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Neuromethods 211

Jaromir Myslivecek
Jan Jakubik Editors

Muscarinic
Receptor
From Structure to Animal Models
Second Edition
NEUROMETHODS

Series Editor
Wolfgang Walz
University of Saskatchewan
Saskatoon, SK, Canada

For further volumes:

http://www.springer.com/series/7657
Neuromethods publishes cutting-edge methods and protocols in all areas of neuroscience as
well as translational neurological and mental research. Each volume in the series offers tested
laboratory protocols, step-by-step methods for reproducible lab experiments and addresses
methodological controversies and pitfalls in order to aid neuroscientists in experimentation.
Neuromethods focuses on traditional and emerging topics with wide-ranging implications to
brain function, such as electrophysiology, neuroimaging, behavioral analysis, genomics,
neurodegeneration, translational research and clinical trials. Neuromethods provides investi-
gators and trainees with highly useful compendiums of key strategies and approaches for
successful research in animal and human brain function including translational “bench to
bedside” approaches to mental and neurological diseases.
 Muscarinic Receptor

From Structure to Animal Models

Second Edition

Edited by

Jaromir Myslivecek
Institute of Physiology, Charles University, Prague, Czech Republic

Jan Jakubik
Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Editors
Jaromir Myslivecek Jan Jakubik
Institute of Physiology Institute of Physiology
Charles University Academy of Sciences of the Czech Republic
Prague, Czech Republic Prague, Czech Republic

ISSN 0893-2336 ISSN 1940-6045 (electronic)

Neuromethods
ISBN 978-1-0716-4014-2 ISBN 978-1-0716-4015-9 (eBook)
https://doi.org/10.1007/978-1-0716-4015-9
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Preface to the Series

Experimental life sciences have two basic foundations: concepts and tools. The Neuro-
methods series focuses on the tools and techniques unique to the investigation of the
nervous system and excitable cells. It will not, however, shortchange the concept side of
things as care has been taken to integrate these tools within the context of the concepts and
questions under investigation. In this way, the series is unique in that it not only collects
protocols but also includes theoretical background information and critiques which led to
the methods and their development. Thus, it gives the reader a better understanding of the
origin of the techniques and their potential future development. The Neuromethods
publishing program strikes a balance between recent and exciting developments like those
concerning new animal models of disease, imaging, in vivo methods, and more established
techniques, including, for example, immunocytochemistry and electrophysiological tech-
nologies. New trainees in neurosciences still need a sound footing in these older methods in
order to apply a critical approach to their results.
Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods
series has been a success since its first volume published through Humana Press in 1985. The
series continues to flourish through many changes over the years. It is now published under
the umbrella of Springer Protocols. While methods involving brain research have changed a
lot since the series started, the publishing environment and technology have changed even
more radically. Neuromethods has the distinct layout and style of the Springer Protocols
program, designed specifically for readability and ease of reference in a laboratory setting.
The careful application of methods is potentially the most important step in the process
of scientific inquiry. In the past, new methodologies led the way in developing new dis-
ciplines in the biological and medical sciences. For example, Physiology emerged out of
Anatomy in the nineteenth century by harnessing new methods based on the newly discov-
ered phenomenon of electricity. Nowadays, the relationships between disciplines and meth-
ods are more complex. Methods are now widely shared between disciplines and research
areas. New developments in electronic publishing make it possible for scientists that
encounter new methods to quickly find sources of information electronically. The design
of individual volumes and chapters in this series takes this new access technology into
account. Springer Protocols makes it possible to download single protocols separately. In
addition, Springer makes its print-on-demand technology available globally. A print copy
can therefore be acquired quickly and for a competitive price anywhere in the world.

Saskatoon, SK, Canada Wolfgang Walz

v
Preface

Muscarinic receptors are involved in physiological events like cognitive processes, motor
coordination, attention, circadian rhythms, food reinforcement, drug addiction, and synap-
tic plasticity. This book provides methodology for the study of muscarinic receptors at the
structural to systemic level. The chapters are primarily intended as a resource for scientists
who want to deploy protocols to study muscarinic receptors quickly, easily, and properly.
One of the possible snags is the lack of subtype selective ligands that makes studies targeted
to specific subtype problematic. One of the methodological approaches for subtype identifi-
cation in tissues and organs is immunohistochemical or Western blot analysis of muscarinic
receptors. Unfortunately, the selectivity of antibodies is usually poor and antibodies also
target nonfunctional and degraded receptors limiting these techniques substantially. Thus,
these methods do not provide assessment of real number of receptors. Moreover, some
artifacts can originate from tissue preparation. Studying receptors in their natural environ-
ment can mitigate this problem.
In this second edition, we bring updated protocols on basic techniques of radioligand
binding, allosteric modulation, visualization, and antibody use, as well as autoradiography
and non-neuronal acetylcholine detection. We bring new protocols on modern advanced
fluorescent techniques including fluorescence anisotropy. Further, we present systemic
measurements of heart contractility and gastrointestinal tract motility as well as telemetric
measurements of animal activity, temperature, and heart rate affected by muscarinic recep-
tors to give a comprehensive picture of current research of muscarinic receptors from
molecular to systemic level.

Prague, Czech Republic Jaromir Myslivecek

Jan Jakubik

vii
Contents

Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 Radioligand Binding at Muscarinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Jan Jakubı́k and Esam E. El-Fakahany
2 Tissue-Segment Binding Method for Detection of Muscarinic Acetylcholine
Receptors in Receptor’s Natural Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Ikunobu Muramatsu, Junsuke Uwada, and Takayoshi Masuoka
3 Use of Antibodies in the Research on Muscarinic Receptor Subtypes . . . . . . . . . . 43
Wisuit Pradidarcheep, Vichununt Kerdput, and Martin C. Michel
4 Allosteric Modulation of Ligand Binding to Muscarinic Receptors . . . . . . . . . . . . 59
Jan Jakubı́k and Esam E. El-Fakahany
5 Allosteric Modulation of Functional Response of Muscarinic Receptors. . . . . . . . 89
Jan Jakubı́k and Esam E. El-Fakahany
6 Exploring Muscarinic Acetylcholine Receptor Binding Kinetics with
Fluorescence Anisotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Tõnis Laasfeld, Maris-Johanna Tahk, Anni Allikalt, Jane Torp,
Lukas Gr€ a tz, Sergei Kopanchuk, and Ago Rinken
7 MultiBacMam Technology for Studying the Downstream cAMP Signaling
Pathway of M2 Muscarinic Acetylcholine Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Anni Allikalt, Santa Veiksina, Maris-Johanna Tahk, Edijs Vavers,
Elen Laanev€ a li, Ago Rinken, and Sergei Kopanchuk
8 Subcellular and Synaptic Distribution of Muscarinic Receptors in Neurons
by Confocal and Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Véronique Bernard
9 Investigation of Muscarinic Receptors by Fluorescent Techniques. . . . . . . . . . . . . 199
Cornelius Krasel and Moritz Bünemann
10 Autoradiography Assessment of Muscarinic Receptors in the Central Nervous
System with a Special Focus on the Selectivity for M1 and M2 Muscarinic
Receptors: Specific Protocol for M1 Muscarinic Receptors Labeling . . . . . . . . . . . 213
Jaromir Myslivecek and Vladimir Farar
11 Detection of Non-neuronal Acetylcholine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Ignaz Karl Wessler and Charles James Kirkpatrick
12 Utilization of Superfused Cerebral Slices in Probing Muscarinic Receptor
Autoregulation of Acetylcholine Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Glenda Alquicer, Vladimı́r Doležal, and Esam E. El-Fakahany

ix
x Contents

13 Evaluation of Acetylcholine Synthesis and Release in Striatal Cholinergic

Interneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Ikunobu Muramatsu, Itsumi Arakawa, Junsuke Uwada,
Noriyuki Matsukawa, and Takayoshi Masuoka
14 Regulation of Heart Contractility by M2 and M3 Muscarinic Receptors:
Functional Studies Using Muscarinic Receptor Knockout Mouse . . . . . . . . . . . . . 281
Takio Kitazawa, Hiroki Teraoka, Nao Harada, Kenta Ochi,
Tatsuro Nakamura, Koichi Asakawa, Shinya Kanegae, Noriko Yaosaka,
Toshihiro Unno, Sei-ichi Komori, and Masahisa Yamada
15 Muscarinic Regulation of Gastrointestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Yasuyuki Tanahashi, Takio Kitazawa, and Toshihiro Unno
16 Systems for a Long-Term Record of Animal Activity, Temperature,
and Heart Rate Affected by Muscarinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Jaromir Myslivecek and Katerina Janisova

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Contributors

ANNI ALLIKALT • Institute of Chemistry, University of Tartu, Tartu, Estonia

GLENDA ALQUICER • Institute of Physiology, Czech Academy of Sciences, Prague, Czech
Republic
ITSUMI ARAKAWA • Division of Genomic Science and Microbiology, School of Medicine,
University of Fukui, Eiheiji, Fukui, Japan; Department of Neurology, Nagoya City
University Graduate School of Medicine, Nagoya, Aichi, Japan
KOICHI ASAKAWA • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
VÉRONIQUE BERNARD • Neuroscience Paris Seine, Institut de Biologie Paris Seine (IBPS)-
Université Pierre at Marie Curie UM 119 – CNRS UMR 8246 – INSERM U1130, Paris,
France
MORITZ BÜNEMANN • Institute of Pharmacology and Clinical Pharmacy, Philipps-University
Marburg, Marburg, Germany
VLADIMÍR DOLEŽAL • Institute of Physiology, Czech Academy of Sciences, Prague, Czech
Republic
ESAM E. EL-FAKAHANY • Department of Experimental and Clinical Pharmacology,
University of Minnesota College of Pharmacy, Minneapolis, MN, USA
VLADIMIR FARAR • Institute of Physiology, 1st Faculty of Medicine, Charles University,
Prague, Czech Republic
LUKAS GRA€ TZ • Department of Physiology and Pharmacology, Section of Receptor Biology and
Signaling, Karolinska Institutet, Stockholm, Sweden
NAO HARADA • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
JAN JAKUBÍK • Institute of Physiology, Academy of Sciences of the Czech Republic, Prague,
Czech Republic
KATERINA JANISOVA • Institute of Physiology, 1st Faculty of Medicine, Charles University,
Prague, Czech Republic
SHINYA KANEGAE • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
VICHUNUNT KERDPUT • Department of Anatomy, Faculty of Medicine, Srinakharinwirot
University, Bangkok, Thailand
CHARLES JAMES KIRKPATRICK • Institute of Pathology, University Medical Center, Johannes-
Gutenberg Universitat (Retired), Mainz, Germany
TAKIO KITAZAWA • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
SEI-ICHI KOMORI • Laboratory of Pharmacology, Faculty of Applied Biological Sciences, Gifu
University, Gifu, Japan
SERGEI KOPANCHUK • Institute of Chemistry, University of Tartu, Tartu, Estonia
CORNELIUS KRASEL • Institute of Pharmacology and Clinical Pharmacy, Philipps-University
Marburg, Marburg, Germany
ELEN LAANEVA€ LI • Institute of Chemistry, University of Tartu, Tartu, Estonia
TÕNIS LAASFELD • Institute of Chemistry, University of Tartu, Tartu, Estonia

xi
xii Contributors

TAKAYOSHI MASUOKA • Department of Pharmacology, School of Medicine, Kanazawa

Medical University, Uchinada, Ishikawa, Japan
NORIYUKI MATSUKAWA • Department of Neurology, Nagoya City University Graduate School
of Medicine, Nagoya, Aichi, Japan
MARTIN C. MICHEL • Department of Pharmacology, Johannes Gutenberg University, Mainz,
Germany
IKUNOBU MURAMATSU • Department of Pharmacology, School of Medicine, Kanazawa
Medical University, Uchinada, Ishikawa, Japan; Division of Genomic Science and
Microbiology, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan
JAROMIR MYSLIVECEK • Institute of Physiology, 1st Faculty of Medicine, Charles University,
Prague, Czech Republic
TATSURO NAKAMURA • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
KENTA OCHI • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido,
Japan
WISUIT PRADIDARCHEEP • Department of Anatomy, Faculty of Medicine, Srinakharinwirot
University, Bangkok, Thailand
AGO RINKEN • Institute of Chemistry, University of Tartu, Tartu, Estonia
MARIS-JOHANNA TAHK • Institute of Chemistry, University of Tartu, Tartu, Estonia
YASUYUKI TANAHASHI • Department of Frontier Life Sciences, Faculty of Life Sciences, Kyoto
Sangyo University, Kita-Ku, Kyoto, Japan
HIROKI TERAOKA • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
JANE TORP • Institute of Chemistry, University of Tartu, Tartu, Estonia; Institute of
Structural Biology, University of Bonn, Bonn, Germany
TOSHIHIRO UNNO • Laboratory of Pharmacology, Faculty of Applied Biological Science, Gifu
University, Gifu, Japan
JUNSUKE UWADA • Department of Pharmacology, School of Medicine, Kanazawa Medical
University, Uchinada, Ishikawa, Japan
EDIJS VAVERS • Institute of Chemistry, University of Tartu, Tartu, Estonia
SANTA VEIKSINA • Institute of Chemistry, University of Tartu, Tartu, Estonia
IGNAZ KARL WESSLER • Institute of Pathology, University Medical Center, Johannes-
Gutenberg Universitat, Mainz, Germany
MASAHISA YAMADA • Common Resources Group, Okinawa Institute of Science and
Technology, Okinawa, Japan
NORIKO YAOSAKA • School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,
Hokkaido, Japan
 Chapter 1

Radioligand Binding at Muscarinic Receptors

Jan Jakubı́k and Esam E. El-Fakahany

Abstract
Five subtypes of muscarinic acetylcholine receptors denoted M1 through M5 mediate a wide array of
physiological functions. Therefore, impairment of muscarinic signaling is involved in numerous diseases
and pathological conditions including Alzheimer’s disease, schizophrenia, and Parkinson’s disease. Musca-
rinic receptors are also pharmacological targets for the treatment of asthma and chronic obstructive
pulmonary disease. Radioligand binding techniques are orthodox but allow a very precise study of the
involvement of individual muscarinic receptor subtypes in the physiology and pathology of muscarinic
signaling, the study of the structure of muscarinic receptors and the structure-activation relationship of
muscarinic ligands. Here we discuss the current state of knowledge of radioligand binding experiments at
muscarinic receptors from the perspective of available radioligands and selective unlabeled muscarinic
ligands. We relate the binding properties of muscarinic ligands to experimental design (e.g., non-specific
binding determination, incubation conditions, buffers, temperature, etc.). We also list tissue/cell sources of
muscarinic receptors suitable for radioligand binding studies and describe procedures of cell and tissue
preparation for radioligand binding experiments. We also discuss several techniques of receptor-bound
ligand separation applicable to muscarinic receptors and provide basic information for binding data analysis.

Key words Muscarinic acetylcholine receptors, Radioligand binding

1 Historical Background

Radioligand binding methods are a cornerstone of receptor phar-

macology, taking muscarinic acetylcholine receptors as an example.
The main principle of the method is to allow a radiolabelled com-
pound specific to a given receptor to incubate with a biological
sample enriched with that receptor, then separate the bound and
free radioligand. Many radiolabelled muscarinic ligands with high
affinity and specific activity are currently available. Development of
a reliable radioligand binding technique at muscarinic receptors
cleared the way for identification, purification and subsequent
sequencing of the first muscarinic receptor [1] that enabled the
ensuing cloning of five subtypes of muscarinic receptors (M1

Jaromir Myslivecek and Jan Jakubik (eds.), Muscarinic Receptor: From Structure to Animal Models, Neuromethods, vol. 211,
https://doi.org/10.1007/978-1-0716-4015-9_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

1
2 Jan Jakubı́k and Esam E. El-Fakahany

through M5) [2] that revolutionized the field of research of musca-

rinic receptor pharmacology. Furthermore, radioligand binding
played a key role in identifying the orthosteric ligand binding site
that is located in a pocket formed by transmembrane helices [3, 4],
receptor subtype-specific ligands, allosteric modulators, and
structure-activation relationships. Radioligand binding may also
be employed in sophisticated experiments to study the kinetics of
drug-receptor interactions or in combination with site-directed
mutagenesis to determine the role of specific residues and domains
in ligand binding. Similar to other receptor targets, radioligand
binding studies at muscarinic receptors are simple and if performed
correctly are very sensitive and highly accurate.

2 Principles of Ligand Binding

2.1 Ligand Binding The interaction of a small molecule (ligand) with a protein (recep-
Definition tor) is mediated by four chemical forces: electrostatic force, hydro-
gen bonding, Van der Waal’s interactions, and hydrophobic bonds.
Electrostatic force mediates attraction between opposed charged
groups or repulsion between similarly charged groups that is pro-
portional to the net sum of charges and inversely proportional to
the square of the distance between charges (as described by Cou-
lomb’s law). Van der Waals interactions are attractive and repulsive
forces between dipoles approximated by the Lennard-Jones func-
tion that has its minimum (strongest attraction) at a certain dis-
tance of the components. A hydrogen bond is a special case of the
electrostatic attractive interaction between polar molecules, in
which hydrogen is bound to a highly electronegative atom like
nitrogen or oxygen. A hydrogen bond is weaker than electrostatic
force but stronger than a Van der Waals interaction. Hydrophobic
bonds are entropy-driven interactions between non-polar groups to
avoid interaction with polar groups, mainly water. Hydrophobic
bonds are stronger than Van der Waals interaction. Because these
forces vary in their strength and dependence on the distance
between components, the combination of all these forces directs
the positioning of a ligand on the receptor binding site with mini-
mal free energy. A measure of attraction of the ligand to the binding
site is termed affinity. Thermodynamic movement does not allow
ligands to stay still at the binding site and makes them associate and
dissociate from the receptor, even at equilibrium. Thus, the proba-
bility with which a ligand is bound to (stays at) the binding site of a
receptor is given by ligand concentration, temperature, and
strengths of interactions. The dependence of the ligand binding
on its concentration (Fig. 1 black curve) is defined by Langmuir
isotherm:
 Radioligand Binding at Muscarinic Receptors 3

Fig. 1 Radioligand binding. Lines represent hypothetical radioligand binding

(blue curve) that consist of saturable specific binding (black curve) defined by
the binding isotherm and non-specific binding (red line) that is linearly
proportional to radioligand concentration and is non-saturable

½L ]
binding = ð1Þ
½L ] þ K D
where square brackets designate concentration and KD is the equi-
librium dissociation constant that is equal to the concentration at
which the binding site is occupied with 50% probability (for
[L] = KD expression is equal to 1/2 while for [L] >> KD it limits
to 1).

2.2 Radioligands Binding experiments aim to quantify ligand binding to the receptor
under given conditions. Labeling the ligand with a radioactive
isotope allows easy and sensitive quantification of binding and
(unlike fluorescent labeling) does not interfere with ligand binding.
High specific radioactivity is required, so ligand binding translates
to a high signal. Low non-specific binding is required for a high
signal-to-noise ratio. Finally, a good radioligand should have a high
affinity for the receptor to prevent ligand dissociation from the
receptor during the separation of free and bound radioligand.
High affinity also affords the use of low concentrations of expensive
radioligands.

2.3 Ligand-Specific Ligand binding to a receptor is a dynamic process of attraction

Binding mediated by chemical forces and disruption of binding by thermal
movement of molecules. As a result, a ligand incessantly associates
with and dissociates from the receptor with time. Ligand (L)
binding to the receptor (R) and formation of ligand-receptor com-
plexes (LR) can be described as a reversible bi-molecular reaction:
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