Mechanosensitive Ion Channels, Part B, Volume 59 (Current Topics in Membranes)

Current Topics in Membranes, Volume 59

Mechanosensitive Ion Channels, Part B


Series Editors

Dale J. BenosDepartment of Physiology and Biophysics

University of Alabama

Birmingham, Alabama

Sidney A. SimonDepartment of Neurobiology

Duke University Medical Centre

Durham, North Carolina


Mechanosensitive IonChannels, Part B

Edited by

Owen P. HamillDepartment of Neuroscience and Cell BiologyUniversity of Texas Medical BranchGalveston, Texas

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Contents

Contributors xiii

Foreword xvii

Previous Volumes in Series xix

CHAPTER 1 Mechanosensitive Ion Channels of Spiders:

Mechanical Coupling, Electrophysiology, and

Synaptic ModulationAndrew S. French and Paivi H. Torkkeli

I. Overview 1II. Introduction 2III. Types of Spider Mechanoreceptors 3IV. Mechanical Coupling 3V. Mechanotransduction in Slit Sensilla 6VI. Dynamic Properties of Mechanotransduction

and Action Potential Encoding 13VII. Calcium Signaling During Transduction by

Spider Mechanoreceptors 14VIII. Synaptic Modulation of

Spider Mechanoreceptors 15IX. Conclusions 17

References 17

CHAPTER 2 Ion Channels for Mechanotransduction in the

Crayfish Stretch ReceptorBo Rydqvist

I. Overview 21II. Introduction 22III. Morphology of the SRO 23IV. Functional Properties 24V. Summary and Discussion of Future

Research Directions 43References 45

v

CHAPTER 3 Mechanosensitive Ion Channels in

Caenorhabditis elegansDafne Bazopoulou and Nektarios Tavernarakis

I. Overview 49II. Introduction 50III. C. elegans Mechanosensitive Behaviors 51IV. C. elegans DEG/ENaCs 55V. C. elegans TRP Ion Channels 66VI. Concluding Remarks 72

References 73

CHAPTER 4 Properties and Mechanism of the Mechanosensitive

Ion Channel Inhibitor GsMTx4, a Therapeutic

Peptide Derived from Tarantula VenomPhilip A. Gottlieb, Thomas M. Suchyna, and Frederick Sachs

I. Overview 81II. Introduction 82III. Properties and Specificity of GsMTx4 85IV. Cellular Sites for GsMTx4 95V. Potential Therapeutic Uses for GsMTx4 97VI. Conclusions 103

References 103

CHAPTER 5 Mechanosensitive Channels in Neurite OutgrowthMario Pellegrino and Monica Pellegrini

I. Overview 111II. Introduction 112III. Encoding of Guidance Cues in

Axon Pathfinding 112IV. Requirement of TRP Channels in

Calcium-Dependent Axon Pathfinding 114V. Physical Guidance Cues and Role of

Mechanosensitive Ion Channels 116VI. Ion Channels as Molecular Integrators 119VII. Concluding Remarks 120

References 122

CHAPTER 6 ENaC Proteins in Vascular Smooth

Muscle MechanotransductionHeather A. Drummond

I. Overview 127II. Introduction 128

vi Contents

III. DEG/ENaC/ASIC Proteins are Members of aDiverse Protein Family Involved inMechanotransduction 129

IV. Involvement of ENaC Proteins in VascularSmooth Muscle Mechanotransduction 137

V. Summary and Future Directions 145References 145

CHAPTER 7 Regulation of the Mechano-Gated K2P Channel

TREK-1 by Membrane PhospholipidsJean Chemin, Amanda Jane Patel, Patrick Delmas, Fred Sachs, Michel

Lazdunski, and Eric Honore

I. Overview 155II. Introduction 156III. TREK-1 Stimulation by Membrane

Phospholipids 158IV. TREK-1 Inhibition by Membrane

Phospholipids 161References 168

CHAPTER 8 MechanoTRPs and TRPA1Andrew J. Castiglioni and Jaime Garca-Anoveros

I. Overview 171II. MechanoTRP Channels 174III. Characteristics of TRPA1 Gene

and Protein 175IV. TRPA1 Expression in

Mechanosensory Organs 176V. Function of TRPA1 177VI. Proposed Biological Roles for TRPA1 185

References 186

CHAPTER 9 TRPCs as MS ChannelsOwen P. Hamill and Rosario Maroto

I. Overview 191II. Introduction 192III. Practical Aspects of Recording

MS Channels 193

Contents vii

IV. Distinguishing Direct vs IndirectMS Channels 195

V. Extrinsic Regulation of Stretch Sensitivity 197VI. Strategies to Identify MS Channel Proteins 197VII. General Properties of TRPCs 198VIII. Evidence for TRPC Mechanosensitivity 203IX. Conclusions 215

References 218

CHAPTER 10 The Cytoskeletal Connection to Ion Channels

as a Potential Mechanosensory Mechanism:

Lessons from Polycystin-2 (TRPP2)Horacio F. Cantiello, Nicolas Montalbetti, Qiang Li,

and Xing-Zhen Chen

I. Overview 234II. Introduction 235III. Role of Actin Cytoskeletal Dynamics in

PC2-Mediated Channel Function 253IV. Identification of Actin-Binding Protein

Interactions with Polycystin-2 261V. EVect of Hydroosmotic Pressure on PC2

Channel Function: Role of the Cytoskeletonin Osmosensory Function 265

VI. The ChannelCytoskeleton Interface:StructuralFunctional Correlates 272

VII. Perspective and Future Directions 281References 282

CHAPTER 11 Lipid Stress at Play: Mechanosensitivity of

Voltage-Gated ChannelsCatherine E. Morris and Peter F. Juranka

I. Overview 298II. The System Components 298III. Big Picture Issues 301IV. Reversible Stretch-Induced Changes in

Particular VGCs 319V. Irreversible Stretch-Induced Gating

Changes in VGCs 325VI. Technical Issues 327VII. Summary Comments 330

References 330

viii Contents

CHAPTER 12 Hair Cell Mechanotransduction: The Dynamic

Interplay Between Structure and FunctionAnthony J. Ricci and Bechara Kachar

I. Overview 339II. Auditory System 340III. Hair Bundle Structure 341IV. MET Involves Mechanically Gated Channels 341V. Where are These Channels? 343VI. The Gating Spring Theory 344VII. How are the Channels Activated? 347VIII. To Be or Not to Be Tethered 349IX. Characterizing Channel Properties? 351X. MET Channel Pore 352XI. Adaptation 354XII. The Dynamic Hair Bundle 361XIII. Summary and Future Directions 365

References 366

CHAPTER 13 Insights into the Pore of the Hair Cell Transducer

Channel from Experiments with Permeant BlockersSietse M. van Netten and Corne J. Kros

I. Overview 376II. Introduction 376III. Ionic Selectivity of the Transducer Channel 377IV. Permeation and Block of Mechanoreceptor

Channels by FM1-43 378V. Permeation and Block of the Hair Cell Transducer

Channel by Aminoglycoside Antibiotics 382VI. Transducer Channel Block by Amiloride

and Its Derivatives 391VII. Conclusions 394

References 396

CHAPTER 14 Models of Hair Cell MechanotransductionSusanne Bechstedt and Jonathon Howard

I. Overview 399II. Introduction 400III. Transduction Channel Properties 401IV. Gating 408V. Active Hair Bundle Motility 415VI. Conclusions 418

References 418

Contents ix

CHAPTER 15 TouchLiam J. Drew, Francois Rugiero, and John N. Wood

I. Overview 426II. Introduction 426III. Structure of Skin and Touch Receptors 427IV. Physiology of Mechanoreceptive

Nerve Fibers 432V. Quantitating Mechanical Responses in

Animal Models 435VI. Electrophysiological Approaches to

Mechanosensation in Rodents 436VII. Mechanosensitive Ion Channels in Cultured

Sensory Neurons 437VIII. Gating MS Ion Channels in DRG Neurons 446IX. Candidate Ion Channels 447X. Voltage-Gated Channels and

Mechanosensation 454XI. Indirect Signaling Between Sensory Neurons and

Nonneuronal Cells 456XII. Conclusions 457

References 457

CHAPTER 16 Mechanosensitive Ion Channels in

Dystrophic MuscleJeffry B. Lansman

I. Overview 467II. Introduction 468III. MS Channel Expression During Myogenesis 469IV. Permeabilty Properties of MS Channels in

Skeletal Muscle 470V. Gating 471VI. Pharmacology 478VII. Conclusions 481

References 482

CHAPTER 17 MscCa Regulation of Tumor Cell Migration

and MetastasisRosario Maroto and Owen P. Hamill

I. Overview 485II. Introduction 486

x Contents

III. DiVerent Modes of Migration 487IV. Ca2 Dependence of Cell Migration 490V. The Role of MscCa in Cell Migration 499VI. Can Extrinsic Mechanical Forces Acting on MscCa

Switch on Cell Migration? 501References 502

CHAPTER 18 Stretch-Activated Conductances in Smooth MusclesKenton M. Sanders and Sang Don Koh

I. Overview 511II. Introduction 512III. Mechanosensitive Conductances that Generate

Inward Currents 514IV. Mechanosensitive Conductances that Generate

Outward Currents 527References 535

CHAPTER 19 Mechanosensitive Ion Channels in Blood

Pressure-Sensing Baroreceptor NeuronsMark W. Chapleau, Yongjun Lu, and Francois M. Abboud

I. Overview 541II. Introduction 542III. BR Sensory Transduction 544IV. Mechanosensitive Channels in BR Neurons 548V. Methodological Limitations and Challenges 558VI. Summary and Future Directions 560

References 561

Index 569

Contents xi

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Contributors

Numbers in parentheses indicate the pages on which the authors contributions begin.

Francois M. Abboud (541), The Cardiovascular Center,Department of Internal Medicine, and Department of MolecularPhysiology & Biophysics, The University of Iowa Carver Collegeof Medicine, Iowa City, Iowa 52242

Dafne Bazopoulou (49), Institute of Molecular Biology andBiotechnology, Foundation for Research and Technology,Heraklion 71110, Crete, Greece

Susanne Bechstedt (399), MaxPlanckInstitute of Molecular CellBiology and Genetics (MPICBG), 01307 Dresden, Germany

Horacio F. Cantiello (233), Renal Unit, Massachusetts GeneralHospital East, Charlestown, Massachusetts 02129; Department ofMedicine, Harvard Medical School, Boston, Massachusetts 02115;Laboratorio de Canales Ionicos, Departamento de Fisicoqumica yQumica Analtica, Facultad de Farmacia y Bioqumica, BuenosAires 1113, Argentina

Andrew J. Castiglioni (171), Departments of Anesthesiology,Physiology, and Neurology, Northwestern University Institute forNeuroscience, Feinberg School of Medicine, NorthwesternUniversity, Chicago, Illinois 60611

Mark W. Chapleau (541), The Cardiovascular Center, Departmentof Internal Medicine, and Department of Molecular Physiology &Biophysics, The University of Iowa Carver College ofMedicine, Iowa City, Iowa 52242; Veterans Affairs MedicalCenter, Iowa City, Iowa 52246

Jean Chemin (155), Institut de Genomique Fonctionnelle, UPR2580 CNRS, F34094 Montpellier cedex 05, France

XingZhen Chen (233), Department of Physiology, University ofAlberta, Edmonton T6G2H7, Canada

xiii

Patrick Delmas (155), Laboratoire de NeurophysiologieCellulaire, Faculte de Medecine, UMR 6150 CNRS, 13916Marseille Cedex 20, France

Liam J. Drew (425), Molecular Nociception Group, BiologyDepartment, University College London, London WC1E 6BT,United Kingdom

Heather A. Drummond (127), Department of Physiology,The Center for Excellence in CardiovascularRenal Research,University of Mississippi Medical Center, Jackson,Mississippi 39216

Andrew S. French (1), Department of Physiology and Biophysics,Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada

Jaime GarcaAnoveros (171), Departments of Anesthesiology,Physiology, and Neurology, Northwestern University Institute forNeuroscience, Feinberg School of Medicine, NorthwesternUniversity, Chicago, Illinois 60611

Philip A. Gottlieb (81), The Department of Physiology andBiophysics, Center for Single Molecule Biophysics,SUNY at Buffalo, Buffalo, New York 14214

Owen P. Hamill (191, 485), Department of Neuroscience andCell Biology, University of Texas Medical Branch, Galveston,Texas 77555

Eric Honore (155), Institut de Pharmacologie Moleculaire etcellulaire, UMR 6097 CNRS, 06560 Valbonne, France

Jonathon Howard (399), MaxPlanckInstitute of Molecular CellBiology and Genetics (MPICBG), 01307 Dresden, Germany

Peter F. Juranka (297), Neuroscience, Ottawa HealthResearch Institute, Ottawa Hospital, Ottawa, Ontario K1Y 4E9,Canada

Bechara Kachar (339), Section of Structural Biology, NationalInstitutes of Deafness and Communicative Disorders, Bethesda,Maryland 20892

Sang Don Koh (511), Department of Physiology and CellBiology, University of Nevada School of Medicine, Reno,Nevada 89557

xiv Contributors

Corne J. Kros (375), School of Life Sciences, University of Sussex,Falmer, Brighton BN1 9QG, United Kingdom

Jeffry B. Lansman (467), Department of Cellular and MolecularPharmacology, School of Medicine, University of California,San Francisco, California 94143

Michel Lazdunski (155), Institut de Pharmacologie Moleculaire etcellulaire, UMR 6097 CNRS, 06560 Valbonne, France

Qiang Li (233), Department of Physiology, University of Alberta,Edmonton T6G 2H7, Canada

Yongjun Lu (541), The Cardiovascular Center and Department ofInternal Medicine, The University of Iowa Carver College ofMedicine, Iowa City, Iowa 52242

Rosario Maroto (191, 485), Department of Neuroscience andCell Biology, University of Texas Medical Branch, Galveston,Texas 77555

Nicolas Montalbetti (233), Laboratorio de Canales Ionicos,Departamento de Fisicoqumica y Qumica Analtica, Facultad deFarmacia y Bioqumica, Buenos Aires 1113, Argentina

Catherine E. Morris (297), Neuroscience, Ottawa Health ResearchInstitute, Ottawa Hospital, Ottawa, Ontario K1Y 4E9, Canada

Amanda Jane Patel (155), Institut de Pharmacologie Moleculaire etcellulaire, UMR 6097 CNRS, 06560 Valbonne, France

Monica Pellegrini (111), Scuola Normale Superiore, Pisa, Italy

Mario Pellegrino (111), Dipartimento di Fisiologia UmanaG. Moruzzi, Universita di Pisa, Pisa, Italy

Anthony J. Ricci (339), Department of Otolaryngology, StanfordUniversity, Stanford, California 94305

Francois Rugiero (425), Molecular Nociception Group, BiologyDepartment, University College London, London WC1E 6BT,United Kingdom

Bo Rydqvist (21), Department of Physiology and Pharmacology,Karolinska Institutet, SE171 77 Stockholm, Sweden

Frederick Sachs (81, 155), The Department of Physiology andBiophysics, Center for Single Molecule Biophysics,SUNY at Buffalo, Buffalo, New York 14214

Contributors xv

Kenton M. Sanders (511), Department of Physiology andCell Biology, University of Nevada School of Medicine, Reno,Nevada 89557

Thomas M. Suchyna (81), The Department of Physiology andBiophysics, Center for Single Molecule Biophysics,SUNY at Buffalo, Buffalo, New York 14214

Nektarios Tavernarakis (49), Institute of Molecular Biology andBiotechnology, Foundation for Research and Technology,Heraklion 71110, Crete, Greece

Paivi H. Torkkeli (1), Department of Physiology and Biophysics,Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada

Sietse M. van Netten (375), Department of Neurobiophysics,University of Groningen, 9747AG, Groningen, The Netherlands

John N. Wood (425), Molecular Nociception Group, BiologyDepartment, University College London, London WC1E 6BT,United Kingdom

xvi Contributors

Foreword

Mechanosensitive Ion Channels, Part B

Owen P. Hamill

Department of Neuroscience and Cell Biology,

University of Texas Medical Branch,

Galveston, Texas

One of the great challenges in studying mechanotransduction (MT)

has been to identify the mechanisms that underlie the exquisite sensitivity

and highfrequency response of specific animal mechanotransducersa

spider detects substrate vibrations within thermal noise limits, whereas a bat

generates and detects ultrasounds of frequencies up to 100 kHz in echolocat-

ing flying prey. Part B of this volume on mechanosensitive (MS) channels

covers the diversity of MS channels and MT mechanisms evident in diVerent

invertebrate and vertebrate mechanotransducers. The combined chapters

highlight the integration ofMS channels into signaling complexes that interact

with ancillary structures and other channels that are critical in shaping the

specific inputoutput relations of mechanotransducers. The opening chapters

describe MT in the slit sensilla in the spiders leg, the stretch receptor organ

in crayfish muscle, and specific touch receptors in the nematode worm,

Caenorhabditis elegans. The studies indicate at least twomajor channel families,

the epithelial Na channel (ENaC) and the transient receptor potential (TRP)

channels, are involved in MT in lower invertebrates. Subsequent chapters

review the roles for ENaC and various TRP channels, and also the MS two

poredomain K channels andMS voltagegated channels in mediatingMT in

mammalian cells.Oneof themajor hurdles in studyingMThasbeen the absence

of specific agents that selectively target MS channelsthe potential for the

tarantula spider venom peptide GsMTx4 to serve this role is discussed in one

chapter. Perhaps one of the interesting actions of GsMTx4 is that it strongly

potentiates neurite outgrowth presumably via block of anMS channel that acts

as a negative regulator of neurite outgrowth first demonstrated in the leech and

reviewed in another chapter. Several chapters highlight diVerent aspects of the

most intensely studied of all biological mechanotransducers, namely those

mediatingvertebratehearing and touch.These two formsofMThavepresented

xvii

the greatest challenge in identifying the membrane proteins forming MS

channels, and each chapter provides new information and diVerent approaches

that should help in completing this goal. The last part of the volume includes

chapters that address the properties of MS channels in cell types where

abnormalities inMTcontribute to significant humanpathologies, including the

elevated stretchinduced Ca2 influx that contributes to muscle fiber degener-

ation in muscular dystrophy, abnormalities in the regulation of smooth muscle

tone, and baroreception that lead to hypertension, and the alterations in MS

channel functional expression that may contribute to increased tumor cell

motility and invasion during cancer progression.

As indicated in Part A of this volume, I would like to thank Dale Benos for

his original invitation to submit the proposal to Elsevier. I would also like to

thank all those involved in the production of the volume and, in particular,

Phil Carpenter for his continual and patient eVorts during the compilation

phase. Finally, I would like to thank all the scientists for presenting their

discoveries regarding MS channels.

xviii Foreword

Previous Volumes in Series

Current Topics in Membranes and Transport

Volume 23 Genes and Membranes: Transport Proteins and Receptors*(1985)Edited by Edward A. Adelberg and Carolyn W. Slayman

Volume 24 Membrane Protein Biosynthesis and Turnover (1985)Edited by Philip A. Knauf and John S. Cook

Volume 25 Regulation of Calcium Transport across Muscle Membranes(1985)Edited by Adil E. Shamoo

Volume 26 NaH Exchange, Intracellular pH, and Cell Function*(1986)Edited by Peter S. Aronson and Walter F. Boron

Volume 27 The Role of Membranes in Cell Growth and Differentiation(1986)Edited by Lazaro J. Mandel and Dale J. Benos

Volume 28 Potassium Transport: Physiology and Pathophysiology*(1987)Edited by Gerhard Giebisch

Volume 29 Membrane Structure and Function (1987)Edited by Richard D. Klausner, Christoph Kempf, and Josvan Renswoude

Volume 30 Cell Volume Control: Fundamental and ComparativeAspects in Animal Cells (1987)Edited by R. Gilles, Arnost Kleinzeller, and L. Bolis

Volume 31 Molecular Neurobiology: Endocrine Approaches (1987)Edited by Jerome F. Strauss, III, and Donald W. Pfaff

*Part of the series from the Yale Department of Cellular and Molecular Physiology.

xix

Volume 32 Membrane Fusion in Fertilization, Cellular Transport, andViral Infection (1988)Edited by Nejat Duzgunes and Felix Bronner

Volume 33 Molecular Biology of Ionic Channels* (1988)Edited by William S. Agnew, Toni Claudio, and Frederick J. Sigworth

Volume 34 Cellular and Molecular Biology of Sodium Transport* (1989)Edited by Stanley G. Schultz

Volume 35 Mechanisms of Leukocyte Activation (1990)Edited by Sergio Grinstein and Ori D. Rotstein

Volume 36 ProteinMembrane Interactions* (1990)Edited by Toni Claudio

Volume 37 Channels and Noise in Epithelial Tissues (1990)Edited by Sandy I. Helman and Willy Van Driessche

Current Topics in Membranes

Volume 38 Ordering the Membrane Cytoskeleton Trilayer* (1991)Edited by Mark S. Mooseker and Jon S. Morrow

Volume 39 Developmental Biology of Membrane Transport Systems(1991)Edited by Dale J. Benos

Volume 40 Cell Lipids (1994)Edited by Dick Hoekstra

Volume 41 Cell Biology and Membrane Transport Processes* (1994)Edited by Michael Caplan

Volume 42 Chloride Channels (1994)Edited by William B. Guggino

Volume 43 Membrane ProteinCytoskeleton Interactions (1996)Edited by W. James Nelson

Volume 44 Lipid Polymorphism and Membrane Properties (1997)Edited by Richard Epand

Volume 45 The Eyes Aqueous Humor: From Secretion to Glaucoma(1998)Edited by Mortimer M. Civan

xx Previous Volumes in Series

Volume 46 Potassium Ion Channels: Molecular Structure, Function, andDiseases (1999)Edited by Yoshihisa Kurachi, Lily Yeh Jan, and Michel Lazdunski

Volume 47 AmilorideSensitive Sodium Channels: Physiology andFunctional Diversity (1999)Edited by Dale J. Benos

Volume 48 Membrane Permeability: 100 Years since Ernest Overton(1999)Edited by David W. Deamer, Arnost Kleinzeller, andDouglas M. Fambrough

Volume 49 Gap Junctions: Molecular Basis of Cell Communication inHealth and DiseaseEdited by Camillo Peracchia

Volume 50 Gastrointestinal Transport: Molecular PhysiologyEdited by Kim E. Barrett and Mark Donowitz

Volume 51 AquaporinsEdited by Stefan Hohmann, Sren Nielsen and Peter Agre

Volume 52 PeptideLipid InteractionsEdited by Sidney A. Simon and Thomas J. McIntosh

Volume 53 CalciumActivated Chloride ChannelsEdited by Catherine Mary Fuller

Volume 54 Extracellular Nucleotides and Nucleosides: Release,Receptors, and Physiological and Pathophysiological EffectsEdited by Erik M. Schwiebert

Volume 55 Chemokines, Chemokine Receptors, and DiseaseEdited by Lisa M. Schwiebert

Volume 56 Basement Membrances: Cell and Molecular BiologyEdited by Nicholas A. Kefalides and Jacques P. Borel

Volume 57 The Nociceptive MembraneEdited by Uhtaek Oh

Volume 58 Mechanosensitive Ion Channels, Part AEdited by Owen P. Hamill

Previous Volumes in Series xxi

CHAPTER 1

Mechanosensitive Ion Channels ofSpiders: Mechanical Coupling,Electrophysiology, andSynaptic Modulation

Andrew S. French and Paivi H. Torkkeli

Department of Physiology and Biophysics, Dalhousie University, Halifax,

Nova Scotia B3H 1X5, Canada

I. Overview

II. Introduction

III. Types of Spider Mechanoreceptors

IV. Mechanical Coupling

V. Mechanotransduction in Slit Sensilla

A. The Ionic Selectivity of Spider Mechanosensitive Channels

B. The Location of VS3 Mechanosensitive Channels

C. Mechanosensitive Channel Conductance, Density, and pH Sensitivity

D. Temperature Sensitivity of Mechanosensitive Channels

E. Molecular Characterization of Spider Mechanosensitive Channels

VI. Dynamic Properties of Mechanotransduction and Action Potential Encoding

VII. Calcium Signaling During Transduction by Spider Mechanoreceptors

VIII. Synaptic Modulation of Spider Mechanoreceptors

IX. Conclusions

References

I. OVERVIEW

Arthropods have provided several important mechanoreceptor models

because of the relatively large size and accessibility of their primary sensory

neurons. Three types of spider receptors: tactile hairs, trichobothria, and slit

sensilla have given important information about the coupling of external

Current Topics in Membranes, Volume 59 1063-5823/07 $35.00Copyright 2007, Elsevier Inc. All right reserved. DOI: 10.1016/S1063-5823(06)59001-5

mechanical stimuli to the neuronal membrane, transduction of mechanical

force into receptor current, encoding of aVerent action potentials, and eVerent

modulation of peripheral sensory receptors. Slit sensilla, found only in spiders,

have been particularly important because they allow intracellular recording from

sensory neurons during mechanical stimulation. Experiments on slit sensilla

have shown that their mechanosensitive ion channels are sodium selective,

blocked by amiloride, and open more at low pH. This evidence suggests that

the channels are members of the same molecular family as degenerins, acid

sensitive ion channels, and epithelial sodium channels. Slit sensilla have also

yielded evidence about the location, density, singlechannel conductance, and

dynamic properties of the mechanosensitive channels. Spider mechanoreceptors

are modulated in the periphery by eVerent neurons, and possibly by circulating

chemicals. Mechanisms of modulation, intracellular signaling, and the role of

intracellular calcium are areas of active investigation.

II. INTRODUCTION

Humans inhabit a sensory world dominated by vision, but we also use

mechanotransduction to provide the senses of hearing, vestibular sensation,

touch, and vibration, as well as chemotransduction for the senses of taste and

smell. In contrast to our visual world, a spiders life is dominated by vibration

and other mechanical inputs, even in those spider species that have relatively

good vision.Waiting for prey to land on aweb, hunting along the ground or on a

plant, and negotiating a vibratory mating ritualin all their daily activities the

mechanical senses are vitally important. In addition, both humans and spiders

detect a variety of internally generated mechanical signals from their musculo-

skeletal systems and internal organs that allow feedback regulation ofmovement

and many internal physiological processes.

Although mechanotransduction is such an important sense for humans,

spiders, and most other animals, its fundamental mechanisms have been

diYcult to unravel, mainly due to the small size and complex morphology of

most mechanoreceptor endings. Arthropods (insects, arachnids, and crus-

taceans) not only possess large arrays of diVerent mechanoreceptors, but

the relatively large sizes of some of their sensory neurons, and the close ass-

ociation of many mechanosensory neurons to the external cuticle have pro-

vided several model systems for investigating fundamental mechanisms of

mechanotransduction.

The most crucial step in mechanotransduction is a change in cell mem-

brane potential, the receptor potential, produced by the application of a

mechanical stimulus to the cell. To study this phenomenon ideally requires

a preparation where the electrical event can be directly observed during

2 French and Torkkeli

accurately controlled mechanical stimulation. This is possible in several

spider preparations, and the information thus obtained will be the major

subject here.

III. TYPES OF SPIDER MECHANORECEPTORS

The hairiness of spiders is well known, but what are the functions of the

thousands of hairs covering a typical spider? Many provide nonsensory fun-

ctions. These include adhesion to the substrate via surface tension, combing

of silk threads from spinnerets, supporting the air bubbles of water spiders,

providing attachment sites for spiderlings clinging to a female, and deterring

predators by intense skin irritation (reviewed by Foelix, 1996). However,

most of the surface hairs are sensory structures. Two major types of sensory

hairs are the trichobothria, or filiform hairs, and the shorter tactile hairs

(Fig. 1). Each of these hair structures is innervated by multiple neurons, typi-

cally four inCupiennius salei, although it is not clear that all these neurons are

mechanically sensitive. This situation contrasts somewhat with insects, which

typically have only one sensory neuron per hair, but the general structures are

otherwise similar.

In addition to hairs that extend beyond the cuticle, embedded in spider

cuticle are numerous mechanoreceptors of a type that is not found in other

arthropods, the slit sensilla (Figs. 1 and 2). These are widely distributed in

the exoskeleton, including the legs, pedipalps, and body (Barth and Libera,

1970; Barth, 1985, 2001; Patil et al., 2006). They detect mechanical events in

the cuticle, primarily strains imposed by normal movements of the animal

and vibrations due to predators, prey, and mates.

Spiders also possess a range of mechanoreceptors deeper within the animal,

particularly the joint receptors and muscle receptors, but spiders apparently

lack the chordotonal structures that are widespread in insects and crusta-

ceans, serving particularly as vibration and auditory receptors (Seyfarth,

1985; Barth, 2001).

IV. MECHANICAL COUPLING

The first functional stage of any mechanoreceptor is mechanical coupl-

ing from the initial stimulus to the mechanically sensitive membrane of

the sensory neuron. A large contribution to overall function is suggested,

although not yet proven, by the wide range of accessory structures found in

mechanoreceptors of both vertebrates and invertebrates, which are assumed

1. Mechanotransduction in Spiders 3

to serve a mechanical coupling role. Detailed quantitative understanding of

this coupling function is limited by the relatively small sizes of most receptors

and the unknown mechanical properties of the materials used to construct

the structures surrounding the sensory endings. The dynamic properties of

coupling structures are particularly diYcult to elucidate because it is hard to

10 mm

Cuticle

Sensorydendrites

Slit

Lymph space

Supportingcells

To cellbodies

50 mm

FIGURE 1 Major types of spider cuticular mechanoreceptors. Top left: hair sensilla at the

joint between the tibia (left) and the femur of a leg of Cupiennius salei. Longer, vertical hairs are

trichobothria, typically about 1mm long, surrounded by numerous shorter tactile hairs. Top

right: scanning electron micrograph of a lyriform organ consisting of approximately parallel slit

sensilla from a leg of C. salei. Dark circles are the sockets of broken hair sensilla. Lower drawing

shows the arrangement of sensory neurons and surrounding tissues at a typical slit sensillum.

Pairs of sensory dendrites, up to 200m long terminate in a ciliary enlargement that leads to a

tubular body surrounded by a dense dendritic sheath. Supporting cells produce a lymph space

surrounding the terminal dendrites that has a diVerent ionic composition than the normal

extracellular fluid. One of the two sensory dendrites proceeds further into the slit structure,

but the functional reason for this diVerence is unknown. On the basis of data from Barth, 2001,

2004; Widmer et al. (2005).


measure the individual movements of each component as the sensillum is

mechanically stimulated.

Barth (2001, 2004) has discussed in depth the available evidence about

mechanical coupling of spider trichobothria, hair sensilla, and slit sensilla.

This work also builds on a substantial base of comparable studies in insect

cuticular sensilla. Tactile hairs, as the name implies, are thought to serve as

touch detectors. They can bend, as well as rotate within their sockets, prov-

iding a reduction of movement estimated to be about 1:750, so that relatively

Stimulatorprobe

100 mm

Slits

Patella cuticle

1 mm

500 pA

200 ms

FIGURE 2 Intracellular recording from VS3 neurons. The approximately tubular patella is

split in two along its length and the muscle tissues removed to reveal the mechanosensory

neurons lying in the hypodermal membrane. A glass microelectrode is used to penetrate the

soma of a neuron while a mechanical probe is raised from below to indent the appropriate slit

from the outside. Step indentations under voltage clamp produce inward receptor currents that

saturate at a few micrometers. The receptor currents have an adapting component, but most of

the current adapts relatively slowly and incompletely. On the basis of data from Hoger et al.

(1997).


large external movements can be detected without damaging the hair.

The longer trichobothria are specialized to detect air movements, and their

varying lengths appear to be tuned to the fluid dynamics of air flow over

the spider surface, especially considering the boundary layer eVect. Estim-

ates of their sensitivity indicate that they can detect movements carrying

energy equivalent to a single photon of visible light and that they operate

close to the level of baseline thermal noise. They seem designed optimally to

detect turbulent air flow produced by rapidly moving prey, such as flying

insects, and their varying lengths and diameters provide tuning to diVerent

stimulation frequencies.

Slit sensilla are distributed in a wide range of patterns over the spider

body, from single, isolated slits to complex arrangements of multiple slits,

forming lyriform structures (Fig. 1). It is clear that slit sensilla respond to

strain in the exoskeleton, produced by the animals movements or by vibra-

tions conducted through the substrate. Measurements in models of spider leg

cuticle indicate that the slits are optimally positioned to detect strain at the

locations where it is maximized by normal loading and that slit orientations

are matched to the directions of maximum natural stress. Most compound

lyriform organs occur near the leg joints, while individual slits are often

found at points of muscle attachment to the cuticle (Barth, 2001). The fine

structure of an individual slit allows cuticular stress to apply a levered com-

pression to the tips of the sensory dendrites. This arrangement has some sim-

ilarities to the campaniform sensilla of insects, which seem to serve a similar

stressdetecting function but use singly innervated, circular structures.

The varying lengths of the slits in a lyriform organ (typically 8 to 200 mlong by 1 to 2 m wide) immediately suggest tuning to diVerent temporalfrequencies, as in the eponymous lyre. There is some evidence that this

occurs, but the varying lengths may also serve functions such as measuring

the relative intensity of the strain by progressive recruitment of diVerent slits

as strain increases (Barth, 2001).

V. MECHANOTRANSDUCTION IN SLIT SENSILLA

Spider slit sensilla have provided important experimental preparations

for research into mechanotransduction because of the following advantages.

(1) Their mechanical structures, while complex, are approximately two

dimensional and relatively amenable to analysis and stimulation. (2) The

exposed location of the sensory neurons inside the surface cuticle has

allowed the development of preparations in which simultaneous mechani-

cal stimulation and stable intracellular recording, including voltageclamp


recording can be conducted. (3) The sensory neurons are located within

a hypodermal membrane that allows them to be removed from the animal

intact. This has been particularly useful for studying their voltageactivated

conductances. (4) A complex eVerent innervation of the peripheral parts

of sensory neurons promises to shed new light into understanding how

mechanosensation is modulated.

The remainder of this chapter will focus on major findings about me-

chanotransduction, sensory encoding, and eVerent modulation of these

processes that have emerged from research on spider lyriform organs and

trichobothria.

A. The Ionic Selectivity of Spider Mechanosensitive Channels

Intracellular recording during mechanical stimulation has been achieved

in two spider leg lyriform organs, VS3 on the patella (Juusola et al., 1994)

and HS10 on the metatarsus (Gingl et al., 2006). In each case, all neurons

innervating the slits were found to be mechanosensitive. Voltageclamp

recording from the neuron cell bodies of VS3 revealed an inward, depolar-

izing receptor current with both adapting and longlasting components that

saturated with slit indentations of about 3 m (Fig. 2). Note that the slitindentation used in these experiments does not represent a natural stimulus.

Although the major functions of VS3 remain unclear, normal slit compres-

sion is presumably produced by cuticle strains. However, more natural

stimulation of HS10 was achieved by moving the tarsus and this gave very

similar results to the VS3 slit indentation.

The receptor current in VS3 neurons could not be reversed, even with

strong depolarization, and was completely eliminated when external sodium

was replaced by choline (Fig. 3). Further tests with the common monovalent

and divalent cations showed that, other than sodium, only lithium ions had

detectable, but much lower, permeation (Hoger et al., 1997). These experi-

ments indicate that spider mechanosensitive channels are highly selective for

sodium ions.

Further support for this selectivity comes from measurements of the ionic

composition of the solution in the lymph space that surrounds the dendrite

tips (Fig. 1). Comparable insect mechanoreceptors have a high concentration

of potassium ions in this region, as well as a potential that is positive

compared to the normal extracellular space (Thurm and Kuppers, 1980;

Grunert and Gnatzy, 1987), but in spiders this region not only lacks the high

potassium and positive potential but also has a relatively high concentration

of sodium ions (Rick et al., 1976).


B. The Location of VS3 Mechanosensitive Channels

The bipolar structure of arthropod cuticular mechanoreceptor neurons

(Fig. 2) has led to a long history of attempts to find the location of the mechan-

osensitive channels, as well as the location of the action potentialinitiating

region. Although the obvious location for transduction would seem to be at

the distal tips of the dendrites because of the close apposition to the initial

mechanical stimulus and the specialized electrochemical gradient of the lymph

space (Fig.1), there have also been theories that transduction occurs near the

ciliarybasalbodyand thatactionpotentialsmightarise in theaxosomatic region

(reviewed by French, 1988).

A direct test of the location of mechanotransduction was performed by

applying small punctate stimuli to diVerent locations along the dendrites of

VS3 neurons (Hoger and Seyfarth, 2001). Only stimuli applied to the distal

dendrites, close to the inner surface of the slits, produced electrical activity in

the neurons, suggesting a distal location.

The general direction of signal flow in a sensory receptor from distal to

proximal implies that transduction should occur either at the site of action

potential initiation or possibly distal to it. Gingl and French (2003) used

several techniques to locate the site of action potential initiation in VS3

neurons, including the voltage jumpmethod that measures collisions between

50 ms

50 pA

Control

Choline100 pA

100 pA

200 pA

300 pA

100 mV 100 mV

FIGURE 3 Receptor current is carried by sodium ions in VS3 neurons. Graph shows

typical peak receptor currents produced by step slit indentations of 3 m while the neuronalmembrane was held at diVerent potentials. Note the failure to reverse, even at strong positive

potentials. Replacement of the sodium ions in spider saline with the large choline cation

completely eliminated the receptor current, but it returned when the normal saline solution

was restored (control). On the basis of data from Hoger et al. (1997).


voltage waves started by the receptor potential and an artificially created

potential step at the soma. These measurements all indicated that transduc-

tion and action potential initiation both start at the distal end of the dendrite.

More recent work has directly observed action potentials flowing along the

dendrite from the distal tips (Gingl et al., 2004).

Although all these experiments support a distal location for the mechan-

osensitive channels, they cannot provide a more accurate position than

somewhere within about 50 m from the end of the dendrite. The basalbody occurs at the distal end of the dendritic enlargement in VS3 neurons

(Fig. 1), which is close to the lymph space. More accurate localization will

probably have to wait for better anatomical evidence such as antibodies to

the mechanosensitive channels.

C. Mechanosensitive Channel Conductance, Density, and pH Sensitivity

Singlechannel recordings of the mechanosensitive channels have not yet

been achieved. Patch clamp recording from VS3 neurons is complicated

by their locationwithin a hypodermalmembrane and extensive glial wrappings.

The probable location of the channels near the tip of the sensory dendrite adds

further diYculty. An alternative approach is to measure the variance, or noise,

of the total receptor current to estimate the singlechannel conductance and

number of channels (Traynelis and Jaramillo, 1998). This approach requires

current variance measurements over a range of diVerent current amplitudes,

which can be achieved by varying the stimulus used to open the channels being

investigated. In VS3 neurons the receptor current adapts slowly after a step

indentation of the slit, and this natural change in current was used to estimate

the mechanosensitive channel properties.

For a single group of identical ion channels, the total variance, s2, of the

current flowing through a membrane is given by:

s2 s20 IV Eg I2=N 1

where s02 is the background variance due to other sources, I is total mem-

brane current, V is the voltage across the membrane, E is the equilibrium

potential of the ions flowing through the channel, is the singlechannelconductance, and N is the number of channels in the membrane. Given the

singlechannel conductance and number of channels, the open probability of

the channels can be calculated from:

Po I

NV Eg2


Hoger and French (1999a) showed that the mechanosensitive channels

were almost completely open at the start of a step indentation, but then

closed with several time constants over a period of several minutes (Fig. 4).

Their singlechannel conductance estimate was about 7 pS and the number

of channels per neuron was about 470. Neither of these parameters was

sensitive to pH (Hoger and French, 2002). However, acid conditions signifi-

cantly raised the open probability of the channels, and hence the overall

receptor current.

From the estimated singlechannel conductance and number of channels,

total mechanosensitive conductance was calculated to be about 3.5 nS in a

single VS3 neuron. However, independent estimates of total charge flowing

during a step indentation gave a significantly higher estimate of about 15 nS

(Gingl and French, 2003). A possible cause of this diVerence lies in the cable

properties of the sensory dendrite. The measured length constant of the

sensory dendrites is about 200 m, which is comparable to the physicallength of the dendrites (Gingl and French, 2003). Although the noise mea-

surements were made at the neuronal resting potential to minimize the

current requirements of the voltage clamp, it is possible that the current

flowing through the mechanosensitive channels at the dendrite tip could

depolarize the membrane beyond the control of the voltage clamp in the

soma. This would reduce the estimated receptor current and its variance.

*

1.0

0.0

1.0

0.0

Popen

0 40Time (s)

pH 8 pH 5

n = 9 n = 23

FIGURE 4 Noise analysis and pH sensitivity of VS3 receptor current. Step indentations of

the slits lasting 40 s produced a slowly adapting receptor current. Noise analysis was used to

estimate the number of mechanosensitive ion channels, singlechannel conductance, and

channel open probability (Popen) during the step. Traces show Popen for a typical neuron at

pH 8 (approximately normal conditions) and at pH 5. Inset shows mean values of Popen at 36

s after the step under normal and acid conditions.Asterisk indicates p < 0.05. On the basis of data

from Hoger and French (2002).


Therefore, the singlechannel conductance of the mechanosensitive channels

could be 20 pS or more. This would be in better agreement with estimates

from mammalian auditory hair cells based on singlechannel recordings,

which are as high as 100 pS (Fettiplace et al., 1992).

D. Temperature Sensitivity of Mechanosensitive Channels

Mechanotransduction has been found to be more thermally sensitive than

would be predicted from simple ion channel conductance in a range of

vertebrate and invertebrate sensory receptors (reviewed in Hoger and

French, 1999b). Most of these measurements were made on the action

potential signals from sensory receptors so that the location of temperature

sensitivity could not be clearly established. The VS3 organ provided the first

direct measure of temperature sensitivity in the receptor current (Hoger and

French, 1999b). These data were wellfitted by the Arrhenius rate equation

to give a mean activation energy of 23 kcal/mol (97 kJ/mol or Q10 3.2 at20C). This is the highest activation energy measured for mechanotransduc-

tion, although close to measurements in other systems (Hoger and French,

1999b). It confirms the general finding that mechanotransduction involves a

significant energy barrier, comparable to the energy required to break a

covalent chemical bond. The reason for this relatively high activation energy

is not clear but is probably associated with the mechanism that links mecha-

nical stimulus to channel opening. It is much higher than the activation

energy required for ionic movement through a waterfilled channel or for the

production of action potentials by voltageactivated ion channels.

E. Molecular Characterization of Spider Mechanosensitive Channels

Two major groups of ion channel molecules have been associated with

sensory mechanotransduction. Members of the transient receptor poten-

tial (TRP) family of channels have been implicated in a range of sensory

functions of both vertebrates and invertebrates, including phototransduc-

tion, thermal transduction, mechanotransduction, pain, and osmosensation

(Minke and Cook, 2002; Corey, 2003; Maroto et al., 2005; Montell, 2005;

Dhaka et al., 2006; Kwan et al., 2006). TRP channels have been strongly

linked to hearing and touch in Drosophila (Kim et al., 2003; Gong et al.,

2004) and to touch in Caenorhabditis elegans (Goodman and Schwarz, 2003;

Li et al., 2006). TRP1 channels have been found in vertebrate pain receptors

(Kwan et al., 2006), as well as mouse, bullfrog, and zebrafish inner ear hair

receptors (Corey, 2003), appearing at the same embryonic stage as sound


sensitivity in mice (Lewin and Moshourab, 2004). However, a knockout

mouse lacking TRP1 had an impaired response to painful stimuli but its

hair cell transduction was not aVected (Kwan et al., 2006). None of the

evidence yet gives clear proof that these channels are the primary source of

the receptor current.

The other channel family associated with mechanotransduction are the

degenerin/acidsensitive/epithelial sodium channels (DEG/ASIC/ENaC),

best known for the amilorideblockable epithelial sodium channels that con-

duct sodium flux through a wide range of epithelia (Bianchi and Driscoll,

2002). In C. elegans, two of the four proteins found only in mechanoreceptor

cells are DEG molecules that have been proposed to form the core of the

mechanotransduction channel, and the receptor current was carried by

sodium ions (Goodman and Schwarz, 2003; Syntichaki and Tavernarakis,

2004). A DEG gene family was also associated with mechanosensitivity in

Drosophila larvae (Adams et al., 1998). In rodents, several members of the

DEG family have been found in dorsal root ganglia and in fine nerve endings

surrounding tactile hairs (Price et al., 2000). Knockout animals for one

channel, BNC1, showed reductions, but not elimination, of mechanosensa-

tion (Price et al., 2000), and none of these molecules have yet been identified

in well known skin mechanoreceptors, such as Pacinian corpuscles or RuYni

endings.

Although the molecular evidence favors TRP channels in Drosophila

mechanosensation (Kim et al., 2003), all the data from spider slit sensilla is

more supportive of ASIC channels. The receptor current is highly selective

for sodium and blocked by amiloride (Hoger et al., 1997). Mechanosensitive

channel open probability is strongly increased at low pH (Hoger and French,

2002). These are all characteristic properties of ASIC channels. In contrast,

TRP channels are quite strongly associated with calcium signaling, and at

least some sensory TRP channels are calcium permeable (Montell, 2005),

whereas spider mechanosensitive channels are probably not permeable to

calcium (Hoger et al., 2005).

Two other commonly proposed features of sensory mechanically acti-

vated channels are heteromeric construction and connections to extracellular

and intracellular structural proteins. Evidence from several preparations

indicates that multiple proteins are required to form functioning eukary-

otic mechanically activated channels, and this may explain the diYculty of

demonstrating mechanosensitivity from proteins expressed in oocytes or

other systems (Hamill and McBride, 1996; Emtage et al., 2004; Syntichaki

and Tavernarakis, 2004). Mechanical connections to cytoskeletal and extra-

cellular matrix structures have been proposed by several lines of evidence,

including the amino acid sequences of proposed channel molecules (Emtage

et al., 2004). It has also been argued that lipid membrane alone could not


provide enough force to open a protein channel (Sachs, 1997). Microtubules

are often prominent in mechanoreceptor endings, and in some cases have

been suggested to form a cytoskeletal anchor (Gillespie and Walker, 2001).

Spider slit sensilla, like other arthropod cuticular mechanoreceptors, contain

prominent arrangements of microtubules in the sensory dendrites that ex-

tend to the distal tips, but mechanotransduction in VS3 neurons and some

insect cuticular mechanoreceptors persists after pharmacological destruction

of microtubules (French, 1988; Hoger and Seyfarth, 2001).

VI. DYNAMIC PROPERTIES OF MECHANOTRANSDUCTION AND

ACTION POTENTIAL ENCODING

Recordings of action potentials from spider tactile hairs and trichobothria

show neurons that are normally silent, signaling brief touching or vibration

(Barth, 2004). Slit sensilla neurons are also silent in their resting condition and

respond preferentially to rapid changes. Each slit is innervated by two neu-

rons that have diVerent dynamic properties. Type A neurons are very rapidly

adapting, giving only one or two action potentials at the start of a step

indentation, while Type B neurons give a longer burst of action potentials

(Fig. 5). This pattern has been observed in both VS3 and HS10 lyriform

organs (Seyfarth and French, 1994; Gingl et al., 2006) so it probably

generalizes to most or all of the slit sensilla.

50 mV

10 mV

100 msType A Type B

FIGURE 5 Spider slit sensilla are innervated by pairs of functionally diVerent neurons.

Intracellular recordings are shown from the two neuron types in a VS3 preparation receiving

step indentations of 150ms duration. Upper traces show normal action potential responses

from Types A (left) and B (right) neurons. Lower traces show receptor potentials produced by

similar steps after action potentials were blocked by treatment with tetrodotoxin. On the basis of

data from Juusola and French (1998).


Recordings of the receptor current (Fig. 2) or the receptor potential

(Fig. 5) do not show such strong adaptation or such a diVerence between

the two neuron types (Juusola and French, 1998). Receptor potential in the

Type A neurons does adapt more rapidly than in Type B neurons, but the

diVerence is less dramatic than the firing behavior. This diVerence in action

potential encoding can also be seen with direct electrical stimulation of the

neurons, and can be explained by diVerences in the inactivation properties of

the voltageactivated sodium channels that cause the initial phase of the

action potentials (Torkkeli and French, 2002).

The time course of the receptor current and potential must be controlled

by the combination of mechanical coupling components and mechanosensi-

tive ion channels. However, little is known about the dynamic properties of

either. Somatic measurements indicate that the receptor current decays with

at least two time constants (Fig. 2), and voltage jump experiments indicated

that there are larger, very transient components occurring in the distal

dendrites (Gingl and French, 2003). It is possible that these diVerent time

constants represent separate filtering by the mechanical components and the

mechanosensitive ion channels. The existing evidence is compatible with

the most parsimonious model of transduction, that is, that a single type of

mechanosensitive channel is present in both Types A and B neurons.

VII. CALCIUM SIGNALING DURING TRANSDUCTION BY

SPIDER MECHANORECEPTORS

The membranes of VS3 neurons contain lowvoltageactivated calcium

selective ion channels (Sekizawa et al., 2000). Measurements of intracellular

calcium concentration during mechanical stimulation of the slits showed

that calcium rises from a resting level of about 400 nM to a maximum level

of about 2 M during rapid action potential firing (Hoger et al., 2005).These experiments failed to show any change in calcium concentration with-

out action potentials, even when there was a receptor potential of 10 mV

amplitude or more, confirming that the mechanosensitive ion channels are

not significantly permeable to calcium. They also failed to show any release

of calcium from internal stores. The amount of calcium entering during

action potential firing was compatible with the estimated conductance via

voltageactivated calcium channels.

These data raise the question of what role the elevation of calcium plays

during normal sensory transduction. There are no known calciumsensitive

ion channels in VS3 neurons, and blockade of calcium entry does not reliably

aVect action potential firing. Calcium rose by similar amounts throughout

the VS3 neurons, but with diVerent time courses in diVerent regions


(Fig. 6), suggesting that calcium channels are distributed throughout the cells.

One possible role for calcium would be regulation of the mechanosensitive

channels. Calcium ions play major roles in controlling the dynamic properties

of auditory hair cells, and at least some of the time constants involved seem to

depend on intracellular actions of calcium on mechanosensitive ion channels

(Ricci et al., 2005).

VIII. SYNAPTIC MODULATION OF SPIDER MECHANORECEPTORS

An interesting feature of arachnid mechanoreceptors is that even their

most peripherally located parts receive extensive and complex eVerent inner-

vation (Foelix, 1975; FabianFine et al., 2002), allowing an early modulation

of the neuronal responses to mechanical stimuli. Several fine eVerent fibers

in the legs of C. salei extend along the sensory nerves all the way to the tips

of the sensory dendrites. They form many types of synaptic contacts with

the sensory neurons, the glial cells that enwrap the sensory neurons, and they

also synapse with other eVerents (FabianFine et al., 2002). The eVerent

fibers have been shown to contain a variety of transmitters, including

aminobutyric acid (GABA), glutamate, acetylcholine (ACh), and octopa-mine (Fig. 7; FabianFine et al., 2002; Widmer et al., 2005), and the mechan-

osensory neurons respond to these transmitters (Panek et al., 2002; Panek

500 nM

50 s

Distaldendrite

Middendrite

Soma Soma

FIGURE 6 Calcium concentration rises significantly in VS3 neurons when they are firing.

Traces show calcium elevations in diVerent regions during stimulation at 10 action potentials per

second. Resting calcium concentration was about 400 nM in all regions and the increases in

diVerent regions were not significantly diVerent. However, the time course of elevation was

significantly slower in the soma, as shown by the traces. On the basis of data from Hoger et al.

(2005).


and Torkkeli, 2005; Widmer et al., 2005, 2006). In addition, antibodies

against transmitter receptors labeled specific sites on the sensory neurons

(Panek et al., 2003, 2005;Widmer et al., 2005, 2006; Fig. 7).

GABA and glutamate both act on inhibitory ionotropic receptors that are

Clgated ion channels. Although both transmitters blocked VS3 neurons

responses to mechanical stimuli, GABA had a significantly stronger eVect

than glutamate (Panek and Torkkeli, 2005). However, GABA only inhibited

axonal action potentials while the glutamate eVect involved both dendritic

and axonal action potentials and it also reduced the receptor current ampli-

tude (Gingl et al., 2004; Panek and Torkkeli, 2005). Thus, glutamatergic

eVerents may control the cellular response to mechanical stimuli at earlier

stages than GABAergic eVerents. The VS3 neurons also have metabotropic

GABAB receptors, concentrated on the most distal parts of the cell bodies

and on the dendrites (Panek et al., 2003). Agonists of these receptors

modulated voltageactivated calcium and potassium currents, allowing a

longer lasting modulatory eVect.

Inhibitory glutamate receptor

GABAB receptor

Octopamine receptor

Inhibitory GABA receptor

DendriteAxon

GABA

Octopamine

Glutamate

Soma

ACh

mACh receptor

Inhibitory ACh receptor

?

?

?

??

?

?

?

AChE

+

+

Inhibitory+ Excitatory

?

+

????

FIGURE 7 Schematic illustration of the arrangement of eVerent neurons and transmitter

receptors on a Type A spider VS3 neuron based on immunocytochemical and electrophysio-

logical evidence. The eVerent fibers contain GABA, glutamate, octopamine, and ACh. The

sensory neurons have inhibitory ionotropic GABA and glutamate receptors and excitatory

octopamine receptors. Type A neurons also have inhibitory ionotropic ACh receptors and they

express acetylcholine esterase (AChE) activity. In addition, metabotropic GABAB and musca-

rinic ACh receptors are found in all VS3 neurons, but their physiological functions are

unknown. Glutamate and mACh receptors are also present in the eVerent fibers. On the basis

of data from FabianFine et al. (2002), Panek et al. (2002, 2003, 2005), Gingl et al. (2004), Panek

and Torkkeli (2005), Widmer et al. (2005, 2006).


Application of octopamine, the invertebrate analogue of noradrenaline,

enhanced trichobothria neuron sensitivity to mechanical stimuli (Widmer

et al., 2005). Immunocytochemical evidence indicated that one eVerent fiber

containing octopamine innervated each mechanosensory neuron in the spider

leg and that octopamine receptors were concentrated at and close to the axon

hillock (Widmer et al., 2005). These findings suggest that octopamine acts as

a transmitter rather than a neurohormone on spider mechanoreceptors,

controlling each sensory neuron individually.

These recent findings only unravel a small part of the complex synaptic

mechanisms that control the sensitivity and gain of spider mechanosensory

neurons. For example, we still know very little about the cholinergic inner-

vation that involves both muscarinic ACh receptors and ionotropic inhibi-

tory receptors and is distinctly diVerent in the two diVerent types of VS3

neurons.

IX. CONCLUSIONS

Spider mechanoreceptors have yielded a great deal of information about

their mechanosensitive ion channels and their mechanisms of activation and

modulation. However, much remains to be discovered. The electrophysio-

logical data from slit sensilla suggest that the channel molecules are related

to ASIC channels and they are probably located near the tips of the sensory

dendrites. The relatively low numbers of channel molecules per cell are one

reason why molecular characterization has so far proved elusive as it has in

other mechanoreceptor systems. However, the spider preparations should

continue to provide useful models for identifying the molecular basis of

mechanosensation and this knowledge can be expected to assist the broader

investigation of this crucial sense in animals and humans.

AcknowledgmentsWe thank Ewald Gingl, Ulli Hoger, Mikko Juusola, Izabela Panek, ShannonMeisner, Ernst

August Seyfarth, and Alexandre Widmer for all their contributions to work described here.

Research in our laboratories has been funded by the Canadian Institutes of Health Research,

the Natural Sciences and Engineering Council of Canada, NATO, the Canadian Foundation

for Innovation, the Nova Scotia Research and Innovation Trust, and the Dalhousie Medical

Research Foundation.

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CHAPTER 2

Ion Channels for Mechanotransductionin the Crayfish Stretch Receptor

Bo Rydqvist

Department of Physiology and Pharmacology, Karolinska Institutet,

SE171 77 Stockholm, Sweden

I. Overview

II. Introduction

III. Morphology of the SRO

IV. Functional Properties

A. General Behavior

B. Viscoelastic Properties of the Receptor Muscles

C. MSCs in the Receptor Neurons

D. Macroscopic Receptor Currents in the Stretch Receptor Neurons

E. Pharmacology of the Crayfish MSCs

F. VoltageGated Ion Channels and the Generation of Impulse Response

G. Adaptation: A Multifactor Property

V. Summary and Discussion of Future Research Directions

References

I. OVERVIEW

Mechanosensitivity is found in almost every cell in all organisms from

bacteria to vertebrates and covers a wide spectrum of function from osmo-

sensing to mechanical sensing in the specialized receptors like the hair cells of

the cochlea. The molecular substrate for such mechanosensitivity is thought

to be mechanosensitive ion channels (MSCs). Since most development

regarding the molecular aspects of the MSC has been made in nonsensory

or sensory systems which have not been accessible to recordings from ion

channels, it is important to focus on mechanosensitivity of sensory organs

where their functional importance is undisputed. The stretch receptor organ

Current Topics in Membranes, Volume 59 1063-5823/07 $35.00Copyright 2007, Elsevier Inc. All right reserved. DOI: 10.1016/S1063-5823(06)59002-7

(SRO) of the crustaceans is a suitable preparation for such studies. Each

organ contains two receptors: one slowly and one rapidly adapting receptor

neurons. The primary mechanosensitivity is generated by two types of MSC

of hitherto unknown molecular type located in the neuronal dendrites, which

are inserted into a receptor muscle fiber. In addition to the MSCs, the

neurons contain voltagegated Na channels which seem to be diVerently

located in the slowly and rapidly adapting neurons. Finally, at least three

types of voltagegated K channels are present in the sensory neurons, the

location of which is not known. The spatial distribution of ion channels

and the kinetics of the channels, together with the viscoelastic properties

of the receptor muscles, determine the overall transducer properties and

impulse firing of the two receptor neurons including their typical adaptive

characteristics.

II. INTRODUCTION

The crustacean SRO has been amajor preparation for the study ofmechan-

otransduction both on the macroscopic and on the ion channel levels. The

SRO is considered to be an organ analogous to themammalianmuscle spindle

organ that is instrumental for proper skeletal muscle function. The receptor

organ was first described in the lobster by the PolishBritish zoologist

Alexandrowicz (1951, 1967). Later, Florey and Florey (1955) described the

same type of organ in the crayfish (Astacus fluviatilis presently namedAstacus

astacus). Identical and similar muscle receptor organs can also be found in

a number of other invertebrate phyla such as Mollusca, Chelicerata, and

Uniramia (for a review see Rydqvist, 1992). The importance of this organ,

and its accessibility relative to the human muscle spindle, and mechanotrans-

duction in general, was soon acknowledged and triggered a number of electro-

physiological studies in several laboratories (Wiersma et al., 1953; KuZer,

1954; Eyzaguirre and KuZer, 1955a,b; Edwards and Ottoson, 1958).

The mechanosensory neurons of the SRO of the crayfish are of the

nonciliated type and are diVerent from the ciliated type represented by the

classical hair cells in the hearing organs. Most investigations regarding

the molecular aspects of the MSC have been made in nonsensory systems,

and it is thus important to focus on mechanosensitivity of sensory organs

where the functional importance of these channels is undisputed. The SRO

of the crustaceans is a suitable preparation for such studies. The SRO is

experimentally accessible to mechanical manipulation and electrophysiolog-

ical recordings using intracellular microelectrodes or patch clamp techniques

for ion channel analysis, although the latter technique is not without prob-

lems since the sensory neuron is covered by supporting glial cells. It is,

22 Bo Rydqvist

however, relatively easy to inject substances into the neuron, which makes

the neuron accessible to measurements using fluorescent probes.

In the present chapter, I have focused on the overall function of the

SRO in the crayfish stretch receptor including results obtained with struc-

tural techniques, classical electrophysiology, and patch clamp techniques.

The main emphasis will be on the mechanotransduction processes and the

ion channels involved in the SRO of the species A. astacus, Pacifastacus

leniusculus, Procambarus clarkii, and Orconectes limosus.

III. MORPHOLOGY OF THE SRO

Since the crayfish stretch receptor is a genuine mechanosensory organ

with several ion channels involved in the overall mechanotransduction, a

brief description of the SRO seems relevant. The SRO has two sensory

neurons, each connected to a receptor muscle, located in the extensor

muscles of the abdomen (Florey and Florey, 1955; Purali, 2005). The sensory

neuron is of the multipolar type (Fig. 1) with its dendrites inserted into the

central (intercalated) part of the receptor muscle which consists of only one

muscle cell (TaoCheng et al., 1981). The receptor muscles insert on consec-

utive segments and the aVerent axons from the neurons join the dorsal

segmental nerve to the ventral ganglion. The SROs also receive eVerent

innervations: (1) one or two motor axons to the receptor muscle cell and

(2) two or three accessory axons conveying inhibitory signals to the receptor

muscle and the sensory neurons (Alexandrowicz, 1951, 1967; Elekes and

Florey, 1987a,b). Functionally, the receptors are activated when stretched by

flexion of the abdomen or contraction of the receptor muscles (KuZer, 1954)

and the SROs are involved in the control of the extensor muscles.

In the crayfish, both the slowly and the rapidly adapting receptor muscles

consist of a single muscle fiber that is divided by invagination of the cell

membrane into numerous cytoplasmic processes in the central region of the

muscle, the intercalated tendon, which is mainly made up of collagen. Some

of the myofibrils insert in the intercalated tendon but some pass this region.

The slowly adapting muscle is in the order of 3080 mm in the central region

but considerably thinner in the distal ends. The rapidly adapting muscle has

a more even diameter and is thicker 70150 mm (Komuro, 1981).

The sensory neurons are large (30100 mm) multiterminal cells of mainly

pyramidal or fusiform shape. They contain a nucleus (ca. 10 mm) with a clear

nucleolus. The dendrites branch about four to five times and intermingle

with the connective tissue and muscle strands in the intercalated tendon. The

fine terminal branches are about 2mm long and about 0.l mm in diameter and

are devoid of mitochondria (TaoCheng et al., 1981). The axon is in the

2. Stretch Receptor Mechanotransduction 23

order of 30 mm in diameter. The receptor neurons of the crayfish have several

layers of sheet cells that surround them except for the dendritic tips.

The fine structure of the inhibitory synapses has been investigated by

several authors (Elekes and Florey, 1987a,b) using serial sectioning and

immunohistochemical technique, which have revealed a complex array of

GABAergic inhibitory synapses on the axon, neuron, and muscle fibers and

also reciprocal synapses on the inhibitory axon.

IV. FUNCTIONAL PROPERTIES

A. General Behavior

The receptors are activated (stretched) by flexion of the abdomen or

contraction of the receptor muscle. The receptors are involved in the motor

control of the abdominal muscles and the physiological range is up to 40%

of resting length (Alexandrowicz, 1951). The first measurements from the

FIGURE 1 (A) Abdomen and thorax of crayfish. (B) Slowly (top) and rapidly (bottom)

adapting receptor with typical action potential firing pattern as a result of a ramp and hold

extension of the receptor muscle. (C) Confocal microscopic image of a slowly adapting neuron

injected with Fluo4 (Bruton and Rydqvist, unpublished data), a recurrent fiber is present at left.

(D) Drawing of stretch receptor neuron with proposed channel distribution. (A, B) Adapted

with kind permission from Springer Science and Business Media (Rydqvist, 1992).

24 Bo Rydqvist

receptor neuron were done by Wiersma et al. (1953) and KuZer (1954)

and subsequent studies by Eyzaguirre and KuZer (1955a,b) and Edwards

and Ottoson (1958) on lobster and crayfish showed that stretching the re-

ceptor organs gave rise to a distinctive pattern of impulse discharge from the

neurons. It was found that the firing properties of the two neurons were

clearly diVerent, one neuron maintained firing as long as the stretch was

applied (slowly adapting) whereas the other neuron generated a short high

frequency discharge (rapidly adapting) at the onset of the stretch (Fig. 1B).

The chain of events that leads from extension of the receptor muscle to

action potential generation in the stretch receptor is represented by the steps

outlined in Fig. 2. In a first step, the extension of the receptor muscle is

Actionpotential

50 mV

100 nA

100 kPa

Na+

K+

cm gL

100 ms

ReceptorpotentialTTX

Receptorcurrent

SA-channels

ViscoelasticReceptormuscle

Voltage-gatedchannels

Passivemembrane

DendritesMuscletension

Extension

ReceptorpotentialTTX, 4AP,TEA

FIGURE 2 Transduction processes in a stretch receptor neuron. Left: recorded responses of

muscle tension, receptor current, receptor potential, and action potentials in response to a ramp

and hold extension of the muscle. The receptor potential is seen both after block of Na

channels with tetrodotoxin (TTX) and after additional block of K channels with tetraethyl-

ammonium chloride (TEA) and 4aminopyridine (4AP). Right: functional blocks in transduc-

tion. Stretchactivated channels; SA channels, MSC. Adapted from Swerup and Rydqvist, 1992

with permission from Elsevier.

2. Stretch Receptor Mechanotransduction 25

converted to tension in the muscle, which leads to deformation of the

dendritic membrane of the sensory neuron. This opens nonselective mechan-

osensitive (gated) ion channels (MSCs) permeable to Na, K, and Ca2

ions producing an inward generator current (Erxleben, 1989). The transfor-

mation from generator current to impulse response is a complex process

determined by the passive membrane properties, that is capacitance (cm) and

membrane resistance (rm or leak conductance gL), and the voltagegated ion

conductances (gion) present in the neuron. At present only voltagegated K

and Na channels and a Ca2activated K channel have been observed.

Figure 2 shows the receptor potential after block by tetrodotoxin (TTX),

4aminopyridine (4AP), and tetraethylammonium chloride (TEA), and the

receptor potential after block with TTX only. In addition, the geometry of

the cell and the spatial distribution of the diVerent ion channels will contrib-

ute to the type of impulse response seen in the cell. The diVerence in respon-

ses reflects the relativity of the concept of receptor potential (see discussion

in Swerup and Rydqvist, 1992).

B. Viscoelastic Properties of the Receptor Muscles

In most mechanosensory cells, the accessory structures contribute to the

overall behavior of the transducer function. In particular, it has been dis-

cussed to what extent the accessory structures contribute to the adaptation

in sensory cells. This is obvious in, for example, the Pacinian corpuscle. The

two neurons in the SRO have diVerent adaptive properties and this could

arise solely from possible diVerences in passive mechanical properties in the

two receptor muscles. Earlier studies (KuZer, 1954) observed that the con-

tractile properties indeed diVered; the rapidly adapting muscle had proper-

ties resembling a fast twitch fiber, whereas the slowly adapting muscle

behaved as a slo

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Mechanosensitive Ion Channels, Part B, Volume 59 (Current Topics in Membranes)