Pumps, Channels and Transporters: Methods of Functional Analysis

Describes experimental methods for investigating the function of pumps, channels and transporters

Ion–transporting membrane proteins, which include ion pumps, channels, and transporters, play crucial roles in all cellular life forms. Not only do they provide pathways linking the extracellular medium with the cytoplasm and the cytoplasm with the contents of intracellular organelles, they are also intimately involved in energy transduction in all cells. 

Pumps, Channels and Transporters: Methods of Functional Analysis covers the analytical techniques used for studying the function of ion transporting membrane proteins. The emphasis of this book is, therefore, on experimental methods for resolving the kinetics and dynamics of pumps, channels, and transporters. Structural methods, such as x–ray crystallography or electronmicroscopy, although clearly important for a complete understanding of membrane protein function down to the atomic level, are specifically excluded.

The experimental methods treated in the book are divided into three main groups: electrical (Chapters 2–6), spectroscopic (Chapters 7–12), and radioactivity–based and atomic absorption–based flux assays (Chapters 13 and 14). Finally, the book concludes with a chapter on computational techniques (Chapter 15).

Pumps, Channels and Transporters features:
A wide range of electrophysiological techniques and spectroscopic methods used to analyze the function of ion channels, ion pumps and transporters New and emerging analytical methods used to study ion transport membrane proteins such as single–molecule spectroscopy State–of–the art analytical methods to study ion pumps, channels and transporters

Readers interested in the dynamic aspects of membrane protein function should find the book interesting and of value for their own research.

Ronald J. Clarke, Ph.D. is an Associate Professor in the School of Chemistry, University of Sydney, Australia. In 2010 he was awarded the McAulay–Hope Prize for Original Biophysics by the Australian Society for Biophysics.

Mohammed A. A. Khalid, Ph.D. is an Associate Professor in the Department of Chemistry, College of Applied Medical and Sciences at Taif University, Turabah, Saudi Arabia.

Preface

List of Contributors

Acknowledgements

Chapter 1: Introduction
Mohammed A. A. Khalid and Ronald J. Clarke

1.1 History

1.2 Energetics of transport

1.3 Mechanistic considerations

1.4 Ion channels

1.4.1 Voltage–gated

1.4.2 Ligand–gated

1.4.3 Mechanosensitive

1.4.4 Light–gated

1.5 Ion pumps

1.5.1 ATP–activated

1.5.2 Light–activated

1.5.3 Redox–linked

1.6 Transporters

1.6.1 Symporters and antiporters

1.6.2 Na+–linked and H+–linked

1.7 Diseases of ion channels, pumps and transporters

1.7.1 Channelopathies

1.7.2 Pump dysfunction

1.7.3 Transporter dysfunction

1.8 Conclusion

Chapter 2: Study of Ion Pump Activity using Black Lipid Membranes
Hans–Jürgen Apell and Valerij S. Sokolov

2.1 Introduction

2.2 Formation of black lipid membranes

2.3 Reconstitution in black lipid membranes

2.3.1 Reconstitution of Na+,K+–ATPase in black lipid membranes

2.3.2 Recording transient currents with membrane fragments adsorbed to a black lipid membrane

2.4 The principles of capacitive coupling

2.4.1 Dielectric coefficients

2.5 The gated–channel concept

2.6 Relaxation techniques

2.6.1 Concentration–jump methods

2.6.2 Charge–pulse method

2.7 Admittance Measurements

2.8 The investigation of cytoplasmic and extracellular ion access channels in the Na+,K+–ATPase

2.9 Conclusions

Chapter 3: Analysing Ion Permeation in Channels and Pumps using Patch–Clamp Recording
Andrew J. Moorhouse, Trevor M. Lewis and Peter H. Barry

3.1 Introduction

3.2 Description of the patch–clamp technique

3.2.1 Development of whole–cell dialysis with voltage–clamp

3.3 Patch–clamp measurement and analysis of single channel conductance

3.3.1 Conductance and Ohm s law

3.3.2 Conductance of channels versus pumps

3.3.3 Fluctuation analysis

3.3.4 Single channel recordings

3.4 Determining ion selectivity and relative permeation in whole cell recordings

3.4.1 Dilution potential measurements

3.4.2 Bi–ionic potential measurements

3.4.3 Voltage and solution control in whole–cell patch–clamp recordings

3.4.4 Ion shift effects during whole–cell patch–clamp experiments

3.4.5 Liquid junction potential corrections

3.5 Influence of voltage corrections in quantifying ion selectivity in channels

3.5.1 Analysis of counterion permeation in glycine receptor channels

3.5.2 Analysis of anion–cation permeability in cation–selective mutant glycine receptor channels

3.6 Ion permeation pathways through channels and pumps

3.6.1 Ion permeation pathway in pentameric ligand–gated ion channels

3.6.1.1 Extracellular and intracellular components of the permeation pathway

3.6.1.2 The TM2 pore is the primary ion selectivity filter

3.6.2 Ion permeation pathways in pumps identified using patch–clamp

3.6.2.1 Palytoxin uncouples the occluded gates of the Na+,K+–ATPase

3.7 Conclusions

Chapter 4: Probing Conformational Transitions of Membrane Proteins with Voltage Clamp Fluorometry (VCF)
Thomas Friedrich

4.1 Introduction

4.2 Description of the VCF technique

4.2.1 Generation of single–cysteine reporter constructs, expression in Xenopus laevis oocytes, site–directed fluorescence labeling

4.2.2 VCF instrumentation

4.2.3 Technical precautions and controls

4.3 Early measurements on voltage–gated K+ channels

4.3.1 Results obtained with VCF on voltage–gated channels

4.3.2 Probing the environmental changes: fluorescence spectra, anisotropy and the effects of quenchers

4.4 VCF applied to P–type ATPases

4.4.1 Structural and functional aspects of Na+,K+– and H+,K+–ATPase

4.4.2 The N790C sensor construct of Na+,K+–ATPase 1–subunit

4.4.2.1 Probing electrogenic steps in the Post–Albers transport cycle

4.4.2.2 Influence of intracellular Na+ concentration

4.4.3. The gastric H+,K+–ATPase S806C sensor construct

4.4.3.1 Voltage–dependent conformational shifts of the H+,K+–ATPase sensor construct S806C during the H+ transport branch

4.4.3.2 Intra– or extracellular access channels of the H+,K+–ATPase?

4.4.3.3 Effects of extracellular ligands on the proton pump: K+ and Na+

4.4.4 Probing intramolecular distances by double labeling and FRET

4.5 Conclusions and Perspectives

Chapter 5: Patch–Clamp Analysis of Transporters via Pre–Steady–State Kinetic Methods
Christof Grewer

5.1 Introduction

5.2 Patch clamp analysis of secondary–active transporter function

5.2.1 Patch clamp methods

5.2.2 Whole–cell recording

5.2.3 Recording from excised patches

5.3 Perturbation methods

5.3.1 Concentration jumps

5.3.2 Voltage jumps

5.4 Evaluation and interpretation of pre–steady–state kinetic data

5.4.1 Integrating rate equations that describe mechanistic transport models

5.4.2 Assigning kinetic components to elementary processes in the transport cycle

5.5 Mechanistic insight into transporter function

5.5.1 Sequential binding mechanism

5.5.2 Electrostatics

5.5.3 Structure–function analysis

5.6 Case studies

5.6.1 Glutamate transporter mechanism

5.6.2 Electrogenic charge movements associated with the electroneutral amino acid exchanger ASCT2

5.7 Conclusions

Chapter 6: Recording of Pump and Transporter Activity using Solid–Supported Membranes (SSM–based Electrophysiology)
Francesco Tadini–Buoninsegni and Klaus Fendler

6.1 Introduction

6.2 The instrument

6.2.1 Rapid solution exchange cuvette

6.2.2 Set–up and flow protocols

6.2.3 Protein preparations

6.2.4 Commercial instruments

6.3 Measurement procedures, data analysis and interpretation

6.3.1 Current measurement, signal analysis and reconstruction of pump currents

6.3.2 Voltage measurement

6.3.3 Solution exchange artifacts

6.4 P–type ATPases investigated by SSM–based electrophysiology

6.4.1 Sarcoplasmic reticulum Ca2+–ATPase

6.4.2 Human Cu+–ATPases ATP7A and ATP7B

6.5 Secondary active transporters

6.5.1 Anti–port: Assessing the forward and reverse modes of the NhaA Na+/H+ exchanger of Escherichia coli

6.5.2 Co–Transport: A sugar induced electrogenic partial reaction in the lactose permease LacY of Escherichia coli

6.5.3 The glutamate transporter EAAC1: a robust electrophysiological assay with high information content

6.6 Conclusions

Chapter 7: Stopped–flow Fluorimetry using Voltage–Sensitive Fluorescent Membrane Probes
Ronald J. Clarke and Mohammed A. A. Khalid

7.1 Introduction

7.2 Basics of the stopped–flow technique

7.2.1 Flow cell design

7.2.2 Rapid data acquisition

7.2.3 Dead time

7.3 Covalent versus noncovalent fluorescence labeling

7.3.1 Intrinsic fluorescence

7.3.2 Covalently bound extrinsic fluorescent probes

7.3.3 Non–covalently bound extrinsic fluorescent probes

7.4 Classes of voltage–sensitive dyes

7.4.1 Slow dyes

7.4.2 Fast dyes

7.5 Measurement of the Kinetics of the Na+,K+–ATPase

7.5.1 Dye concentration

7.5.2 Excitation wavelength and light source

7.5.3 Monochromators and filters

7.5.4 Photomultiplier and voltage supply

7.5.5 Reactions detected by RH421

7.5.6. Origin of the RH421 response

7.6 Conclusions

Chapter 8: Nuclear Magnetic Resonance Spectroscopy
Philip W. Kuchel

8.1 Introduction

8.1.1 Definition of nuclear magnetic resonance

8.1.2 Why so useful?

8.1.3 Magnetic polarization

8.1.4 Larmor equation

8.1.5 Chemical shift

8.1.6 Free induction decay

8.1.7 Pulse excitation

8.1.8 Relaxation times

8.1.9 Splitting of resonance lines

8.1.10 Measuring membrane transport

8.2 Covalently–induced chemical shift differences

8.2.1 Arginine transport

8.2.2 Other examples

8.3 Shift–reagent–induced chemical shift differences

8.3.1 DyPPP

8.3.2 TmDTPA and TmDOTP

8.3.3 Fast cation exchange

8.4 pH–induced chemical shift differences

8.4.1 Orthophosphate

8.4.2 Methylphosphonate

8.4.3 Triethylphosphate 31P shift reference

8.5 Hydrogen–bond–induced chemical shift differences

8.5.1 Phosphonates DMMP

8.5.2 HPA

8.5.3 Fluorides

8.6 Ionic–environment–induced chemical shift differences

8.6.1 Cs+ transport

8.7 Relaxation time differences

8.7.1 Mn2+ doping

8.8 Diffusion coefficient differences

8.8.1 Stejskal–Tanner plot

8.8.2 Andrasko s method

8.9 Some subtle spectral effects

8.9.1 Scalar (J) coupling differences

8.9.2 Endogenous magnetic field gradients

8.9.2.1 Magnetic induction and magnetic field strength

8.9.2.2 Magnetic field gradients across cell membranes, and CO treatment of red blood cells

8.9.2.3 Exploiting magnetic field gradients in membrane transport studies

8.9.3 Residual quadrupolar ( ÞQ) coupling

8.10 A case study: The stoichiometric relationship between the number of Na+ ions transported per molecule of glucose consumed in human red blood cells

8.11 Conclusions

Chapter 9: Time–resolved and Surface–enhanced Infrared Spectroscopy
Joachim Heberle

9.1 Introduction

9.2 Basics of infrared spectroscopy

9.2.1 Vibrational spectroscopy

9.2.2 Fourier–transform infrared spectroscopy

9.2.3 IR spectra of biological compounds

9.2.4 Difference spectroscopy

9.3 Reflection techniques

9.3.1 Attenuated total reflection (ATR)

9.3.2 Surface–enhanced IR absorption spectroscopy (SEIRAS)

9.4 Applications to electron transferring proteins

9.4.1 Cytochrome c

9.4.2 Cytochrome c oxidase

9.5 Time–resolved IR spectroscopy

9.5.1 The rapid–scan technique

9.5.2 The step–scan technique

9.5.3 Tunable quantum cascade lasers

9.6 Applications to retinal proteins

9.6.1 Bacteriorhodopsin

9.6.2 Channelrhodopsin

9.7 Conclusions

Chapter 10: Analysis of Membrane–Protein Complexes by Single Molecule Methods
Katia Cosentino, Stephanie Bleicken, and Ana J. García–Sáez

10.1 Introduction

10.2 Fluorophores for single particle labeling

10.3 Principles of fluorescence correlation spectroscopy (FCS)

10.3.1 Analysis of molecular complexes by two–color FCS

10.3.2 FCS variants to study lipid membranes

10.3.3 FCS applications to membranes

10.4 Principle and analysis of single molecule imaging

10.4.1 Total internal reflection fluorescence (TIRF) microscopy

10.4.2 Single–molecule detection

10.4.3 Single particle tracking (SPT) and trajectory analysis

10.5 Complex dynamics and stoichiometry by single molecule microscopy

10.5.1 Application to single molecule stoichiometry analysis

10.5.2 Application to kinetics processes in cell membranes

10.6 FCS versus SPT

Chapter 11: Probing Channel, Pump and Transporter Function Using Single Molecule Fluorescence
Eve E. Weatherill, John S. H. Danial, and Mark I. Wallace

11.1 Introduction

11.1.1 Basic principles

11.2 Practical considerations

11.2.1 Observables

11.2.2 Labels

11.2.3 Bilayers

11.3 Single molecule fluorescence imaging

11.3.1 Fluorescence co–localization

11.3.2 Conformational changes

11.3.3 Super–resolution microscopy

11.4 Single molecule Förster resonance energy transfer

11.4.1 Interactions/stoichiometry

11.4.2 Conformational changes

11.5 Single molecule counting by photobleaching

11.6 Optical channel recording

11.7 Simultaneous techniques

11.8 Summary

Chapter 12: Electron Paramagnetic Resonance Site Directed Spin Labeling
Louise J. Brown and Joanna E. Hare

12.1 Introduction

12.1.1 Development of Electron Paramagnetic Resonance (EPR) as a tool for structural biology

12.1.2 Site–Directed Spin–Labeling (SDSL)–EPR – a complementary approach to determine structure–function relationships

12.2 Basics of the EPR method

12.2.1 Physical basis of the EPR signal

12.2.2 Spin labeling

12.2.3 EPR instrumentation

12.3 Structural and dynamic information from SDSL–EPR

12.3.1 Mobility measurements

12.3.2 Solvent accessibility

12.4 Distance measurements

12.4.1 Interspin distance measurements

12.4.2 Continuous wave

12.4.3 Pulsed methods Double Electron Electron Resonance (DEER)

12.5 Challenges

12.5.1 New labels

12.5.2 Spin label flexibility

12.5.3 Production and reconstitution challenges – nanodiscs

12.6 Conclusions

Chapter 13: Radioactivity–based Analysis of Ion Transport
Ingolf Bernhardt and J. Clive Ellory

13.1 Introduction

13.2 Membrane permeability for electroneutral substances and ions

13.3 Kinetic considerations

13.4 Techniques for ion flux measurements

13.4.1 Conventional methods

13.4.2 Alternative method

13.5 Kinetic analysis of ion transporter properties

13.6 Selected cation transporter studies on red blood cells

13.6.1 K+,Cl– cotransport (KCC)

13.6.2 Residual transport

13.7 Combination of radioactive isotope studies with methods using fluorescent dyes

13.8 Conclusions

Chapter 14: Cation Uptake Studies with Atomic Absorption Spectrophotometry (AAS)
Thomas Friedrich

14.1 Introduction

14.2 Overview of the technique of atomic absorption spectrophotometry (AAS)

14.2.1 Historical account of AAS with flame atomization

14.2.2 Element–specific radiation sources

14.2.3 Electrothermal atomization in heated graphite tubes

14.2.4 Correction for background absorption

14.3 The expression system of Xenopus laevis oocytes for cation flux studies practical considerations

14.4 Experimental outline of the AAS flux quantification technique

14.5 Representative results obtained with the AAS flux quantification technique

14.5.1 Reaction cycle of P–type ATPases

14.5.2 Rb+ uptake kinetics, inhibitor sensitivity

14.5.3 Dependence of Rb+ transport of gastric H+,K+–ATPase on extra– and intracellular pH

14.5.4 Determination of Na+,K+–ATPase transport stoichiometry and voltage dependence of H+,K+–ATPase Rb+ transport

14.5.5 Effects of C–terminal deletions of the H+,K+–ATPase –subunit

14.5.6 Li+ and Cs+ uptake studies

14.6 Concluding remarks

Chapter 15: Long Timescale Molecular Simulations for Understanding Ion Channel Function
Ben Corry

15.1 Introduction

15.2 Fundamentals of molecular dynamics simulation

15.2.1 The main idea

15.2.2 Force fields

15.2.3 Other simulation considerations

15.2.4 Why do molecular dynamics simulations take so much computational power?

15.2.4.1 Force calculations

15.2.4.2 Time step

15.3 Simulation duration and simulation size

15.4 Historical development of long molecular dynamics simulations

15.5 Limitations and challenges facing on MD Simulations

15.5.1 Force field and algorithm accuracy

15.5.2 Sampling problems

15.6 Example simulation studies of ion channels

15.6.1 Simulations of ion permeation

15.6.2 Simulations of ion selectivity

15.6.3 Simulations of gating and conformational changes

15.7 Conclusions