European Biophysics Journal

, Volume 34, Issue 5, pp 403–412

The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity

  • I-Jung Tsai
  • Zhen-Wei Liu
  • John Rayment
  • Christel Norman
  • Allan McKinley
  • Boris Martinac
Article

Abstract

The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively (Maurer and Dougherty J. Biol. Chem. 278(23):21076–21082, 2003). We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (I24C-SL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin–spin interaction with the neighboring I24C-SL residues in the MscL channel pentamer (Perozo et al.Nature 418:942–948, 2002). The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.

Keywords

Mechanosensitive channel Electron paramagnetic resonance Lysophosphatidylcholine Patch-clamp Osmoregulation 

Abbreviations

ANOVA

Analysis of variance

D/R

Dehydration/rehydration

EPR

Electron paramagnetic resonance

GOF

Gain of function

GST

Glutathione S-transferase

Hepes

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

ID

Inner diameter

IPTG

Isopropyl-β-D-thiogalactoside

LB

Luria–Bertani

LOF

Loss of function

LPC

Lysophosphatidylcholine

MS

Mechanosensitive

MscK

Potassium-regulated mechanosensitive channel

MscL

Mechanosensitive channel of large conductance

MscS

Mechanosensitive channel of small conductance

OD

Outer diameter

PBS

Phosphate-buffered saline

SD

Standard deviation

SDSL

Site-directed spin labeling

TM1

First transmembrane domain

TM2

Second transmembrane domain

TPX

Specially designed polymethylpentene

WT

Wild type

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Copyright information

© EBSA 2005

Authors and Affiliations

  • I-Jung Tsai
    • 1
    • 2
  • Zhen-Wei Liu
    • 1
    • 3
  • John Rayment
    • 1
    • 2
  • Christel Norman
    • 1
  • Allan McKinley
    • 2
  • Boris Martinac
    • 1
    • 3
  1. 1.School of Medicine and PharmacologyUniversity of Western AustraliaNedlandsAustralia
  2. 2.School of Biomedical and Chemical SciencesUniversity of Western AustralianCrawleyAustralia
  3. 3.School of Biomedical SciencesUniversity of QueenslandBrisbaneAustralia

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