Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli

Paul Blount, Sergei I. Sukharev, Matthew J. Schroeder, Scott K. Nagle, Ching Kung

Research output: Contribution to journalArticle

169 Citations (Scopus)

Abstract

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

Original languageEnglish (US)
Pages (from-to)11652-11657
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number21
DOIs
StatePublished - Oct 15 1996

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Escherichia coli
Proteins
Amino Acids
Mechanical Stress
Glutamine
Mutagenesis
Lysine
Cell Membrane
Genes

ASJC Scopus subject areas

  • Genetics
  • General

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Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. / Blount, Paul; Sukharev, Sergei I.; Schroeder, Matthew J.; Nagle, Scott K.; Kung, Ching.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 93, No. 21, 15.10.1996, p. 11652-11657.

Research output: Contribution to journalArticle

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AU - Nagle, Scott K.

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