Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment

Min Cheung, Myles Akabas

Research output: Contribution to journalArticle

94 Citations (Scopus)

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both α-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 Å to allow the MTS reagents to reach these residues.

Original languageEnglish (US)
Pages (from-to)2688-2695
Number of pages8
JournalBiophysical Journal
Volume70
Issue number6
StatePublished - Jun 1996
Externally publishedYes

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Cystic Fibrosis Transmembrane Conductance Regulator
Cysteine
Membranes
Chloride Channels
Xenopus
Oocytes
Membrane Proteins
Adenosine Triphosphate
Phosphorylation
Ions
Water
methanethiosulfonate

ASJC Scopus subject areas

  • Biophysics

Cite this

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title = "Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment",
abstract = "The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both α-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 {\AA} to allow the MTS reagents to reach these residues.",
author = "Min Cheung and Myles Akabas",
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T1 - Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment

AU - Cheung, Min

AU - Akabas, Myles

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N2 - The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both α-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 Å to allow the MTS reagents to reach these residues.

AB - The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both α-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 Å to allow the MTS reagents to reach these residues.

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