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

Electrochemical noise analysis to probe ion transport mechanisms in a membrane channel Maria Queralt-Martin , M. Lidón López and Antonio Alcaraz Laboratory of Molecular Biophysics, Department of Physics Universitat Jaume I, Castellón, Spain July 13, 2015 1

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

The OmpF channel • Protein that forms trimeric channels • Located at the outer membrane of E. coli 1. Introduction • Wide pores with multiionic transport OmpF Narrow channels 3

Experimental technique Planar Bilayer Electrophysiology Bilayer formed by apposition of monolayers Measurement of 1. Introduction single-channel current V (mV) → I (pA) Detailed study of the transport properties of protein channels 4

Noise analysis in OmpF channel 2. Noise analysis in OmpF channel Study of current fluctuations Sampling rate: 50 kHz Low-pass Bessel filter at 10 kHz Inset: 500 Hz Bessel filter 5

Noise analysis in OmpF channel 2. Noise analysis in OmpF channel Previous studies of current fluctuations in OmpF Lorentzian curve Two-state Markov process Simple model for first-order protonation reactions Problem Current steps between substates ( D i ) are not constant Nestorovich et al. Biophys. J . 2003 Bezrukov et al. Phys. Rev. Lett . 1993 6

Noise analysis in OmpF channel 2. Noise analysis in OmpF channel Alternative approach for the study of current fluctuations in OmpF Conductance fluctuations S G : The PSDs of the voltage-dependent noise sources scale as V 2 For an Ohmic system (G=I/V) The electrical current through 𝐓 𝐇 S I = S G · V 2 = the channel reveals fluctuations · I 2 𝐇 𝟑 in conductance Parabolic coefficient 7

Noise analysis in OmpF channel 2. Noise analysis in OmpF channel Alternative approach for the study of current fluctuations in OmpF Parabolic coefficient S G /G 2 Conductance We evaluate conductance fluctuations S G : fluctuations without knowing the S G · G 2 exact mechanism behind them S G = G 2 8

Exploring different conditions 2. Noise analysis in OmpF channel Different electrolytes: KCl, LiCl, MgCl2, CaCl2 Different concentrations: 10 mM – 6 M Mg 2+ ion bound 1 M MgCl 2 Yamashita et al. EMBO J. 2008 Noise analysis as a complement of previous studies with conductance and selectivity 9

Exploring different conditions Different electrolytes: KCl, LiCl, MgCl2, CaCl2 Different concentrations: 10 mM – 6 M 3. Results obtained Conductance Parabolic coefficient a -1/4 a -3/2 10 1 10 -5 a 3/2 a 3/4 S G / G 2 (Hz -1 ) 10 -6 G (nS) 10 0 10 -7 KCl KCl KCl 10 -8 10 -1 LiCl LiCl LiCl 10 -5 a -1/4 a -3/2 10 1 S G / G 2 (Hz -1 ) a 3/4 10 -6 G (nS) 10 0 a 3/2 10 -7 MgCl2 MgCl 2 MgCl 2 10 -1 CaCl2 CaCl 2 CaCl 2 10 -8 10 1 10 2 10 3 10 4 10 5 10 1 10 2 10 3 10 4 Activity (mM) Activity (mM) 10

Exploring different conditions Similar pattern for all Two characteristic & salts under study transport regimes 3. Results obtained Conductance Parabolic coefficient a -1/4 a -3/2 10 1 10 -5 a 3/2 a 3/4 S G / G 2 (Hz -1 ) 10 -6 G (nS) 10 0 10 -7 KCl KCl KCl 10 -8 10 -1 LiCl LiCl LiCl 10 -5 a -1/4 a -3/2 10 1 S G / G 2 (Hz -1 ) a 3/4 10 -6 G (nS) 10 0 a 3/2 10 -7 MgCl2 MgCl 2 MgCl 2 10 -1 CaCl2 CaCl 2 CaCl 2 10 -8 10 1 10 2 10 3 10 4 10 5 10 1 10 2 10 3 10 4 Activity (mM) Activity (mM) 11

Exploring different conditions Similar pattern for all Two characteristic & salts under study transport regimes 3. Results obtained Theoretical model Conductance Channel fixed charges control ion transport 10 1 G / G min Donnan Equilibrium Equations KCl 10 0 LiCl Model 10 1 G / G min MgCl 2 MgCl2 10 0 CaCl 2 CaCl2 Model Existence of a binding site for cations 10 1 10 2 10 3 10 4 10 5 Langmuir Adsorption Isotherm Activity (mM) 12

Exploring different conditions Similar pattern for all Two characteristic & salts under study transport regimes 3. Results obtained Parabolic coefficient Conductance a -3/2 10 -5 fluctuations S G : a 3/2 S G / G 2 (Hz -1 ) 10 -6 S G 10 -7 · G 2 S G = KCl KCl G 2 10 -8 LiCl LiCl 10 -5 a -3/2 S G / G 2 (Hz -1 ) 10 -6 Parabolic coefficient a 3/2 10 -7 MgCl 2 CaCl 2 10 -8 Conductance 10 1 10 2 10 3 10 4 Activity (mM) 13

Exploring different conditions Similar pattern for all Two characteristic & salts under study transport regimes 3. Results obtained Low activity regime Conductance fluctuations S g ~ a 0 a 0 10 -5 S G (nS 2 /Hz) 10 -6 Controlled by channel 10 -7 fixed charges 10 -8 a 1 KCl 10 -9 LiCl High activity regime a 0 10 -5 S g ~ a 1 S G (nS 2 /Hz) 10 -6 10 -7 Binding of cations 10 -8 a 1 increases the fluctuating MgCl 2 10 -9 CaCl 2 particles at the pore walls 10 1 10 2 10 3 10 4 Activity (mM) 14

Summarizing… 4. Conclusions and open questions Use of a complimentary Extract information from the approach for the analysis PSDs without knowing much of current fluctuations about the sources of noise Overall view of the transport mechanisms of the channel Functional evidence of the existence of 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 15

Acknowledgements David P. Hoogerheide, PhD Joint Postdoctoral Fellow Acknowledgements Sergey M. Bezrukov , PhD Section on Molecular Transport Laboratory of Molecular Biophysics Program in Physical Biology Department of Physics Antonio Alcaraz, PhD M. Lidón López, PhD 16

Acknowledgements David P. Hoogerheide, PhD Joint Postdoctoral Fellow Acknowledgements Thanks for your attention! Sergey M. Bezrukov , PhD Section on Molecular Transport Laboratory of Molecular Biophysics Program in Physical Biology Department of Physics Antonio Alcaraz, PhD M. Lidón López, PhD 17