Expression, Localisation and Function of Truncated
Forms of the Voltage-Dependent Calcium Channel
a1B
Ayesha Raghib
A thesis submitted in fulfillment of the requirements of the University of London for the degree of Doctor of Philosophy
2002
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ABSTRACT
Voltage gated calcium channels (VDCC) exist minimally as a complex containing an a1
subunit and at least two accessory subunits p and o2-ô. The a1 subunit forms the pore of the
channel, and consists of four homologous domains connected by intracellular loops. Several
mutations in the a1 subunit of VDGGs have been identified that predict the generation of
truncated proteins. This study focused on the expression and localisation of truncated a1B
subunits. Gonstructs containing domain I, domain l-ll and domain lll-IV of a1B were
constructed using standard molecular biology techniques, and their subcellular localisation
was studied using'antibody to an intracellular epitope of a1B and an extracellular epitope of
o2-ôi, which was assumed to form a complex with the Domain l-ll and Domain lll-IV. The
constructs were also tagged with GFP or its spectral variants (XFP), in order to permit direct
visualisation of the proteins. All the truncated constructs were expressed successfully in Gos-7
cells. Using laser scanning confocal microscopy, it was shown that both the untagged and
XFP-tagged Domain l-ll were plasma membrane localised in the absence and presence of
accessory subunits, although the presence of p subunits appeared to increase plasma membrane localisation. However, the XFP-Domain l-ll construct showed pronounced ER
retention, which was not evident when the construct was visualised using antibodies. The
subcellular localisation of untagged Domain lll-IV also varied when compared to the tagged
Domain lll-IV EGFP construct. Antibodies to untagged Domain lll-IV and the Domain
III-IV/a2-5i complex revealed plasma membrane and cytoplasmic localisation. Plasma
membrane localisation was not observed in the absence of accessory subunits. In contrast.
Domain lll-IV EGFP showed pronounced ER retention and no conclusive plasma membrane
localisation, both in the absence and presence of accessory subunits. Thus, it appeared that
the addition of the XFP-tag was altering the subcellular localisation of the truncated proteins.
Goexpression of domains I II and domains lll-IV in the absence and presence of accessory
subunits showed no detectable increase in plasma membrane localisation, although this
combination did form functional channels. The XFP-tagged Domain I construct showed
cytoplasmic and ER localisation, and was not detectable at the plasma membrane. The
localisation of the untagged Domain I could not be investigated due to lack of antibodies.
Additional studies were also performed to investigate the effect of the truncated proteins on
the expression of GFP-tagged a1B. Goexpression of GFP-a1B with untagged Domain I and
Domain I II dramatically reduced the expression of GFP-a1B, as evidenced by the absence of
fluorescence from GFP-a1B. In contrast, untagged Domain lll-IV trapped GFP-a1B in the ER.
These results indicate that truncated proteins of a1B may have a suppresive effect on the
ABSTRACT
CONTENTS
LIST OF FIG U R ES_____________________________________________ 14 LIST OF TABLES_______________________________________________ 20 LIST OF ABBREVIATIONS ______________________________________ 21
CHAPTER 1
GENERAL INTRODUCTION
1.1 INTRODUCTION____________________________________________ 24
1.2 STRUCTURE OF VOLTAGE-GATED CALCIUM CHANNELS _______ 24
1.2.1 T heal Subunit ___________________________________________ 26
1.3 CLASSIFICATION OF VD D C s_________________________________ 29 1.4 THE N-TYPE (a lB ) CALCIUM CHANNEL_______________________ 29
1.4.1 Structure and Biophysical Properties of the N-Type Channel ________ 31 1.4.2 Localisation and Function of Native N-Type Currents_______________ 31 1.4.3 The Cloned a lB (N-Type) Channel Subunit _____________________ 33 1.4.4 Modulation of N-Type Calcium Channels________________________ 34 1.4.5 Involvement of the N-Type Channel in Neurotransmitter Release_____ 34 1.4.6 Splice-Variants of a1 B _____________________________________ 37
1.5 IDENTIFICATION AND CLONING OF HIGH-VOLTAGE ACTIVATED CALCIUM CHANNELS________________________________________ 39
1.5.1.1 The a l S Subunit _____________________________________ 39 1.5.1.2 The a ie Subunit _____________________________________ 40 1.5.1.3 The a l D Subunit _____________________________________ 41 1.5.1.4 The a l F Subunit _____________________________________ 42 1.5.2 The P/Q-Type Channels ____________________________________ 42 1.5.2.1 The a lA Subunit ______________________________________ 43 1.5.3 The R-Type Current _______________________________________ 45 1.5.3.1 The a l E Subunit ______________________________________ 45
1.6 IDENTIFICATION AND CLONING OF LOW-VOLTAGE ACTIVATED CALCIUM CHANNELS________________________________________ 46
1.6.1 The T-Type Channels ______________________________________ 46 1.6.1.1 The alG , a lH and a11 Subunits ___________________________ 47
1.7 ACCESSORY SUBUNITS OF VOLTAGE-GATED CALCIUM CHANNELS ____________________________________________________________ 48
1.8 MODULATION OF VDCCs_____________________________________ 55
1.8.1 G-Protein Modulation ______________________________________ 55 1.8.2 Phosphorylation of VDCCs___________________________________ 57
1.9 TRUNCATED FORMS OF VOLTAGE-DEPENDENT CALCIUM
CHANNELS_________________________________________________ 59
1.9.1 Episodic Ataxia-2 (EA-2) ____________________________________ 59 1.9.2 Other Truncations__________________________________________ 61 1.9.3 Protein Folding and Membrane Integration_______________________ 62 1.9.4 Quality Control of Proteins in the ER____________________________ 63 1.9.5 Protein Degradation________________________________________ 64
1.10 GREEN FLUORESCENT PROTEIN (GFP)_______________________ 65
1.10.1 Cloning of the GFP Gene _________________________________ 65 1.10.2 Crystal Structure of G FP__________________________________ 65 1.10.3 Composition and Characteristics of the GFP Chromophore _______ 66 1.10.3.1 Proposed Mechanism of Formation of the GFP Chromophore 68 1.10.4 Biophysics of GFP Fluorescence ___________________________ 68 1.10.5 Classification of GFP ____________________________________ 71 1.10.6 Expression and Detectability of GFP ________________________ 76 1.10.7 Spectral Variants of GFP Used in this Study___________________ 77 1.10.7.1 GFP Mut3_________________________________________ 77 1.10.7.2 Enhanced Yellow Fluorescent Protein (EYFP)_____________ 77 1.10.7.3 Enhanced Cyan Fluorescent Protein (ECFP)______________ 78
CHAPTER 2
MATERIALS AND GENERAL METHODS
2.1 CELL CULTURE_____________________________________________ 81
2.1.1 Cos-7 C ells______________________________________________ 81 2.1.2 Cell Culture of Cos-7 Cells___________________________________ 81
2.2 TRANSFECTION_____________________________________________ 83
2.2.1 Electroporation ___________________________________________ 83 2.2.2 Lipofection_______________________________________________ 83 2.2.3 Coverslips_______________________________________________ 84
2.3 LASER SCANNING CONFOCAL MICROSCOPY__________________84
2.3.1 The Laser Scanning Confocal Microscope (LCSM) ________________ 84 2.3.2 Optical Sectioning________________________________________ 85 2.3.3 Immunofluorescence of Cos-7 Cells for Confocal Microscopy ______ 88 2.3.3.1 Indirect Immunofluorescence Using Antibodies_____________88 2.3.3 2 Direct Immunofluorescence Using XFP-Fusion Proteins______ 89 2.3.3 3 Organelle Markers___________________________________ 93 2.3.3 4 Protein Synthesis Inhbitors_____________________________ 95
2.4 GENERAL METHODS FOR MOLECULAR BIOLOGY_____________ 95
2.4.3 DNA Isolation and Purification from Agarose Gels______________ 99 2.4.4 DNA Isolation and Purification from Solutions_________________ 99 2.4.5 Restriction Digests____________________________________ 100 2.4.6 Ligations___________________________________________ 100 2.4.7 Sequencing of DNA___________________________________ 101 2.4.7.1 Manual Sequencing of DNA_________________________ 101 2.4.7 2 Automated Sequencing of DNA_____________________ 101 2.4.8 Preparation of cDNA_____________________________________ 102 2.4.8.1 Transformation of Competent Cells______________________ 102 2.4.9 Purification of cDNA_____________________________________ 103 2.4.9.1 Minipreps- Small Scale Production of cDNA_______________ 103 2.4.9 2 Maxipreps- Large Scale Production of cDNA______________ 104
CHAPTER 3
PREPARATION OF TRUNCATED CALCIUM CHANNEL cDNA
3.1 INTRODUCTION___________________________________________ 107
3.1.1 The pMT2 Vector _______________________________________ 107 3.1.2 General Methods for the Preparation of Truncated Calcium Channels _ 107 3.1.2.1 PCR Reactions______________________________________ 109 3.1.2.2 Isolation of the Clones Containing the Required Constructs_____ 109
3.2 PREPARATION OF TRUNCATED CONSTRUCTS OF a l B _______ 110
3.2.2 Construction of the Domain l-ll Construct______________________ 113 3.2.2.1 Preparation of Vector________________________________ 114 3.2.2 2 Preparation of Insert ________________________________ 114 3.2.2.3 Preparation of PCR Product___________________________ 116 3.2.2 4 Isolation of the Clone Containing the Domain l-ll Construct 116 3.2.3 Construction of the Domain lll-IV Construct__________________ 118 3.2.3.1 Preparation of Vector_______________________________ 118 3.2.3 2 Preparation of Insert________________________________ 118 3.2.3 3 Preparation of PCR Product__________________________ 120 3.2.3 4 Isolation of the Clone Containing the Domain lll-IV Construct _ 122
3.3 PREPARATION OF TRUNCATED a1B PROTEINS FUSED TO GREEN, YELLOW, OR CYAN FLUORESCENT PROTEINS ______________ 122
3.3.3 4 Isolation of the Clone Containing the Domain lll-IV ECFP
Construct__________________________________________ 136 3.3.4 Construction of the a1 B-ECFP Construct_____________________ 136 3.3.4.1 Preparation of Vector_________________________________ 136 3.3 4.2 Preparation of Insert _________________________________ 137 3.3.4 3 Isolation of the Clone Containing the alB-ECFP Construct 137 3.3.5 Construction of the ECFP Domain lll-IV Construct______________ 139 3.3.5.1 Preparation of Vector_________________________________ 139 3.3.5 2 Preparation of Insert_________________________________ 139 3.3.5.3 Preparation of PCR Product___________________________ 141 3.3.5 4 Isolation of the Clone Containing the ECFP Domain lll-IV
Construct_________________________________________ 144 3.3.6 Construction of the EYFP Domain I Construct___________________ 144 3.3.6.1 Preparation of Vector________________________________ 145 3.3.6.2 Preparation of PCR Product___________________________ 145 3.3.6 3 Isolation of the Clone Containing the EYFP Domain I Construct __ 149 3.3.7 Construction of the EYFP Domain l-ll Construct_________________ 150 3.3.7.1 Preparation of Vector________________________________ 150 3.3.7.2 Preparation of PCR Product___________________________ 152 3.3.7.3 Isolation of the Clone Containing the EYFP Domain l-ll
Construct__________________________________________ 152
CHAPTER 4
4.1 INTRODUCTION___________________________________________ 156 4.2 RESULTS________________________________________________ 156
4.2.1 Expression of Domain l-ll and Domain lll-IV in Cos-7 Cells________ 156 4.2.1.1 Determination of the Subcellular Localisation of Domain l-ll and
Domain lll-IV of a^ B Using the a1 B Antibody________________ 158 4.2.1.2 Determination of the Subcellular Localisation of Domain l-ll/o2-Si/p and
Domain IIl-IV/a2-ôi/p Complex Using the o2 Antibody_________ 162
4.3 DISCUSSION _____________________________________________ 166
4.3.1 Expression and Subcellular Localisation of Domain l-ll and Domain lll-IV Using the a1 B Antibody___________________________________ 166 4.3.2 Expression and Subcellular Localisation of Domain I II and Domain lll-IV
Using the a2 Antibody ___________________________________ 168 4.3.3 Endogenous Production of Truncated Calcium Channel a1 Subunits 168 4.3.4 Other Truncated Proteins_________________________________ 169 4.3.4.1 Sodium Channels_________________________________ 170 4.3.4 2 Cytsic Fibrosis Transmembrane Chloride Conductor (CFTR) 170 4.3.4 3 Na*-Phosphate Cotransporter (Na Pi ll) _________________ 170 4.3.4 4 Human Red Cell Anion Transporter (Band 3 )_____________ 171
4.4 CONCLUSION_____________________________________________ 172 CHAPTER 5
INVESTIGATION OF THE EXPRESSION AND SUBCELLULAR
LOCALISATION OF THE TRUNCATED FORMS OF a lB USING THE GREEN FLUORESCENT PROTEIN AND ITS SPECTRAL VARIANTS
5.1 INTRODUCTION__________________________________________ 174
5.2 RESULTS_________________________________________________ 175
5.2.1 Control Experiments for GFP, EYFP and ECFP__________________ 175 5.2.2 XFP-Fusions of Full-Length a1 B _____________________________ 177
5.2.2.1 Expression and Subcellular Localisation of GFP-alB in
Cos-7 C ells__________________________________________ 177 5.2.2 2 Investigation of the Subcellular Localisation of GFP-alB Using the
Anti-GFP Antibody___________________________________ 184 5.2 2.3 Expression and Subcellular Localisation of a1 B-ECFP in
Cos-7 Cells_________________________________________ 187
5.3 TRUNCATED CONSTRUCTS: GFP DOMAIN I II and
ECFP DOMAIN III- I V _______________________________________ 187
5.3.1 Expression and Subcellular Localisation of GFP Domain I II in Cos-7
Cells__________________________________________________ 187 5.3.2 Expression and Subcellular Localisation of Domain lll-IV ECFP in
Cos-7 Cells ____________________________________________ 191 5.3.2 Expression and Subcellular Localisation of ECFP Domain lll-IV in
Cos-7 Cells ____________________________________________ 193
5.4 CO-LOCALISATION OF EYFP DOMAIN I II AND ECFP DOMAIN III- IV ___________________________________________________________ 197
5.4.1 Expression and Subcellular Localisation of EYFP Domain l-ll in Cos-7 Cells ________________________________________________ 197 5.4.2 Co-expression of EYFP Domain l-ll and ECFP Domain lll-IV________ 200
5.5 DISCUSSION ______________________________________________ 206
5.5.1 Location of the XFP-Fusion Tag _____________________________ 206 5.5.2 Subcellular Localisation of GFP Domain l-ll and ECFP Domain lll-IV
5.5.3 Co-localisation Studies of EYFP Domain l-ll and ECFP Domain lll-IV
209 5.5.4 Effect of Accessory Subunits on the Subcellular Localisation of
GFP Domain l-ll and ECFP Domain lll-IV ______________________ 210
5.6 CONCLUSION 211
CHAPTER 6
DOMINANT-NEGATIVE SUPPRESSION OF a lB INDUCED BY TRUNCATED CONSTRUCTS
6.1 INTRODUCTION___________________________________________ 213
6.1.2 Aims of Study In This Chapter______________________________ 214
6.2 RESULTS________________________________________________ 214
6.2.1 Expression and Subcellular Localisation of EYFP Domain_I_________ 214 6.2.2 Coexpression of the Truncated Proteins of a lB with Full-Length a lB
_______________________________________________ 217 6.2.2.1 Effect of Domain I on the Expression of Full-Length a1 B _______ 217 6.2.2 2 Effect of Domain l-ll on the Expression of Full-Length a l B ______ 219 6.2 2.3 Effect of Domain lll-IV on the Expression of Full-Length a lB ____ 219 6.2.2 4 Effect of the N-terminus on the Expression of Full-Length a l B ___ 222 6.2 2.5 Effect of the l-ll Loop on the Expression of Full-Length a l B _____ 225 6.2.2.6 Inhibition of the Proteasomal Degradation Pathway by Lactacystin
226
6.3.1 Dominant-Negative Suppression of Potassium Channels___________ 227 6.3.2 Mechanisms of Dominant-Negative Suppression_________________ 229 6.3.3 is Membrane Integration of the Truncated Proteins a Prerequiste for
Dominant-Negative Suppression?____________________________ 231
6.4 CONCLUSION____________________________________________ 232 CHAPTER 7
GENERAL DISCUSSION________________________________________ 234 FUTURE DIRECTIONS__________________________________________ 238 AKNOWLEDGEMENTS________________________________________ 240 BIBLIOGRAPHY_______________________________________________ 241
ADDITIONAL MATERIAL
Publication
Raghib, A., Bertaso, F., Davies, A., Page, K. M., Meir, A., Bogdanov, Y., Dolphin, A. C. (2001). Dominant-Negative Synthesis Suppression of Voltage-Gated Calcium Channel Cav2.2 Induced by Truncated
LIST OF FIGURES
CHAPTER 1
Figure 1.1 Schematic View of the Subunit Composition
Of the VDCC 25
Figure 1.2 Schematic Diagram of the Neuronal a1 Subunit Showing Structural Elements and Regions of Interaction with Other
Proteins 27
Figure 1.3 Truncations in Voltage-Gated Calcium Channels 60
Figure 1.4 Structure of GFP 67
Figure 1.5 Proposed Mechanism of Formation of the Mature GFP
Chromophore 69
Figure 1.6 Locations of Mutations that Improve GFP Folding 72
CHAPTER 2
Figure 2.1 Diagram of the Illumination Path in a Confocal Microscope 86
CHAPTER 3
Figure 3.1 The pMT2 Vector 108
Figure 3.2 Strategy for the Construction of the Domain I Construct 111
Figure 3.4 Strategy for the Construction of the Domain l-ll Construct 115
Figure 3.5 PCR Strategy for the Domain l-ll Construct 117
Figure 3.6 Strategy for the Construction of the Domain lll-IV Construct 119
Figure 3.7 PCR Strategy for the Domain lll-IV Construct 121
Figure 3.8 Strategy for the Construction of the GFP- a1 B Construct 123
Figure 3.9 PCR Strategy for the GFP- a1 B and GFP Domain l-ll 126 Constructs
Figure 3.10 Strategy for the Construction of the GFP Domain l-ll
Construct 129
Figure 3.11 Strategy for the Construction of the Domain lll-IV ECFP 132 Construct
Figure 3.12 PCR Strategy for the Domain lll-IV ECFP Construct 134
Figure 3.13 Strategy for the Construction of the a1 B-ECFP Construct 138
Figure 3.14 Strategy for the Construction of the ECFP Domain lll-IV
Construct 140
Figure 3.15 PCR Strategy for the ECFP Domain lll-IV Construct 142
Figure 3.16 Strategy for the Construction of the EYFP Domain I
Construct 146
Figure 3.18 Strategy for the Construction of EYFP Domain l-ll 151 Construct
Figure 3.19 PCR Strategy for the EYFP Domain l-ll Construct 153
CHAPTER 4
Figure 4.1 Schematic View of the Subunit Composition of the VDCC Showing the Target Epitopes for the Anti-al B and Anti-o2
Antibodies 157
Figure 4.2 Expression of Domain l-ll and Domain lll-IV in Cos-7 Cells
in the Presence of Accessory Subunits 159
Figure 4.3 Expression of Domain l-ll and Domain lll-IV in Cos-7 Cells
in the Absence of Accessory Subunits 161 Figure 4.4 Expression of a2-6i and a2-5i/p2a in Permeabilised Cos-7
Cells Transfected with a2-5i 164
Figure 4.5 Expression of Domain l-ll and Domain lll-IV in
Non-Permeablised Cos-7 Cells 165
CHAPTER 5
Figure 5.1 Expression and Subcellular Localisation of GFP, EYFP and
ECFP in Cos-7 Cells 176
Figure 5.2 Expression and Subcellular Localisation of GFP-a1 B in the
Presence of pi b and a2-ôi 178
Presence of pi b and a2-5i 179
Figure 5.4 Line Scan of Cell Transfected with GFP-a1 B/p1 b/a2-5i
Showing Plasma Membrane Localisation of GFP-a1 B 181
Figure 5.5 Expression and Subcellular Localisation of GFP-a1 B in the
Absence of p1 b and o2-ôi 182
Figure 5.6 Optical Sections of a Cos-7 Cell Expressing GFP-a1 B 183
Figure 5.7 Expression of GFP and its Variants Using the Anti-GFP
Antibody (GFP-Ab) 185
Figure 5.8 Expression and Subcellular Localisation of GFP-a1 B in the
Presence of pi b and a2-5i Using the Anti-GFP Antibody 186
Figure 5.9 Expression and Subcellular Localisation of GFP Domain I II
in the Presence of pi b and o2-5i 188
Figure 5.10 Optical Sections Showing Plasma Membrane Localisation of
GFP Domain l-ll in the Presence of p ib and a2-5i 189
Figure 5.11 Expression and Subcellular Localisation of GFP Domain I II in
the Absence of pi b and o2-ôi 190
Figure 5.12 Optical Sections Showing Subcellular Localisation of GFP
Domain l-ll in the Absence of a2-5i and pib 192
Figure 5.13 Expression and Subcellular Localisation ECFP Domain lll-IV
in the Presence of o2-ôi and pib 194
andplb 195
Figure 5.15 Excitation and Emission Spectra for (E)GFP, EYFP, ECFP
and Other Spectral Variants 196
Figure 5.16 Expression and Subcellular Localisation of EYFP
Domain l-ll in the Presence of a2-5i and pib 198
Figure 5.17 Optical Sections Showing Plasma Membrane Expression of EYFP Domain l-ll in the Presence of a2-Si and pib 199
Figure 5.18 Expression and Subcellular Localisation of EYFP Domain l-ll and ECFP Domain lll-IV in the Presence of
a2-0i and pib 201
Figure 5.19 Optical Sections Showing Expression and Subcellular Localisation of EYFP Domain l-ll and ECFP Domain lll-IV in
the Presence of aP-Si and Rib 202
Figure 5.20 Expression and Subcellular Localisation of EYFP Domain l-ll
and ECFP Domain lll-IV in the Absence of a2-5i and Bib 203 Figure 5.21 Optical Sections Showing Subcellular Localisation of EYFP
Domain l-ll and ECFP Domain lll-IV in the Absence of a2-5i
and p ib 204
CHAPTER 6
Figure 6.1 Expression and Subcellular Localisation of EYFP Domain I in
the Presence of a2-6i and p ib 215
Figure 6.2 Optical Sections of the Subcellular Localisation of EYFP
Figure 6.3 Effect of Coexpression of GFPa1 B and Domain I 218
Figure 6.4 Effect of Coexpression of GFP-a1 B and Domain l-ll 220
Figure 6.5 Effect of Coexpression of GFP-a1 B and Domain lll-IV, and
a1B and Domain lll-IV 221
Figure 6.6 Effect of Coexpression of GFP-a1 B and the N-terminus 223
Figure 6.7 Effect of Coexpression of GFP-a1 B and the myc l-ll loop 224
CHAPTER 7
Figure 7.1 Schematic Diagram Representing the Evolution of the VDCC 235
Figure 7.2 Schematic Diagram Showing Possible Effects of Truncated
Proteins of a1 B 237
LIST OF TABLES
CHAPTER 1
Table 1.1 Properties of Native Calcium Channels
Table 1.2 Spectral Characteristics of the Classes of GFP Table 1.3 Amino Acid Substitutions in EYFP and ECFP
30 73 37
CHAPTER 2
Table 2.1 Culture Media 82
Table 2.2 Laser Lines Used For Excitation of GFP, GFP Spectral
Variants, and Fluorophores 87
Table 2.3 Optical Section Thickness for Different Objective Lenses Using the Biorad MRC600 Laser Scanning Confocal
Microscope 88
Table 2.4 Primary Antibodies 90
Table 2.5 IgG-ConJugates 91
Table 2.6 Fluorophores 92
Table 2.7 Organelle Markers 94
Table 2.8 Example of PCR Conditions 97
Table 2.9 Constituents of PCR Reactions 98
Table 2.10 Buffers Supplied with Maxiprep and Miniprep Kits 105
CHAPTER 3
List of Abbreviations
Ab Antibody
co-Aga IVA co-Agatoxin IVA (o-CTX GVIA co-ConotoxIn GVIA
AID Alpha Interaction Domain BID Beta Interaction Domain
Bp base pairs
cDNA complementary DNA
ddH20 Double distilled water (d)dNTP (di)deoxy nucleotide
DHP di hydropyridine
DMEM Dulbeccos Modified Eagle Medium
DNA Deoxyribonucleic acid
Dorn Domain
DRG Dorsal Root Ganglion
EA Episodic Ataxia
ECFP Enhanced Cyan Fluorescent Protein EDTA Ethyllenediaminetetra-acetic acid
ER Endoplasmic Reticulum
EYFP Enhanced Yellow Fluorescent Protein FHM Familial Hemiplegic Migraine
FITC Fluorescein Isothiocyanate GFP Green Fluorescent Protein
G-protein Guanosine triphosphate binding protein
HVA High voltage activated
Kb Kilobase
kDa kilo Dalton
LCSM LVA mRNA PBS PCR PKA PKC RNA SNAP-25 TAE TBS UV VDCC
Laser Scanning Confocal Microscope Low voltage activated
messenger RNA
Phosphate buffered saline Polymerase Chain Reaction Protein Kinase A
Protein Kinase C Ribonucleic acid
Synaptosome Associated Protein of 25 kOA Tris-buffered acetate
Tris buffered saline Ultraviolet
Chapter 1
1.1 INTRODUCTION
The first description of voltage-dependent calcium channels (VDDCs) was by Fatt and Ginsborg (1958) who demonstrated the presence of these channels in crustacean muscle. Since then, VDCCs have been shown to be located at the plasma membrane of almost all excitable cells. These channels exist as
heteromultimers, and are activated by depolarisation of the plasma membrane in response to action potentials. They are a major route of calcium entry from the extracellular space, and are involved in a multitude of cellular processes including control of membrane excitability, neurotransmitter release, gene expression and muscle contraction. Thus, VDDCs behave as transducers, coupling electrical activity to cellular biochemical function.
1.2 STRUCTURE OF VOLTAGE-GATED CALCIUM CHANNELS
Extensive studies have indicated that neuronal VDCCs exist minimally as heteromultimeric complexes containing a pore-forming a1 subunit and at least two accessory subunits, p and a2-8 (Fig 1.1). An additional y subunit is
associated with skeletal muscle VDCCs. The basic structure and composition of VDCCs was elucidated by studies on the skeletal muscle L-type calcium
channel, a lS . Because of its high concentration in skeletal muscle T-tubules and high affinity for DHPs (G o u ld À a/., 1984), many of the early studies used skeletal muscle to investigate VDCCs (Sanchez et al., 1978, Beaty et al., 1976). Consequently, the first calcium channels to be purified were from rabbit skeletal muscle T-tubules (Tanabe et al., 1987). The a i subunit was found to form functional DHP sensitive L-type channels, and was identified as the pore- forming protein from reconstitution studies in lipid bilayers (Flockerzi et al.,
oc1
rS-S-,
AID
BID
p
The molecular weight of this complex is approximately 390 kDa, suggesting that the subunits exist in 1:1 stoichiometry. The a1 and y subunit are
transmembrane, whereas the p subunit is entirely intracelluar. Further studies were needed to clarify the topology of the a2 subunit, which is now known to be extracellular (Brickley et al., 1995; Jay et al., 1991), and is anchored to the membrane via the transmembrane 5 subunit.
1.2.1 The a1 Subunit
The a1 subunit is a large protein that forms the ion-conducting pore of all voltage-gated calcium channel subtypes. This subunit is non-glycosylated in all VDCCs with the exception of the T-type channels, which are thought to be glycosylated. Both the pharmacology and biophysical properties are defined by the a1 subunit which is the structural and functional core of the channel. To date, 10 a1 subunits have been cloned and expressed (a1A-l, S) (Birnbaumer
etal., 1994; Perez-Reyes et a i, 1998). The amino acid sequence shares considerable sequence homology with voltage-gated Na"" channels, suggesting that both channels are derived from a common ancestral gene (Strong et al.,
1993). Hydropathy plots suggest that the pore-forming a1 subunit consists of four homologous domains linked by intracellular loops (Figure 1.2). Each domain consists of 6 transmembrane regions (81-86) and a P-loop between transmembrane regions 5 and 6 of each domain (Tanabe et al., 1987). This is very similar to the voltage-dependent Na"^ channel that shares similar topology. The VDCCs also share some similarities with voltage-dependent channels, which are comprised of four independent subunits, each composed of six transmembrane regions with P-loops. Each of these Kvi subunits is structurally similar to one domain of the Na^ and Ca^ pore-forming a1 subunits.
Voltage-sensor (S4) P-loop (ion selectivity filter)
rx
r^ f O
-\ j r ^
GPy binding
domain
Syntaxin/SNAP-25/ Synaptotagmin binding
p-binding| GPy site
PKA Fig 1.2
Schematic Diagram of the Neuronal a1 Subunit Showing Structural Elements and Regions
of Interactions with Other Proteins
Each domain of the a1 Subunit has six transmembrane segments (labelled 81-86 in Domain l).The individual
domains each posses a voltage-sensor (84) and a P-loop (85-86. The diagram also shows the intracellular
loops connecting domains l-IV and the proteins they interact with, as well as binding sites for the and G|iy
four domains. These transmembrane a helices contain positively charged amino acids (arginine or lysine) in every third to fourth position in each region
\
(Varadi etal., 1995). On depolarisation, these residues are thought to move in response to the altered electric field, thereby changing the arrangement of the tertiary structure and opening the channel. The extracellular pore (P) loops are believed to form the ion-selectivity filter (Figure 1.2). These loops link
transmembrane regions S5 and S6 of each domain. They are thought to exist as a hairpin loop bent back into the membrane, forming the mouth and lining of the pore. This structure has been supported by evidence from amino acid mutations in this region (Heinèmann et al., 1992). Each P-loop contains a negatively charged glutamate residue (aspartate in some L-type channels) which is essential for the selectivity of the pore. These glutamate residues
2+
coordinate Ca ions during their passage through the pore (Ellinor et al., 1995), and also underlie channel block by heavy metal ions such as Cd^^ and Co^^. Studies have shown that mutation of any one of these glutamate residues to either a positively charged or neutral amino acid abolished calcium selectivity completely or significantly reduced it (Yang etal., 1993a). Unlike K"” channels, the positions of the residues are hypothesised to be slightly asymmetrical with respect to each other (Miller et al., 1992). This asymmetric pattern is thought to provide an imbalance of contribution, allowing the simultaneous interaction with the two calcium ions and therefore permitting larger conductance through the channel (Yang etal., 1993a) due to electrostatic repulsion.
1991; Nakai et al., 1998). The same loop in a lA and a lB interacts with syntaxin 1A and possibly synaptosomal associated protein (SNAP-25).
1.3 CLASSIFICATION OF VDCCs
Two distinct groups of native VDCCs have been found, the low voltage
activated (LVA) and the high voltage activated (HVA) channels (Table 1.1). The primary distinction is between the relative activation potentials of each group. The LVA current is rapidly inactivating, and is activated by weak
depolarisations, whereas HVA currents display variable inactivation and are activated by strong depolarisations (Carbone & Lux, 1984). Consequently, the LVA channels were named T ’ or transient type and the HVA channels in heart and skeletal muscle were named the L-type channels from long-lasting. The single channel conductance of the LVA channels is 9-11 pS in 110 mM Ba^^, as compared to the larger conductance of HVA channels (20-24 pS). The HVA channels were then found to include the L, N, P/Q, and R type channels, classified on the basis of their pharmacology, gating and single channel conductance profiles. These channels are primarily involved in excitation- secretion and excitation-contraction coupling. The LVA channels include the a lG , a lH and a l l channels. These channels were identified very recently, and much work remains to be done to determine their functions, though it is thought
(o(o
that a lG underlies the classic T-type current in thalamic neurones (Huguenard
et a!., 1996). A lack of selective antagonists for the T-type channels has also prevented further dissection of this class.
1.4 THE N-TYPE (alB ) CALCIUM CHANNEL
Table 1.1 Properties of Native Calcium Channels
Classification
Properties T L N P / Q R
(LVA) (HVA) (HVA) (HVA) (HVA)
alG , H, 1 a1S,C, D,F,
a lB a lA a lE ?
Inactivation fast slow intermediate slow intermediate fast
rate (ms) (20-50) (>500) (50-500) (>500) (>100) (20-30)
Relative conductance
Ba^*=Ca^ B a ^ > C a ^ B a ^ > C a ^ ? ? Ba^*>Ca^*
Conductance (pS)
7-10 9-26 10-20 10-20 10-20 15
Selective pharmacology
kurtoxin (<350nM)
DHP? m-CgTx
GVIA (<100nM)
cù-Aga IVA (<100nM)
co-Aga IVA (>100nM)
1.4.1 Structure and Biophysical Properties of the N-Type Channel
Like other neuronal VDCCs, the N-type channels exist as a complex containing
C
the a1, p, a2-0 subunits (McEnery etal., 1991; Witcher etal., 1993a; Witcher et al., 1993b; DeWaard etal., 1994; McEnery ef a/., 1994). Neuronal N-type channels have also been found to co-purify with proteins involved in neurotransmitter release, including syntaxin, synaptotagmin and HPC-1 (Leveque etal., 1992; Yoshida etal., 1992; Leveque etal., 1994). In
pharmacological terms, these channels are defined by their high selectivity for (0- CO notoxin GVIA (co-CTX-GVIA), which was used to purify the N-type
channels (McEnery etal., 1993), and also their insensitivity to dihydropyridines (Fox etal., 1987a and b). In terms of current kinetics, N-type channels are activated at more depolarised potentials and show greater inactivation than the L-type channels (Fox et al., 1987a and b). Their single channel conductance is between 10-20 pS (Dolphin, 1995), lower than the L-type channels. In addition, many studies have consistently demonstrated G-protein modulation of N-type channels in many different cell types (Menon-Johansson et al., 1993; Kuo & Bean, 1993; Mynlieff & Bean, 1994; Zhu & Ikeda, 1994; Ehrlich & Èlmsiie, 1995).
1.4.2 Localisation and Function of Native N-Type Channels
The N-type calcium channel is widely expressed throughout the brain, with the highest concentration in the cerebellar cortex, hippocampus, olfactory bulb, hypothalamus and thalamus, and the cerebellar cortex (Fortier et al., 1991; Westenbroek etal., 1992; Du bel W a/., 1992; Fujita etal., 1993; Volsen etal.,
7f\
1995; Tanaka et al., 1995). Many studies have found that the expression of the N-type channel is neurone-specific, as no expression was detected in the testis, kidney, spleen, adrenal gland, liver, lung or pituitary (Dubel et al., 1992;
pituitary cells (Suzuki & Yoshioka, 1987, Lievano, 1994), testicular cells (D' agostino'ef a/., 1992), endocrine cells (Dunlap et al., 1995) and chromaffin cells (Carbone et a/., 1993/ ^
Two forms of the N-type channel have been identified in the rat brain, of size
p'
240 and 210 kDa. Antibodies that recognise both these forms showed that the channel is localised predominantly in dendrites. The large terminals of the mossy fibers of the dentate granule cells were also heavily labelled in a punctate pattern. There was also some staining in the cell bodies of certain cells of the dorsal cortex and Purkinje cells (Westenbroek et a!., 1992). Consistent with their role in neurotransmitter release, N-type channels have been found diffusely on noninnervated cell bodies of hippocampal neurons, whereas following innervation, the channels were concentrated and immobilised at synaptic contact sites (Owen et a!., 1989).
Additionally, N-type channels have been found clustered at the presynaptic active zones of the frog NMJ (Robitaille etal., 1990; Cohen etal., 1991; Torri- Tarelli et al., 1991), sites of synaptic contact in hippocampal cultures (Jones ef
al., 1989) and the climbing fibers of cerebellar Purkinje cells (Regehr & Mintz, 1994). It has also been found on the presynaptic membrane of the calyx in chick ciliary ganglia (Stanley & Atrakchi, 1990; Haydon etal., 1994), and in the
rCî
growing neurite processes of P C I2 cells (Rebef & Reuter, 1990; Usowicz et al.,
1990). However, in the mammalian NMJ, the N-type channel is concentrated mostly in nerve-terminal associated Schwann cells (Day et^al., 1997), though some reports suggest that co-CTX-GVIA has some effects at this site (Rossoni
et a/., 1994).
N-type channels have also been identified along dendritic trees and in a
I ii S
subpopulation of dendritic spines (Westenbroek et al., 1992; Miller at a!., 1994).
N-type channels have also been found in the leading processes of immature cerebellar granule cells where they are involved in the migration of these cells, prior to establishing synaptic contacts (Komuro & Rakic, 1992). Lastly, studies in foetal rat primary sensory neurones indicate that N-type channels can induce neuronal gene expression in response to phasic membrane depolarisation
Go
(Brosenitsch & Katz, 2001). This is in contrast to L-type channels, which
G I
regulate gene expression in response to chronic depolarisation (Brosenitsch at a!., 1998). Prior to synaptogenesis, N-type channels are expressed in a diffuse pattern over the entire neuronal surface (Jones at a!., 1989). This suggests that these channels fulfill multiple functions in immature neurons such as neuronal migration (Komuro & Rakic, 1992), axon outgrowth (Doherty etal., 1991) and activity-dependent-gene expression. In rat hippocampus, some N-type channels exist before birth, consistent with a role in migration. However, the majority of expression is postnatal, and coincides with synaptogenesis (Owen etal., 1997). This suggests a role of a lB in the development of the nervous system.
1.4.3 The Cloned a lB (N-Type Channel) Subunit
The a lB cDNA has been cloned from human (Williams at al., 1992b), rat (Dubel
6>S
etal., 1992) rabbit, (Fujita etal., 1993) and mouse (Coppola etal., 1994)
neuronal tissues. It has also been cloned from rabbit and human brain (Fujita at al., 1993; Williams at a/.,1992b). The a lB clone has been firmly attributed to the native N-type channel due to its selective block by co-CTX GVIA. The deduced amino acid sequence is very similar to the a lA sequence (82%), but shares less homology with a lS (Fujita etal., 1993).
co-CTX-GVIA (Williams et al., 1992b; Fujita et al., 1993; Stea et al., 1993). In
Xenopus oocytes, the activation kinetics were relatively slow (xact = 32 ms when expressed with a2-ô/p1), and 65 ms when expressed alone (Stea et al.,
1993). However, in mammalian expression systems activation kinetics were much faster (xact= app. 2 ms (HEK 203 cells) (Bleakman et al., 1995). These discrepancies may reflect differences in post-translational modifications
between the expression systems, different splice variants used or inaccuracies associated with two-electrode voltage-clamp in oocytes.
1.4.4 Modulation of N-Type Calcium Channels
Accessory subunits have also been shown to modulate cloned a lB currents. For example, coexpression of p ib with a lB increased the currents four-fold as well as slowing activation kinetics (Stea et al., 1993). In both oocytes and HEK 293 cells, coexpression of a2-6 and p ib with a lB led to the largest currents and to the most co-CTX receptors (Stea et al., 1993; Brust et al., 1993) (see Section
1.7.2).
Additional mechanisms of modulation involve G-proteins and phosphorylation. As described later, G-proteins bind to the I II loop of neuronal VDCCs, and compete with binding of the accessory p subunits. Phosphorylation of the a l subunit of the N-type channel is thought to be mediated by cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC), and calcium-calmodulin kinase (CaM kinase II) (Dolphin,
1995). Both mechanisms are almost certainly involved in the regulation of neurotransmitter release (Man-Son-Hing et al., 1989; Hille, 1992; Toth et al.,
1993; Dolphin, 1995).
Neurotransmitter release is initiated by the influx of calcium through VDCCs within 200 fis of the arrival of the action potential at the presynaptic nerve terminal (Robitaille et al., 1990). Here, clusters of VDCCs are thought to supply calcium to initiate neurotransmitter release (Pumplin etal., 1981; Pumplin, 1983; Zucker, 1993). The Ca^^ concentrations necessary for exocytosis of synaptic vesicles can be as low as 5-10 pM or as high as 100-200 pM
(Augustine, 2001; Cohen etal., 1991; Sheng etal., 1996; Wheeler etal., 1994). As the intracellular calcium concentration declines steeply as a function of distance away from presynaptic VDCCs, it is likely that the brief rise in Ca^^ necessary for exocytosis occurs in close proximity to^calcium channels (Cope and Mendell, 1982; Llinas etal., 1991; Stanley, 1993). Thus, it is plausible that there is a direct physical association between the exocytosis machinery and presynaptic VDCCs, and that a critical intermolecular distance must be maintained between these components for efficient neurotransmitter release.
Synaptic vesicle docking and fusion with the presynaptic plasma membrane are mediated by a complex of proteins, including the vesicle protein VAMP/
synaptobrevin (Trimble et al., 1988) and the plasma membrane proteins
syntaxin and SNAP-25 (Bennett et al., 1992; Yoshida et al., 1992; Sollner et al.,
/ V
1993; Calakos et al., 1994). Synaptotagmin is a synaptic vesicle protein that binds Ca^^ (Pehn et al., 1990; Li etal., 1995), and also interacts with syntaxin in
2+
a Ca -dependent manner (Li ^ a /., 1995b; Chapman et al., 1995). It is thought to function as a Ca^^ - sensor for Ca^^-dependent neurotransmitter release (Elferink et al., 1993; Geppert etal., 1994). Several studies have indicated that there is an intimate association of both syntaxin and snyaptotagmin with N-type channels. These studies suggest that the N-type channel (and the P/Q-channel) is also a component of the synaptic vesicle/docking and fusion complex. This role is supported by the co-localisation of high-density clusters of N-type channels and synataxin in presynaptic terminals (Westenbroek etal., 1992;
^ Ù 1 I
organ NMJ exist within a complex containing cytosolic spectrin and a4piy1 laminin, found in the synaptic cleft. The authors suggest that a4piy1 laminin serves to anchor VDCCs to the presynaptic membrane.
Sheng et al. (1994, 1996) have identified a specific site in the ll-lll loop of the N- type channel that interacts with syntaxin. This region, termed the ‘synprint site’ consists of amino acids 718-963 of the ll-lll loop, and contains two adjacent binding domains. The second domain displays tighter binding than the first. Studies have also shown that the C-terminal one-third of syntaxin interacts with
A
the synprint site (Sheng etal., 1994; Rettig etal., 1996). The vesicle protein synaptotagmin has also been shown to interact with N-type channels (Sheng et
Z.' %
al., 1997), similar to syntaxin and SNAP-25 ( L ^ y e g u e ^ ^ ^ 1992, 1994; Vance
efa/., 1999), BiS
Mochida et al. (1996) demonstrated that injection of peptides containing the synprint site of a lB into superior cervical ganglion neurones reduced synaptic transmission by 23-42%, by disrupting the interaction of native N-type channels with syntaxin. A similar study in cultured Xenopus embryonic neuromuscular junctions revealed that the Ca^^ -dependence of neurotransmitter release was
shifted to higher values, so that at physiological Ca^^ concentrations a 50% t y o
reduction in transmitter release was observed (Rettig^ ef a/., 1997). This
suggests that the injected synprint peptides disrupted the physical association of a lB and the synaptic vesicle complex, thus displacing pre-docked synaptic vesicles and reducing the efficiency of neurotransmitter release. Both the above studies point to a direct physical association of N-type channels and the
synaptic vesicle docking/fusion machinery.
In addition, it has also been found that the interaction of the N-type channel synprint site with syntaxin is Ca^^ -dependent and occurs in the same
concentration range as the threshold for neurotransmitter release (Sheng et al.,
channels and syntaxin are phosphorylated by either Cam-kinase II, PKC, PKA or PKG, all of which are expressed in presynaptic nerve terminals (Hell et al.,
1994; Miming and Scheller, 1996; Shimazaki et a!., 1996). Studies have shown that phosphorylation of the synprint site of a lB by CaM KM and PKA, but not PKA or PKG, strongly inhibited the binding of synatxin and SNAP-25
(Yokoyama et a!., 1997). This provides another avenue for the modulation of neurotransmitter release.
A recent study suggests that in addition to anchoring N-type channels via their synprint site, syntaxin also modulates these channels. Bezprovanny et ai.
(2000) have shown that syntaxin reduced the size of currents elicited by a lB in
Xenopus oocytes, by promoting the slow inactivation of these channels. The authors suggest that the modulatory role of syntaxin is completely separate from its anchoring role, and may serve to stabilize calcium channel inactivation until reincorporation into a new pre-fusion SNARE complex. Further effects of syntaxin include a hyper-polarising shift in the steady-state inactivation curve and promotion of tonic inhibition of the channel by Gpy (Jarvis et ai., 2000). Both these effects are mediated by separate molecular determinants in syntaxin (Jarvis and Zamponi, 2001). Cysteine string proteins have also been shown to interact with both G-proteins and N-type channels (Magga et ai., 2000), and may also modulate N-channels during neurotransmitter release.
1.4.6 Splice-Variants of a1B
Many splice-variants of the N-type channel have been identified. In general, splicing regions include the intracellular I II and ll-lll loops, and the extracellular loops connecting IIIS3-IIIS4, and IVS3-IVS4. Splicing in these regions may affect binding to p and Gpy subunits, and may also affect channel kinetics such as the voltage dependence of activation. However, co-CTX GVIA cannot
Two forms of the N-type channel have been identified in the rat brain, of size 240 and 210 kDa (Westenbroek et al., 1992; Williams at a!., 19921^ which
iOL)
possess different C-termini (Hell etal., 1993). They also differ in terms of phosphorylation, with PKA and PKC phosphorylating both forms, and CaM Kinase II only phosphorylating the longer form (Hell etal., 1994). More recently, two more forms of a l B message have been identified in the rat nervous system and are thought to arise from the use of alternative polyadenylation signal
1 oT
sequences in the 3’-untranslated region (Schorge etal., 1999). The authors also showed that the isoform containing the long 3’ untranslated region, is stabilised by Ca^^ entry in response to depolarisation, and leads to increased N-channel expression and current density in neurones. Due to the short time- course of this effect, the authors concluded that this was a transcription
independent event. In addition, dendrites appear to express a unique variant of N-type channels (Delmas ^eial., 2000). These N-type currents are more
sensitive to inhibition by neurotransmitters and G-proteins than their somatic counterparts, and also show lower single conductance.
io n
Lin et al. (1999) have shown that an isoform of a l B containing an ‘SFMG’ (Dom IIIS3-S4) tetrapeptide sequence is expressed in rat brain (rna1B-d), whereas an isoform containing the ‘ET’ didpeptide (DIVS3-S4) sequence is predominant in
\ \ 0
sympathetic ganglia (rna1B-b)(Lin et al., 1997). The sympathetic ganglia dominant form activates 1.5 times slower and at potentials 7 mV more
depolarised than the brain dominant form (Lin et al., 1997), due to the presence of the ET sequence (Lin etal., 1999). However, the inactivation kinetics of these two variants are identical (Lin et al., 1999).
were shifted to more depolarised potentials, but the rate or voltage-dependence of channel opening was unaffected (Qian & Lipscombe, 2000).
Finally, Mittman and Agnew (2000) have identified a splice variant of a1B, which would introduce a stop codon near the end of the ll-lll loop. The expression of this splice variant would give rise to a protein consisting of domains I and II of a1B (see later).
1.5 IDENTIFICATION AND CLONING OF HIGH- VOLTAGE ACTIVATED CALCIUM CHANNELS
1.5.1 The L-Type Channels
The L-type channels are the most widely distributed of the HVA channels and are found in skeletal, cardiac and smooth muscle, endocrine glands and neurons. The transverse- tubules (T-tubules) of skeletal muscle contain the highest density of dihydropyridine (DHP) -binding sites and were thus used for the purification of L-type channels (Catterall et a/., 1988). A long and short form have been described in skeletal muscle (Catterall at al., 1988; De Jongh at a!.,
1991) and both function as voltage-sensors in excitation-contraction coupling (Beam at a/., 1992). Hosey at al. (1989) were the first to identify a cardiac L- type channel. Further studies led to the identification of the four accessory subunits which were found to co-purify with the skeletal muscle L-type channel (Catterall at al., 1989). The L-type channels have a single channel conductance of 25 pS in 110 mM Ba^"", and are activated at a potential of approximately -1 0 to 0 mV (Fox at al., 1987). In contrast to the N-type^hannels, these channels are not thought to be G-protein modulated (Zhang at al., 1996). However, some native L-type channels have been described to be G-protein modulated
(Carbone efa/., 2001; Carabelli efa/., 2001; Gilon at al., 1997). oc^cA \-o
The a1S subunit is found only in skeletal muscle and belongs to the DHP sensitive class of a1 subunits. It was the first a1 subunit to be isolated, due to its high abundance in skeletal muscle and high affinity for DHRs (Tanabe et al.,
1987). It has a predicted MW of 212 kDa. This subunit is alternatively spliced
ivS)- wp ^
after the IVS3 region, and other splice-sites similar to those in a lC and a l D o h -
have also been identified (Perez-Reyes at a!., 1990). Furthermore, a two- , i?, domain isoform of a lS has been identified (Malouf at a!., 1992). The function of this truncated protein, if any, is unknown. Subsequent studies by Chaudhari (1994) failed to confirm the presence of this particular splice-variant. The a lS subunit is unique in its crucial role in excitation-contraction coupling, which involves the binding of its ll-lll loop to the ryanodine receptor in the
l ? o
sarcoplasmic reticulum (Tanabe at a!., 1990; Catterall, 1991).
1.5.1.2 The a lC Subunit
The a lC clone is the isoform of the L-type channel found most predominantly in cardiac tissue, although it has also been detected in a number of tissues
\ V \
including brain (Snutch at a!., 1991), lung (Biel efa/., 1990); aorta (Koch at a!.,
iZ.5 e+^>- '2^
1990), pancreas (Iwashima at a!., 1993) and kidney (Yu, 1995). It was first cloned from rabbit heart (Mikami at a!., 1989). The human form of cardiac a lC
^ b has also been cloned and its gene mapped to chromosome 12p13 (Schultz at al., 1993). It has 66% overall homology to the a lS subunit and a predicted molecular mass of 243 kDa. The a lC subunit has also been used to identify the amino acids important in DHP binding. Studies have shown that amino acids in IIIS5, IIIS6 and IVS6 and the pore-forming region are critical for DHP activity (Grabner efa/., 1996; Sinnegger at a!., 1997; Bodi at a!., 1997).
At least six sites for alternative splicing have been reported in the a lC subunit. These include the amino terminus, IS6, the cytoplasmic loop between IS6 and IIS1, IIIS2, IVS3 (flanking regions) and C-terminal cytoplasmic tail (Perez-Reyes
long amino terminal isoform appears to be dominant in the heart (Biel et al.,
1991). Recently, Wielowieyski et ai. (2001) have identified novel splice-variants of a lC generated by alternative splicing in the ll-lll loop which predict two truncated forms of a lC consisting of domains I and II. The functional
significance of all these variants has yet to be elucidated. However, Gao et ai.
(2001) have recently demonstrated that the cleaved C-terminal fragments of a lC can associate with the truncated a l subunit and regulate its conductance. This finding is indicative of a regulatory function of the C-terminally truncated variants of a lC .
Although the a lC subunit is not modulated by G-proteins (Zhang et ai., 1996), it is significantly affected by phosphorylation. It is known that different isoforms have different phosphorylation sites but differences in current kinetics are not always observed. For example, Klockner et ai. (1997) found three splice variants for the C-terminal region of the human cardiac alC , though the difference in the electrophysiological properties of these splice variants was minimal. However, studies by Soldatov et al. (1997) showed significant differences in current inactivation and a current voltage (l-V) shift to more hyperpolarised potentials, for one of the three a lC subunits with alternatively spliced C-terminal tails isolated from human hippocampus. It has not been established if these are similar to those variants identifed by Klockner and colleagues. It remains plausible however, that phosphorylation has a different effect on each splice-variant.
1.5.1.3 The a lD subunit
The a l D clone is another member of the DHP sensitive L-type channels. It was cloned from pancreatic islet cells (Seine et al., 1992) and human brain (Williams
a1C clone, and has a predicted MW of 245 kDa. Like a1C, it has many splice variants, of which many of the splice sites are similar to a1C (Perez-Reyes et
a
al., 1994). The a1D subunit is found primarily in the endocrine system but can also be detected in the brain, heart, kidney and ovaries (Hui at al., 1991; Yu et al., 1992; Yaney ef a/., 1992; Wyatt efa/., 1997; Perez-Reye^ef a/., 1990). It has been suggested that a l D is the only L-type channel that is G-protein modulated (Gilon etal., 1997) although the molecular basis for this is poorly understood. However, Bell et al. (2000) have shown that L-type currents are resistant to modulation by alGi/o -linked G-protein coupled receptors.
1.5.1.4 The a1F Subunit
The a l F subunit is a novel member of the L-type channel family. It is thought to
be expressed exclusively in the retina, and is mutated in patients with X-linked ^ ^ , k:
? K u u x o ^ c . ^ C o v C g P ^
congenital stationary night blindness (Strom et al., 1998; Bechansen et al.;
■ O N ( ; C ■ ' ' & 2_
1998). Functional studies are needed to elucidate the exact role of this subunit. although it possible that it may be involved in retinal neurotransmitter release,
ill 1
which has been shown to be d(ependent on L-type channels (Heidelberger et al., 1994; von Gersdorff and Matthews, 1994).
1.5.2 The P/Q-Type Channel
1 ( 4 3
The P-type channels are sensitive to co-Agatoxin IVA (co-AgalVA) (Usowicz et al., 1992; Mintz etal., 1992) and were initially identified in cerebellar Purkinje neurones. These channels are localised throughout the brain although
functional expression is predominantly at presynaptic terminal in most neurones. P-type channels have also been identified in the kidney,
neuromuscular junction and pancreas (Yu 1995; Hi vert etal., 1995; Day etal.,
1997; Lignon etal./1^98). These channels are characterised kinetically by their
' 1
1992). Q-type channels were initially identified in cerebellar granule cells (Randall and Tsien, 1995). They show greater inactivation as compared to P- type channels, and are less sensitive to co-Aga IVA. Together, the current sensitive to co-Aga IVA is referred to as the P/Q-type current.
1.5.2.1 The a lA Subunit
The cloned a l A subunit is thought to be the counterpart for the native P/Q-type channel (Starr et al., 1991)(rbA-1 MW 257kDa). This subunit was cloned from rabbit and rat brain, using the skeletal muscle a lS cDNA as a probe (Mori at a!., 1991; Starr at a!., 1991). Its sequence is approximately 40% identical to the L-type channel a lS . It is expressed at high levels in Purkinje neurones (Mintz at a!., 1992a) as well as other regions of the brain (hippocampus, spinal cord) and peripheral neurones, kidney and pancreas (Mori at a!., 1991; Dunlap at al.,
1995; Lignon at al., 1998).
When expressed in mammalian cells, the activation kinetics of a lA appear to be similar for the pib, p2a and p3 subunits (time to peak 2.16-2.4 ms) (Moreno
at al., 1997). However, the inactivation kinetics appear to depend on the p- subunits coexpressed with the channel. Co-expression of the p3 subunit
produced an inactivation time-constant (xin ac t) of 214 ms in HEK 293 cells, while p ib produced a Tmact of 225 ms in the same system. In Cos-7 cells, the Xjnact for p ib was 332 ms. p2a either did not produce any inactivation or had a very slow Xinact of 860ms (HEK 293 cells) (Berrow at al., 1997; Moreno at al., 1997).
The pharmacology of the a l A subunit shows more similarity to P/Q-type channels when expressed in mammalian cells, as opposed to expression in
the P-type component in cerebellar granule neurones (Mintz et al., 1992a, b; Pearson etal., 1995; Randall etal., 1995). However, similar expression (a1A/a2-0/p1^) in Xenopus oocyte produced an estimated IC50 of 200 nM (Sather etal., 1993), approximately 100-fold more than native P-type channels in Purkinje neurones (Mintz etal., 1992a,b). Overall, data from experiments in the mammalian expression system was in better agreement to the native channels than the Xenopus oocyte data. Studies by Mori et al. (1991) in oocytes showed that the increase in currents upon coexpression with pi and a2-ô was additive. This indicates that there may be a synergistic action between these two subunits. Single channel analysis detected a channel with 16.5 pS conductance (Mori etal., 1991). However, it was later shown that oocytes contain an endogenous channel with similar conductance (Lacerda et al.,
1994). The expressed channel can be blocked by co-Aga-IVA, which is selective for P/Q -type channels. However, P-type channels in vivo are approximately 100 fold more sensitive than a lA induced currents in oocytes (Sather etal., 1993). In addition, the latter are approximately 10 fold more sensitive to co-CTX-MVMC. These findings led to speculation that a l A represents a novel type of channel, the Q-type channel. There is however evidence to support the designation of a l A to the P-type current. The tissue distribution of the a lA subunit largely correlates to the electrophysiological detection of the P-type current (Gillard ef al., 1997). These authors also demonstrated that treatment of cerebellar Purkinje cells with antisense
oligonucleotides directed against the a lA subunit resulted in both the reduction in a lA detected by immunofluorescence and the measured P-type current.
Taken together, these studies show that the properties of the a l A subunit are not identical to those of the native P- and Q-type currents, and therefore
rat brain (MW 257 kDa) (Starr et al., 1991). Additional splice-variants of a lA have also been identified, with splicing occurring in the C-terminus, the ll-lll loop and the 83-84 loop of domain IV (Yu etal., 1992; Perez-Reyes etal., 1990). In a recent study by Bourinet et al. (1999), it was demonstrated that alternative splicing in the domain IV 83-84 extracellular loop affected the voltage- dependence of activation and reduced the affinity of co-Aga-IVA binding. Splicing in the domain l-ll loop slowed channel inactivation kinetics and also affected steady-state inactivation. Thus, it is possible that alternative-splicing of the primary transcript of a l A accounts for the different biophysical and
pharmacological properties of the P/Q-type current.
1.5.3 The R-Type Current
The R-type current is so named because it was the calcium current remaining after all other calcium channels had be blocked with toxins and DHPs (hence residual or R-type current). It was first identified in cerebellar granule neurones (Zhang et al.^ 1993). Initial studies indicated that the R-type current was rapidly inactivating and sensitive to Ni . However, more recent studies have described this current as more slowly inactivating (Tottene et al., 1996). This suggests heterogeneity among the R-type currents.
1.5.3.1 The a lE Subunit ^
The a lE subunit was cloned from the rat brain by 8obng et al. (1993) with predicted MW of 250 kDa. 8plice-variants for this subunit have also been cloned, with splicing regions in the C-terminus and ll-lll loop (Niidome et al.,
1992). Northern blot analysis has detected mRNA for this subunit only in the brain. There is significant expression in olfactory bulb, cortex, hippocampus and cerebellum (8oong etal., 1993; Niidome etal., 1992). In addition,
neurones and dentate gyrus, cell soma and dendritic trees of the globus pallidus, amygdala and thalamus (Yokoyama et al., 1995). However, by using the more sensitive technique of reverse transcription polymerase chain reaction (RT-PCR), a1E mRNA was also detected in mouse spermatogenic cells and rat atria (Taussig etal., 1993; Piedras-Renteria etal., 1997). This subunit has been expressed in various systems including Xenopus oocytes (Wakamori etal.,
1994) and Cos-7 cells (Stephen efa/., 1997). There is a lot of confusion about the corresponding native channel of the a lE subunit, although it has been suggested that it may contribute to R-type currents. A recent study in a lE knockout mice demonstrated that only a small component if the R-type current is attributable to the a lE subunit, and the authors suggest that the majority of
\2\
the R-type current is produced from other VDCC subunits (Wilson et al., 2000).
iT\^ss\rs9 - iS'J Clearly, further studies are needed to identify the origin of the R-type current.
1.6 IDENTIFICATION AND CLONING OF LOW-VOLTAGE ACTIVATED CALCIUM CHANNELS
1.6.1 The T-Type Channels
T-type currents have been found in many areas of the body including the CMS, cardiac muscle and auditory hair cells. These calcium currents are
characterized by their low activation voltage, with a typical voltage-dependence of activation, V50, in the range -6 0 to -30mV. Owing to the lack of known
different tissues. However, it is possible that traditional HVA channels may contribute to the T-type channels. Meir & Dolphin (1998) showed that HVA a l clones have an LVA state seen in single channel recordings (single channel conductance was 4-7 pS).
1.6.1.1 The a lG , a lH and a l l Subunits
The a lG , a lH and a l I subunits have recently been identified as counterparts of the LVA channels, as identified by expression in Xenopus oocytes and HEK
! - \ 0 \ . ' - . V v C l ■■ ' t ^ ^ '
cells (Cribbs etal., 1998) Perez-Reyes etal., 1998; Lee etal., 1999; Williams et al., 1999; Klugbauer et al., 1999b). All of these subunits encode T-type
channels. Full length a l G and a l I have been cloned from rat and human tissues (Perez-Reyes etal., 1998; Lee etal., 1999; Klugbauer ef a/., 1999b), and a l H has only been cloned from human tissue (Williams et al., 1999; Cribbs
et al., 1998). All three subunits have splice-variants. a lG and a l I are
predominantly expressed in the CNS, although low level expression has been found in the testis and lung (Talley et al., 1999; Kase et al., 1999; Klugbauer et al., 1999b). Conversely, a lH is expressed in the CNS as well as in peripheral neurones (Williams etal., 1999; Cribbs et al., 1998). Of the three subunits, a lG is the most predominantly expressed in the brain. The localisation and
biophysical properties of a l G suggest that this subunit is responsible for the classic thalamic T-type Ca^^ current (Huguenard et al., 1996). In support of this, a recent study in alG -null mice has demonstrated that a lG modulates the intrinsic firing patterns of thalamo-cortical relay neurones, contributing to the generation of absence epilepsy (Kim etal., 2001). Significantly, none of the novel T-type clones possess the full p-binding site in the l-ll loop which is conserved in all other HVA VDCC clones. It is unknown if these subunit
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