., Neuron 16, 123 (1996).","journal-title":"Neuron"},{"key":"e_1_3_1_8_2","doi-asserted-by":"crossref","first-page":"1377","DOI":"10.1016\/0896-6273(94)90452-9","volume":"12","author":"Goldstein S.","year":"1994","unstructured":"Goldstein S., Pheasant D. J., Miller C., Neuron12, 1377 (1994).","journal-title":"Neuron"},{"key":"e_1_3_1_9_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1126\/science.7716527","volume":"268","author":"Hidalgo P.","year":"1995","unstructured":"Hidalgo P., MacKinnon R., Science268, 307 (1995).","journal-title":"Science"},{"key":"e_1_3_1_10_2","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1016\/S0896-6273(00)80057-4","volume":"16","author":"Gross A.","year":"1996","unstructured":"Gross A., MacKinnon R., Neuron16, 399 (1996).","journal-title":"Neuron"},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"6834","DOI":"10.1021\/bi00188a012","volume":"33","author":"Garcia M. L.","year":"1994","unstructured":"Garcia M. L., et al., Biochemistry33, 6834 (1994).","journal-title":"Biochemistry"},{"key":"e_1_3_1_12_2","unstructured":"Three mutations (Q58A T61S and R64D) were introduced into the KcsA K + channel gene to modify its pore region sequence by PCR mutagenesis and confirmed by DNA sequencing. The gene also contained a mutation at the second residue (P2A) to introduce an Nco I restriction endonuclease site and it was lacking the last two carboxyl-terminal residues. This gene was cloned into the pQE60 vector for expression with a carboxyl terminal thrombin and hexahistidine fusion. Channel protein was expressed in XL-1 Blue strain of E. coli (Stratagene) by induction with 1-\u03b2-D-thiogalactopyranoside at a concentration of 1.0 mM. Three hours after induction bacteria were sonicated in 50 mM tris buffer (pH 7.5) 100 mM KCl 10 mM Mg 2 SO 4 25 mg deoxyribonuclease 1 and 250 mM sucrose plus pepstatin leupeptin and PMSF. The channel was extracted in the same solution containing 40 mM decylmaltoside (Anatrace) at room temperature. Following centrifugation the supernatant was bound to cobalt resin (Talon) at a protein to resin ratio that will saturate the resin. The resin was washed and detergent concentration was lowered to 10.0 mM. One-milliliter columns were prepared. The control resin (no channel) was handled in the same manner. The resin preparation was the same for mass spectrometry and binding studies."},{"key":"e_1_3_1_13_2","unstructured":"Forty milligrams of L. quinquestriatus hebraeus venom (Alomone Labs) was suspended in buffer identical to that of the channel (10.0 mM declymaltoside) and applied to the column. After washing channel was eluted with 1.0 M imidazole in the same buffer."},{"key":"e_1_3_1_14_2","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1021\/bi00001a003","volume":"34","author":"Lippens G.","year":"1995","unstructured":"J. A. Debin, J. E. Maggio, G. R. Strichartz,Am. J. Physiol. Soc. 264, C369 (1993); Lippens G., Najib J., Wodak S. J., Tartar A., Biochemistry 34, 13 (1995).","journal-title":"Biochemistry"},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0896-6273(00)80143-9"},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1038\/350232a0","volume":"350","author":"MacKinnon R.","year":"1991","unstructured":"MacKinnon R., Nature350, 232 (1991).","journal-title":"Nature"},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1021\/ac9507956","volume":"68","author":"Cohen S. L.","year":"1996","unstructured":"Cohen S. L., Chait B. T., Anal. Chem.68, 31 (1996).","journal-title":"Anal. Chem."},{"key":"e_1_3_1_18_2","unstructured":"Wild-type and mutant agitoxin2 were prepared (10). Tritiated N -ethylmaleimide (NEN Life Sciences) was conjugated to agitoxin2 D20C (14). Binding was performed in a 300-\u03bcl volume containing 50 mM tris 100 mM KCl 10 mM declymaltoside and 0.3 \u03bcl of cobalt resin saturated with the mutant KcsA K + channel for 30 min at room temperature. After brief centrifugation the supernatant was removed. Resin was applied to a filter and rinsed briefly with ice-cold buffer; radioactivity was then determined in a scintillation counter. All binding measurements were made with a paired control containing a saturating concentration (200 times K D ) of unlabeled wild-type agitoxin2 to determine nonspecific binding. The competition assay was carried out under the same conditions. Labeled agitoxin2 at 0.06 \u03bcM was always present and unlabeled toxin was added to compete with bound labeled toxin."},{"key":"e_1_3_1_19_2","doi-asserted-by":"crossref","unstructured":"A. M. Krezel et al. Prot. Sci. 4 1478 (1995).","DOI":"10.1002\/pro.5560040805"},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1002\/prot.340110407"},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1107\/S0108767390010224"},{"key":"e_1_3_1_22_2","unstructured":"We thank J. Gulbis J. P. Imredy and D. A. Doyle for assistance in preparation of the figures and helpful discussions on the work. Supported by NIH GM43949. R.M. is an investigator in the Howard Hughes Medical Institute."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.280.5360.106","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T04:31:39Z","timestamp":1705120299000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.280.5360.106"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1998,4,3]]},"references-count":21,"journal-issue":{"issue":"5360","published-print":{"date-parts":[[1998,4,3]]}},"alternative-id":["10.1126\/science.280.5360.106"],"URL":"https:\/\/doi.org\/10.1126\/science.280.5360.106","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1998,4,3]]}}}