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probe ion transport mechanisms in a membrane channel Maria - - PowerPoint PPT Presentation

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Electrochemical noise analysis to probe ion transport mechanisms in a membrane channel Maria Queralt-Martin , M. Lidn Lpez and Antonio Alcaraz Laboratory of Molecular Biophysics, Department of Physics Universitat Jaume I, Castelln, Spain

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Maria Queralt-Martin, M. Lidón López and Antonio Alcaraz

Laboratory of Molecular Biophysics, Department of Physics Universitat Jaume I, Castellón, Spain

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Electrochemical noise analysis to probe ion transport mechanisms in a membrane channel

July 13, 2015

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Outline

1.Introduction 2.Noise analysis in the bacterial channel OmpF 3.Results obtained 4.Conclusions and open questions

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  • 1. Introduction

The OmpF channel

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  • Protein that forms trimeric channels
  • Located at the outer membrane of E. coli
  • Wide pores with multiionic transport

OmpF Narrow channels

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  • 1. Introduction

Experimental technique

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Planar Bilayer Electrophysiology Bilayer formed by apposition of monolayers

→ V (mV) I (pA)

Measurement of single-channel current

Detailed study of the transport properties of protein channels

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Study of current fluctuations

  • 2. Noise analysis in OmpF channel

Noise analysis in OmpF channel

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Sampling rate: 50 kHz Low-pass Bessel filter at 10 kHz Inset: 500 Hz Bessel filter

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  • 2. Noise analysis in OmpF channel

Noise analysis in OmpF channel

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Previous studies of current fluctuations in OmpF

Nestorovich et al. Biophys. J. 2003 Bezrukov et al. Phys. Rev. Lett. 1993

Lorentzian curve Two-state Markov process Simple model for first-order protonation reactions Problem Current steps between substates (Di) are not constant

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  • 2. Noise analysis in OmpF channel

Noise analysis in OmpF channel

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Alternative approach for the study of current fluctuations in OmpF Conductance fluctuations SG: The PSDs of the voltage-dependent noise sources scale as V2 For an Ohmic system (G=I/V)

The electrical current through the channel reveals fluctuations in conductance

Parabolic coefficient SI = SG · V2 = 𝐓𝐇 𝐇𝟑 · I2

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Parabolic coefficient SG/G2

Conductance fluctuations SG: SG = SG G2 · G2

We evaluate conductance fluctuations without knowing the exact mechanism behind them

  • 2. Noise analysis in OmpF channel

Noise analysis in OmpF channel

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Alternative approach for the study of current fluctuations in OmpF

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  • 2. Noise analysis in OmpF channel

Different electrolytes: KCl, LiCl, MgCl2, CaCl2 Different concentrations: 10 mM – 6 M

Exploring different conditions

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1 M MgCl2

Yamashita et al. EMBO J. 2008

Noise analysis as a complement of previous studies with conductance and selectivity

Mg2+ ion bound

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Activity (mM) 101 102 103 104 SG / G2 (Hz-1) 10-8 10-7 10-6 10-5 SG / G2 (Hz-1) 10-8 10-7 10-6 10-5 KCl LiCl KCl LiCl a-3/2 a3/2 MgCl2 CaCl2 a3/2 a-3/2 Activity (mM) 101 102 103 104 105 G (nS) 10-1 100 101 MgCl2 CaCl2 a3/4 a-1/4 MgCl2 CaCl2 G (nS) 10-1 100 101 KCl LiCl a3/4 a-1/4

Different electrolytes: KCl, LiCl, MgCl2, CaCl2 Different concentrations: 10 mM – 6 M

  • 3. Results obtained

Exploring different conditions

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Conductance Parabolic coefficient

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Similar pattern for all salts under study

Activity (mM) 101 102 103 104 SG / G2 (Hz-1) 10-8 10-7 10-6 10-5 SG / G2 (Hz-1) 10-8 10-7 10-6 10-5 KCl LiCl KCl LiCl a-3/2 a3/2 MgCl2 CaCl2 a3/2 a-3/2 Activity (mM) 101 102 103 104 105 G (nS) 10-1 100 101 MgCl2 CaCl2 a3/4 a-1/4 MgCl2 CaCl2 G (nS) 10-1 100 101 KCl LiCl a3/4 a-1/4

  • 3. Results obtained

Exploring different conditions

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Conductance Parabolic coefficient

Two characteristic transport regimes &

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Existence of a binding site for cations

Langmuir Adsorption Isotherm

Activity (mM) 101 102 103 104 105 G / Gmin 100 101 MgCl2 CaCl2 Model G / Gmin 100 101 KCl LiCl Model MgCl2 CaCl2

  • 3. Results obtained

Exploring different conditions

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Conductance Channel fixed charges control ion transport

Donnan Equilibrium Equations

Theoretical model Similar pattern for all salts under study Two characteristic transport regimes &

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Activity (mM) 101 102 103 104 SG / G2 (Hz-1) 10-8 10-7 10-6 10-5 SG / G2 (Hz-1) 10-8 10-7 10-6 10-5 KCl LiCl KCl LiCl a-3/2 a3/2 MgCl2 CaCl2 a3/2 a-3/2

  • 3. Results obtained

Exploring different conditions

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Parabolic coefficient

Conductance fluctuations SG:

SG = SG G2 · G2

Similar pattern for all salts under study Two characteristic transport regimes &

Parabolic coefficient Conductance

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Activity (mM) 101 102 103 104 SG (nS2/Hz) 10-9 10-8 10-7 10-6 10-5 SG (nS2/Hz) 10-9 10-8 10-7 10-6 10-5 KCl LiCl a0 a1 MgCl2 CaCl2 a1 a0

  • 3. Results obtained

Exploring different conditions

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Conductance fluctuations

Sg ~ a0 Low activity regime Controlled by channel fixed charges Sg ~ a1 High activity regime Binding of cations increases the fluctuating particles at the pore walls

Similar pattern for all salts under study Two characteristic transport regimes &

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  • 4. Conclusions and open questions

Summarizing…

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Use of a complimentary approach for the analysis

  • f current fluctuations

Extract information from the PSDs without knowing much about the sources of noise Overall view of the transport mechanisms of the channel

Functional evidence of the existence

  • f a binding site for cations

But…

  • We only take into account a

part of the spectrum

  • We do not extract any

characteristic time from the PSDs

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Acknowledgements

Acknowledgements

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Laboratory of Molecular Biophysics Department of Physics

Antonio Alcaraz, PhD

  • M. Lidón López, PhD

David P. Hoogerheide, PhD

Joint Postdoctoral Fellow

Sergey M. Bezrukov , PhD

Section on Molecular Transport Program in Physical Biology

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Acknowledgements

Acknowledgements

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Laboratory of Molecular Biophysics Department of Physics

Antonio Alcaraz, PhD

  • M. Lidón López, PhD

David P. Hoogerheide, PhD

Joint Postdoctoral Fellow

Sergey M. Bezrukov , PhD

Section on Molecular Transport Program in Physical Biology

Thanks for your attention!