Calcium Signaling, Second Edition

by Putney, Jr.; James W.

• ISBN13: 9780849327834
• ISBN10: 0849327830
• Edition: 2nd
• Format: Hardcover
• Copyright: 2005-11-21
• Publisher: CRC Press

Summary

The first edition of James Putney's Calcium Signaling offered readers a comprehensive view of the fascinating diversity of technologies that the new field of calcium signaling employed. And while that work is still regarded as a premier text on the basics of calcium signaling, progress has been so dramatic that an update is now required.In Calcium Signaling, Second Edition, Putney focuses on those processes that generate calcium signals to compile the first comprehensive exploration of calcium signaling research from a methodological standpoint.This new edition deals with methods for studying calcium from a variety of perspectives. Several chapters discuss calcium indicators and other tools, and look at microscopic and electrophysiological techniques, as well as other special methodological aspects of calcium signaling research. Other chapters examine the study of different systems, ranging from those found in yeast to those found in mammals, and several more are devoted to the cellular and molecular basis for calcium signaling, including explorations of receptors, calcium pumps, apoptosis, and drug delivery.Once again, Putney has called upon top researchers from across the globe to contribute their expertise. Several new chapters have been added and in many cases, where chapters from the first edition were retained, new researchers were recruited to offer a fresh perspective.As calcium signaling involves such a breadth of technical approaches and a wide range of applications, this work contains invaluable information for established researchers, as well as those graduate students and scientists just beginning to find a direction in cellular calcium signaling.

Table of Contents

Fluorescent Calcium Indicators Based on BAPTA	1	(50)
Martin Poenie
Introduction	2	(1)
Calcium Indicators Based on BAPTA: General Considerations	3	(1)
High-Affinity Calcium Indicators	4	(16)
High-Affinity Ratiometric Indicators	4	(1)
High-Affinity Ratiometric Indicators Excited by Ultraviolet Light (Fura-2, Benzothiaza-1, Benzothiaza-2, and Indo-1)	5	(2)
High-Affinity Ratiometric Indicators Excited by Visible Light (Fura Red)	7	(1)
Summary of the Ratiometric Indicators	8	(2)
High-Affinity Nonratiometric Calcium Indicators	10	(1)
Indicators Based on Fluorescein Derivatives (Fluo-3, Fluo-4, Calcium Green-1, Calcium Green-2, Oregon Green 488 BAPTA)	10	(6)
Indicators Based on Rhodamine (Rhod-2, X-Rhod-2, Calcium Orange, Calcium Crimson)	16	(2)
Summary of Nonratiometric Indicators	18	(2)
Low-Affinity Calcium Indicators for Measuring Large or Rapid Calcium Transients	20	(11)
The Relationship between Affinity and Kinetics	21	(2)
Design of Low-Affinity Indicators	23	(1)
Low-Affinity Calcium Indicators Based on APTRA	24	(2)
Tetracarboxylate Low-Affinity Calcium Indicators	26	(2)
The 5-Nitro BAPTAs (Fluo-5N, Calcium Green-5N, Calcium Orange-5N, Oregon Green 488 BAPTA-5N)	28	(1)
Benzothiazole Derivatives (Benzothiaza-1 and -2, BTC)	28	(1)
The Halogenated BAPTA Moderate- to Low-Affinity Indicators (BAPTA-4F, -5F, -5CI, -6F, and -FF Derivatives)	29	(1)
Summary of the Low-Affinity Calcium Indicators	30	(1)
Indicators that Resist Leakage and Sequestration	31	(3)
Indicator-Dextran Conjugates	32	(1)
Fura-PE3 and Related Indicators	32	(2)
Summary of the Leakage-Resistant Calcium Indicators	34	(1)
Near-Membrane Calcium Indicators	34	(6)
The C18 Near-Membrane Calcium Indicators (C18-Fura-2, Calcium Green C18)	36	(1)
C18-Fura-2 (Fura-2-C18)	36	(2)
Calcium Green C18	38	(1)
Fura-FFP18 and Related Calcium Indicators	38	(1)
Summary of the Near-Membrane Calcium Indicators	39	(1)
Use of Calcium Indicators with Multiphoton Microscopy	40	(2)
Conclusion	42	(9)
Acknowledgments	43	(1)
References	43	(8)
Fluorescent Indicators---Facts and Artifacts	51	(34)
Gary St. J. Bird
James W. Putney, Jr.
Introduction	52	(1)
Ca2+ Signaling	52	(1)
Anatomy of a Calcium Signal	53	(17)
Monitoring the [Ca2+]i Signal	53	(1)
Fluorescent Indicator Selection	53	(1)
Fura-2	54	(1)
Fura-5F	54	(2)
Fluorescent Indicators and Cytoplasmic Loading	56	(2)
Quantitative Assessment of a [Ca2+]i Response	58	(1)
Dissecting the [Ca2+]i Signal	59	(1)
Resting [Ca2+]i and the ``Calcium Re-Addition'' Protocol	60	(1)
Receptor-Activated Cells and Biphasic Calcium Signaling	61	(2)
Intracellular Ca2+-Release and Ca2+-Pools	63	(1)
Manipulating Intracellular Ca2+-Pools in Intact Cells	63	(5)
Protocol for Defining Intracellular Ca2+-Pools	68	(2)
Ca2+-Entry	70	(7)
Monitoring Ca2+-Entry	70	(1)
Mn2+ -Quench (i.e., Mn2+ -Entry)	71	(1)
Sr2+ -Entry	72	(1)
Ba2+ -Entry	72	(1)
Manipulating Ca2+-Entry	73	(1)
Lanthanides	73	(1)
2-APB (2-Aminoethoxydiphenyl Borane or 2-Aminoethyldiphenyl Borate)	74	(1)
Regulation of Ca2+-Entry	74	(1)
Feedback Inhibition of CCE by [Ca2+]i	75	(1)
Membrane Potential	76	(1)
CCE and Excitable Cells	77	(1)
Conclusions	77	(8)
References	77	(8)
Fluorescence Microplate-Based Techniques for the High-Throughput Assessment of Calcium Signaling: The Highs and Lows for Calcium Researchers	85	(16)
Gregory R. Monteith
Gary St. J. Bird
Introduction	86	(1)
HT and [Ca2+]CYT Measurements	86	(1)
Equipment Options for HT-Based Assessment of [Ca2+]CYT	87	(5)
Excitation Light Sources	88	(1)
Lasers and LEDs	88	(1)
Lamp-Based Illumination	88	(3)
Fluorescence Emission Detection	91	(1)
Plate Formats	91	(1)
Robotics and Solution Handling	91	(1)
Environmental Control	92	(1)
Specific Issues with [Ca2+]CYT Measurements in Microplates	92	(4)
Cell Plating: Adherent Cells and Cell Suspensions	92	(1)
Fluorescence Calcium Indicator Selection	93	(1)
Loading Cells with Fluorescent Calcium Indicators	93	(1)
Addition of Reagents During [Ca2+]CYT Measurements: Options and Limitations	94	(2)
Data Analysis	96	(1)
Potential Applications for the HT Assessment of [Ca2+]CYT	96	(1)
Challenges and Future Opportunities Related to HT Assessment of Free Calcium Using Fluorescence Microplate Readers	97	(1)
Summary	98	(3)
References	98	(3)
Genetically Encoded Fluorescent Calcium Indicator Proteins	101	(12)
Atsushi Miyawaki
Takeharu Nagai
Hideaki Mizuno
Green Fluorescent Protein-Based Fluorescent Indicators for Ca2+	101	(1)
Ca2+ Indicators Using Single GFP Variants	102	(1)
Ca2+ Indicators Using Circularly Permuted GFP Variants	102	(3)
G-CaMP	102	(1)
Pericam	103	(2)
Ca2+ Indicators Utilizing Pairs of GFP Variants That Permit FRET	105	(3)
Procedure for Dual Emission Ratio Imaging of YCs	107	(1)
Monitoring Rodent Neural Activity with GFP-Based Fluorescent Ca2+ Indicators	108	(5)
References	109	(4)
Targeted Aequorins	113	(12)
Andrea Prandini
Rosario Rizutto
Introduction	113	(2)
Targeting the Aequorin cDNA to Cellular Compartments	115	(2)
Expression and Detection of the Probe	117	(2)
Use of Aequorin in Cell Compartments with High Ca2+ Concentrations	119	(3)
Targeting the Aequorin cDNA to Specific Proteins	122	(3)
Acknowledgments	122	(1)
References	122	(3)
Electrophysiological Recordings of Ca2+ Currents	125	(22)
Anant B. Parekh
Introduction	126	(1)
Electrophysiological Recordings of Ca2+ Currents	127	(2)
Basic Considerations	127	(1)
Holding Potential	128	(1)
Voltage Pulses	129	(1)
Store-Operated Ca2+ Current	129	(8)
Isolation of the Store-Operated Current	129	(1)
K+ Currents	130	(1)
Cl- Currents	130	(1)
Ca2+-Activated Currents	130	(1)
MagNuM/MIC	130	(2)
TRPV6	132	(1)
Store Depletion Protocols	133	(2)
Experimental Methods to Increase the Size of the Store-Operated Ca2+ Current	135	(1)
External Solution	135	(1)
Internal Solution	136	(1)
Leak Subtraction Protocols to Isolate the Store-Operated Ca2+ Current	136	(1)
Recording Store-Operated Ca2+ Current under Physiological Conditions	137	(1)
Recording of Voltage-Gated Ca2+ Current	137	(2)
Perforated Patch Recordings	139	(1)
Estimate of Single Ca2+ Channel Conductance	140	(3)
Single-Channel Recording	140	(1)
Voltage-Gated Ca2+ Channels	141	(1)
Store-Operated Ca2+ Channels	141	(1)
Fluctuation Analysis	142	(1)
Total Internal Reflection Fluorescence Microscopy: A New Tool for Studying Ca2+ Channels	143	(4)
Acknowledgments	144	(1)
References	144	(3)
Molecular Biology of Ca2+ Channels: Lessons from the TRP Superfamily	147	(14)
Veit Flockerzi
Thomas Aberle
Marcel Meissner
Christine Jung
Stephan Philippand
Ulrich Wissenbach
Introduction	147	(1)
Drosophila TRP Channel, the Founding Member of the TRP Superfamily	148	(1)
Mammalian TRP-Related Proteins	148	(2)
Strategies to Identify TRP-Related Proteins	150	(1)
Limitations of Database Searches or: What Makes a TRP a TRP?	151	(2)
Analysis of TRPC Expression by Antibodies	153	(4)
Conclusions	157	(4)
Acknowledgments	157	(1)
References	157	(4)
Genetic and Molecular Characterization of Ca2+ and IP3 Signaling in the Nematode Caenorhabditis elegans	161	(26)
Ana Y. Estevez
Kevin Strange
Introduction	161	(1)
C. elegans Biology	162	(2)
Natural History and Life Cycle	162	(1)
Laboratory Culture	163	(1)
Anatomy	164	(1)
Forward and Reverse Genetic Analysis	164	(2)
Forward Genetic Analysis	164	(1)
Reverse Genetic Analysis	165	(1)
Public Resources	166	(1)
Calcium and IP3 Signaling in the C. elegans Hermaphrodite Gonad	167	(3)
Oscillatory Ca2+ Signaling in the C. elegans Intestine	170	(4)
Role of TRP Channel-Mediated Ca2+ Signaling in Sperm Function	174	(1)
Calcium Signaling in C. elegans Neurons and Muscle	175	(5)
Regulation of Pharyngeal Muscle Contraction	175	(2)
Mechanotransduction	177	(2)
Neurodegeneration	179	(1)
Conclusion and Future Perspectives	180	(7)
References	180	(7)
Calcium Signaling Networks in Yeast	187	(16)
Kyle W. Cunningham
Why Study Yeast?	187	(2)
Ca2+ Pumps and Exchangers	189	(4)
SPCA-Type Ca2+ Pumps	189	(2)
PMCA-Type Ca2+ Pumps	191	(1)
H+/Ca2+ Exchangers and Related Enzymes	191	(2)
Missing and Novel Ca2+ Transporters?	193	(1)
Ca2+ Channels	193	(3)
TRPC-Type Ca2+ Channels	193	(1)
VGCC-Type Ca2+ Channels	194	(2)
Other Ca2+ Channels?	196	(1)
Synopsis and Future Directions	196	(7)
References	197	(6)
Nuclear Patch Clamp Electrophysiology of Inositol Trisphosphate Receptor Ca2+ Release Channels	203	(28)
Don-On Daniel Mak
Carl White
Lucian Ionescu
J. Kevin Foskett
Introduction	204	(1)
Molecular Identification of the InsP3R	204	(1)
Development of Nuclear Patch Clamp Electrophysiology to Study Single InsP3R Channels	205	(12)
Xenopus Oocytes	206	(1)
Frog Husbandry	206	(1)
Solutions for Oocytes	207	(1)
Surgical Extraction, Defolliculation, and Maintenance of Oocytes	207	(1)
Oocyte Nucleus Isolation	208	(2)
Oocyte Nucleus Patch Clamping	210	(2)
Solutions Used in Patch Clamp Experiments	212	(1)
Sf9 Cells	213	(1)
Sf9 Cell Culture and Nucleus Isolation	213	(2)
Sf9 Cell Nucleus Patch Clamping	215	(1)
DT40 Cells	215	(1)
DT40 Cell Culture	215	(1)
Generation of Recombinant InsP3R-Expressing DT40-KO Cells	216	(1)
DT40 Cell Nuclear Membrane Patch Clamp Electrophysiology	217	(1)
Ion Channel Properties of the InsP3R in Native ER Membranes by Nuclear Patch Clamping	217	(5)
Basic Conductance Properties of the InsP3R	217	(2)
Gating Regulation of the InsP3R	219	(3)
Conclusions	222	(9)
Acknowledgments	223	(1)
References	223	(8)
Ryanodine Receptors	231	(34)
Xander H.T. Wehrens
Stephan E. Lehnart
Andrew R. Marks
Introduction	231	(2)
Ryanodine Receptor Genes and Isoforms	233	(1)
Differential Expression of RyR Isoforms	233	(1)
Mutations in Ryanodine Receptor Genes	234	(1)
Ryanodine Receptor Macromolecular Channel Complex	234	(5)
Three-Dimensional Structure of Ryanodine Receptors	234	(2)
The Calcium-Channel Stabilizing Subunit ``Calstabin''	236	(1)
Other Regulatory Proteins Associated with RyR	237	(2)
Regulation of Ryanodine Receptors	239	(6)
Modulation of Ryanodine Receptors by Calcium	239	(1)
Phosphorylation-Dependent Regulation of Ryanodine Receptors	240	(2)
Abnormal RyR2 Regulation in Cardiac Arrhythmias	242	(2)
RyR1 Mutations Causing Malignant Hyperthermia	244	(1)
Cellular Basis of Excitation--Contraction Coupling	245	(3)
EC Coupling in Cardiomyocytes	245	(1)
EC Coupling in Skeletal Muscle Fibers	246	(1)
EC Coupling Abnormalities in Cardiac and Skeletal Muscle	247	(1)
Studies of Genetically Altered Mice	248	(2)
Ryanodine Receptor Knockout Mice	249	(1)
Calstabin Knockout Mice	249	(1)
Concluding Remarks	250	(15)
References	251	(14)
Methods in Cyclic ADP-Ribose and NAADP Research	265	(70)
Anthony J. Morgan
Grant C. Churchill
Roser Masgrau
Margarida Ruas
Lianne C. Davis
Richard A. Billington
Sandip Patel
Michiko Yamasaki
Justyn M. Thomas
Armando A. Genazzani
Antony Galione
Introduction	267	(7)
Background to cADPR and NAADP	267	(3)
Mechanism of cADPR-Mediated Ca2+ Release	270	(1)
Channels and Targets	270	(1)
cADPR and RyR Subtypes	271	(1)
Non-RyR Sites of Action?	272	(1)
Mechanism of NAADP-Induced Ca2+ Release	272	(2)
Purification and Synthesis of cADPR and Analogs	274	(8)
HPLC	274	(1)
cADPR Synthesis	275	(1)
Synthesis of [32P]cADPR	276	(1)
Caged cADPR	277	(3)
cADPR Analogs	280	(1)
Agonists and Antagonists	280	(2)
Fluorescent cADPR Analogs	282	(1)
Purification and Synthesis of NAADP and Analogs	282	(7)
Synthesis of NAADP	282	(1)
Synthesis of [32P]NAADP	283	(2)
Caged NAADP	285	(1)
Synthesis	285	(3)
Purification	288	(1)
Synthetic NAADP Analogs	288	(1)
Studying cADPR- and NAADP-Induced Ca2+ Release	289	(6)
The Sea Urchin Egg	289	(1)
Collection of Sea Urchin Eggs	290	(1)
Sea Urchin Egg Homogenates	291	(1)
Preparation of Sea Urchin Egg Homogenates	291	(2)
Fluorimetry of Ca2+ Release from Egg Homogenates	293	(1)
Sea Urchin Egg Fractionation	293	(1)
Preparation of Microsomes	293	(1)
Preparation of NAADP-Responsive Acidic Vesicles	293	(1)
Testing Fractions for Ca2+ Releasing Properties	294	(1)
Pharmacology of cADPR Signaling	295	(2)
Troubleshooting and Practical Considerations	296	(1)
Pharmacology of NAADP Signaling	297	(3)
Targeting Receptors	298	(1)
Targeting Stores	299	(1)
Enzymology of cADPR and NAADP Metabolism	300	(5)
Enzymatic Synthesis and Degradation of cADPR and NAADP	300	(1)
ADP-Ribosyl Cyclases	300	(1)
cADPR Hydrolases	301	(1)
NAADP Metabolism	302	(1)
Subcellular Location	302	(1)
Detection of Metabolism by Discontinuous Bioassay	303	(1)
Fluorescent Analogs	303	(1)
NGD Assay: Continuous Assay for ADP-Ribosyl Cyclase Activity	303	(2)
cIDPR Assay: Continuous Assay for cADPR Hydrolase	305	(1)
HPLC	305	(1)
Measurement of cADPR Levels in Cells and Tissues	305	(5)
Background	305	(1)
Kinetics	306	(1)
Signaling Cascades Modulating ARC	306	(1)
Mammalian Cells: NO and cADPR	307	(1)
Other Pathways	307	(1)
Regulation of cADPR Removal	308	(1)
Preparation and Extraction of cADPR from Cells and Tissues	308	(1)
Bioassay for cADPR	308	(1)
[32P]cADPR Binding and Radioreceptor Assay	309	(1)
cADPR-Binding Sites	309	(1)
cADPR Radioreceptor Assay	310	(1)
Measurement of NAADP in Cells and Tissues	310	(4)
Background	310	(1)
Radioreceptor Assay for NAADP	311	(1)
Acid Extraction	311	(1)
NAADP Radioreceptor Assay	312	(1)
NAADP Receptor Binding	313	(1)
Membrane-Associated Receptors	313	(1)
Detergent-Solubilized Receptors	313	(1)
Investigating cADPR and NAADP Signaling in Single Cells	314	(6)
Techniques for Introducing Cell Impermeant Compounds	314	(1)
Microinjection	314	(3)
Patch Pipette	317	(1)
Permeabilized Cells	318	(2)
Conclusion	320	(15)
References	320	(15)
Methods for Studying Calcium Pumps	335	(52)
Leonard Dode
Luc Raeymaekers
Ludwig Missiaen
Bente Vilsen
Jens P. Andersen
Frank Wuytack
Introduction	336	(1)
Identification of Isoforms, Measurement of Expression Levels, and Subcellular Distribution of Calcium Pumps and Their Regulators	337	(6)
Studies at the mRNA Level	338	(1)
Detecting and Quantifying RNA	338	(2)
Alternative Splicing	340	(1)
Studies at the Protein Level	340	(1)
Non-Discriminating and Isoform-Specific Antibodies	340	(2)
Discrimination of Calcium Pumps by Non-Immunological Techniques	342	(1)
Phospholamban and Sarcolipin	343	(1)
Studies of Calcium Pump Activity and Its Catalytic Mechanism	343	(22)
Catalytic Cycle	344	(2)
Heterologous Expression Systems in Yeast, Insect, and Mammalian Cells	346	(2)
Detection and Quantitation of Overexpressed Calcium Pumps	348	(1)
Overall Reactions: Ion Transport and ATPase Activity	349	(2)
Assays for Ca2+ Transport in Isolated Membrane Vesicles	351	(3)
Assays for Ca2+ and Mn2+ Transport in Permeabilized Cells	354	(2)
Assays of Ca2+-ATPase Activity in Isolated Membrane Vesicles	356	(4)
Steady State and Transient State Kinetic Analyses of Partial Reactions	360	(1)
Phosphorylation Approach	360	(4)
Ca2+ Occlusion Approach	364	(1)
Nucleotide-Binding Approach	365	(1)
Calcium Pump Inhibition as a Tool to Study Function	365	(5)
Inhibition of SERCA by Organic Compounds	365	(1)
Inhibition by Analogues of Inorganic Phosphate	366	(1)
Inhibition by Vanadate	366	(1)
Inhibition by Fluoride Complexes of Aluminum and Beryllium	367	(1)
Inhibition by Lanthanides	368	(1)
Inhibition of SERCA3 by PL/IM430 Antibody	369	(1)
Inhibition by RNAi Experiments	369	(1)
Inhibition of the Plasmodium Falciparum Ca2+ Pump by Artemisinins	370	(1)
Calcium Pumps: Purification and Structure	370	(1)
SERCA: Crystallization and 3D Structure	370	(1)
PMCA: Calmodulin Affinity-Chromatography Purification	371	(1)
Studies of Genetically Altered Mice	371	(1)
Conclusions	372	(15)
Acknowledgments	372	(1)
References	372	(15)
Measuring Single Cell and Subcellular Ca2+ Signals	387	(30)
Sandip Patel
Lawrence D. Gaspers
Nicola Pierobon
Walson Metzger
Andrew P. Thomas
Introduction	387	(3)
Fluorescent Ca2+-Sensitive Indicator Dyes	390	(10)
Properties of Fluorescent Ca2+ Indicator Dyes	390	(2)
Loading Fluorescent Ca2+ Indicator Dyes into Cells	392	(1)
Compartmentalization of Ca2+ Indicator Dyes	392	(3)
Calibration of Fluorescence Signals	395	(5)
Fluorescent Protein-Based Ca2+ Indicators	400	(5)
Advantages of Protein-Based Ca2+ Indicators	400	(1)
Imaging of Photoprotein Signals	400	(1)
GFP-Based Ca2+ Indicators	401	(1)
Using GFP-Based Ca2+ Indicators	402	(3)
Instrumentation	405	(12)
Epifluorescence Microscope	405	(2)
Detectors and Image Acquisition	407	(3)
Confocal Microscopy	410	(1)
Acknowledgments	411	(1)
References	411	(6)
Subcellular Compartmentalization of Calcium Signaling	417	(16)
Nicholas J. Dolman
Michael C. Ashby
Myoung K. Park
Ole H. Petersen
Alexei V. Tepikin
Introduction	417	(1)
Intra-Patch Pipette Uncaging --- Technique for Studying the Distribution of Receptors in Polarized Cells	418	(2)
Local Calcium Signals and the Geography of Cellular Organelles or How to Put Calcium Signals on the Map	420	(4)
Some Considerations about the Instruments	420	(1)
Combinations of Fluorophores for Simultaneous Organellar Labeling and Studies of Calcium Signaling	421	(2)
Measuring Local Calcium Gradients and Organelle Localization in Polarized Secretory Cells	423	(1)
Probing Calcium Signaling with Calcium Uncaging (in the Cytosol and the Lumen of Cellular Organelles)	424	(2)
Measuring Local Calcium Extrusion	426	(7)
References	429	(4)
Apoptosis	433	(22)
Clark W. Distelhorst
Introduction	433	(1)
Apoptosis and Methods for Its Detection	434	(4)
Apoptosis and Necrosis	434	(1)
Cell Death Initiation and Execution: A Brief Overview	434	(2)
Diagnostic Tests and Quantitative Measures of Apoptosis	436	(2)
Alterations of Ca2+ Homeostasis That Signal Apoptosis	438	(4)
Ca2+ Homeostasis and Signaling	438	(1)
Physiological and Nonphysiological Ca2+ Changes That Initiate Cell Death Pathways	439	(1)
Downstream Effectors That Mediate the Execution Phase of Ca2+-Induced Apoptosis	440	(2)
Methods to Detect Alterations in Ca2+ Homeostasis Associated with Apoptosis	442	(6)
Measuring Cytoplasmic Ca2+ Elevation Induced by an Apoptotic Signal	442	(2)
Determining Whether Cytoplasmic Ca2+ Elevation Mediates Apoptosis	444	(1)
Detecting Alterations in Intracellular Ca2+ Distribution	445	(3)
Methods to Investigate the Involvement of IP3 Receptors in Apoptosis	448	(1)
Concluding Remarks	448	(7)
Acknowledgments	449	(1)
References	449	(6)
Calcium Entry Channels and Drug Discovery	455	(32)
Su Li
Anne Dodge
Chris T. Poll
Martin Gosling
Drug Discovery and Calcium Entry Channels: A Historical Perspective	456	(6)
Discovery of L-Type Voltage-Operated Ca2+ Channel Blockers	456	(2)
Discovery of Receptor-Operated Ca2+ Channel Blockers	458	(1)
ROCC Channel Blockers	459	(3)
Evolution of Drug Discovery Approaches: Implications for Ca2+ Channels	462	(1)
Current Drug Discovery Approaches	462	(4)
Overview	462	(1)
Target Selection	462	(2)
Hit-Finding	464	(1)
Hit to Lead	464	(1)
Lead Optimization	465	(1)
Summary	465	(1)
Emerging Ca2+ Channel Targets	466	(2)
TRPV1 Channels	466	(1)
N-Type Voltage-Operated Ca2+ Channels	467	(1)
Assays and Technologies for Ca2+ Channel Drug Discovery	468	(11)
Cell Lines	468	(1)
Radioligand Binding	469	(2)
Radiotracer Flux	471	(1)
Optical Readouts --- Intracellular Ca2+ Concentration and Membrane Potential	472	(4)
Electrophysiology --- Patch Clamp and Oocyte Recording	476	(3)
Concluding Remarks	479	(8)
Acknowledgments	479	(1)
Appendix	480	(1)
References	481	(6)
Index	487