- Email: [email protected]
Use of transgenic mice to study voltage-dependent Ca2+ channels
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TRENDS in Pharmacological Sciences Vol.22 No.10 October 2001
Use of transgenic mice to study voltage-dependent Ca2++ channels James N. Muth, Gyula Varadi and Arnold Schwartz During the past decade a great number of genes encoding high- and lowvoltage-dependent Ca2++ channels and their accessory subunits have been cloned. Studies of Ca2++ channel structure–function relationships and channel regulation using cDNA expression in heterologous expression systems have revealed intricate details of subunit interaction, regulation of channels by protein kinase A (PKA) and protein kinase C (PKC), drug binding sites, mechanisms of drug action, the ion conduction pathway and other aspects of channel function. In recent years, however, we have arrived at the brink of an entirely new strategy to study Ca2++ channels by overexpressing or knocking out genes encoding these channels in transgenic mice. In this article, various models of gene knockout or gene overexpression will be discussed. This new approach will reveal many secrets regarding Ca2++ channel regulation and the control of Ca2++-dependent cellular processes.
James N. Muth Gyula Varadi Institute of Molecular Pharmacology and Biophysics and the Dept of Cell Biology, Neurobiology and Anatomy Arnold Schwartz* Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, PO Box 670828, 231 Albert Sabin Way, Cincinnati, OH 45267-0828, USA. *e-mail: schwara@ email.uc.edu
Voltage-dependent Ca2+ channels (VDCCs) are present in all excitable tissues and in some nonexcitable cell types such as fibroblasts. VDCCs mediate the influx of Ca2+ in response to membrane depolarization and regulate numerous intracellular functions including contraction, secretion, neurotransmitter release and gene transcription. The present nomenclature classifies the VDCC α1-subunit families into three main groups (Cav1.X, Cav2.X and Cav3.X) on the basis of their physiological properties and sequence similarities1. Although a large body of knowledge about VDCCs has accumulated, physiological characteristics of these channels in native tissue and the contribution of the channels to broad scope function are only just being considered. Ca2+ is the most ubiquitous signaling molecule, yet it elicits the most exquisitely specific responses of all signaling molecules. Therefore, proper regulation and control of Ca2+ mobilization is essential to prevent aberrant signaling. In this review, transgenic mice that have been developed to investigate VDCCs will be discussed. Accessory subunits
Ca2+ channels are multi-subunit protein complexes composed of at least four subunits, designated α1, α2–δ and β (Refs 2–6). Depending on the tissue, a γ-subunit might also be present. The α1-subunit harbors the ion-conducting pore, voltage sensor, gating machinery and drug binding sites for molecules such as Ca2+ channel modulators [e.g. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BZTs)] and toxins, and is also a downstream target for intracellular signaling pathways. The α2–δ-, β- and γ-subunits are accessory subunits that modulate the function of the α1-subunit2–6. http://tips.trends.com
Since the initial discovery of the multi-subunit nature of Ca2+ channels and the cloning of the channel subunits, numerous studies have been published on possible roles that the subunits might possess in channel function. The interaction of the subunits is ‘tight’ but not covalently bound, with Kds in the nanomolar range7–9. Most current knowledge concerning the role of the accessory subunits has been obtained in coexpression studies performed in Xenopus oocytes and in various mammalian cell lines. The data suggest that coexpression of accessory subunits in general enhances current amplitude and density, sometimes as high as 2–20-fold2–9, and accelerates activation and inactivation rates (Box 1). The precise interpretation of these data is rendered somewhat cloudy by the fact that heterologous expression systems might or might not possess endogenous accessory subunits. In addition, such systems might be inadequate for studying the processing and trafficking of such a large membrane protein complex. Furthermore, it is difficult to extrapolate data obtained in cells to the whole organism. In attempts to identify the native role of the subunits, a variety of transgenic animals have recently been produced (Table 1). Similarly, several spontaneous Ca2+ channel mutations have been identified that result in defects ranging from slightly aberrant Ca2+ channel function to disease (Box 2). α1C) knockout mice Cav1.2 (α
Gene ablation of the L-type VDCC (Cav1.2, α1C) addressed the ‘flip-side’ of the regulation of Ca2+ cycling in the heart whereby a decrease in Ca2+ entry was created. Not surprisingly, homozygous knockout of these channels was embryonic lethal. However, contraction of transgenic and wild-type embryonic hearts was indistinguishable until day 12.5 postcoitum (p.c.), which suggests the presence of a novel, L-type-like Ca2+ channel10. This channel displayed lower affinity to DHPs and was shown to be different from the Cav1.3. The contribution of this unidentified Ca2+ channel to adult heart contraction or, more importantly, to pathology is unclear. A reversion to fetal gene expression is a phenomenon that occurs when the hypertrophic gene program is initiated. It is tempting to speculate that expression of this unidentified Ca2+ channel might also be elevated in hypertrophy and could contribute to Ca2+ overload.
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Box 1. Ca2++ channel subunit interaction and association with cellular signaling pathways The subunits of Ca2+ channels (α1, α2–δ and β, and sometimes γ) interact tightly with each other (Fig. I). Coexpression studies have provided insights into the roles of Ca2+ channel subunits in channel function, and their interactions with other molecules. Binding sites for dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BZTs) are shown in Fig. I. (1) Ca2+ channel α1-subunits [which each contain four homologous motifs (I–IV), each of which have six transmembrane domains] associate with β-subunits at their intracellular connecting loop between motifs I and II. The association manifests in a broad scope of functional modulation such as enhanced translocation of α1 to the cell membrane, accelerating activation and inactivation kinetics and altering channel voltagedependencea. Recently, a small G protein (Kir/GEM) was shown to interact with the α1-subunit and interfere with its plasma membrane expressionb. (2) P/Q-, N- and R-type Ca2+ channels (Cav2.1, 2.2, 2.3) are modulated by G-protein βγ-subunit complexes. Apparently, the Gβγ interaction site is partly shared with the β-subunit interaction site. The βγ interaction is also modulated by protein kinase C (PKC) phosphorylationc,d. (3) Inactivation of high-voltage-activated Ca2+ channels depends on intracellular Ca2+. Ca2+–calmodulin (CaM) associates with an IQ domain on the large intracellular C-terminal tail and enhances channel inactivation. This interaction is also necessary for facilitation of L- and P/Q-type channels, a positive feedback mechanism that augments Ca2+ currents in response to increased intracellular Ca2+ concentrationse. In addition, CaM bifurcates the local Ca2+ signal and
Fig. I
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I NH2
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IV
1
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2
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Ca
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CaM Ca NH2
COOH
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mediates the Ca2+-dependent inactivation (negative feedback) and facilitation of the channel (positive feedback)f. (4) Phosphorylation of L-type Ca2+ channels by Ca2+–CaM kinase II (CaMKII) is another factor that determines Ca2+ channel current facilitation. Phosphorylation by CaMKII causes frequent and long openings at the single channel levelg. (5) Phosphorylation of cardiac Ca2+ channels at the C-terminal consensus site is one downstream target of protein kinase A (PKA) signalingh. Increases in PKA activation arise from β-adrenoceptor signaling and result in an enhanced Ca2+ influx. References a Bichet, D. et al. (2000) The I-II loop of the Ca2+ channel α1-subunit contains an endoplasmic reticulum retention signal antagonized by the β-subunit. Neuron 25, 177–190
It is also worth mentioning that Cav1.2 heterozygote knockout mice were indistinguishable from wild-type mice with respect to shape, development and behavior10. Interestingly, cardiomyocytes isolated from 12.5 p.c. heterozygotes displayed a significant reduction of Ca2+ channel currents. However, by age 14.5 p.c., Ca2+ channel currents increased to levels equivalent to wild-type animals10. Thus, the ablation of Cav1.2 (heterozygous only) can lead to either an increase in Ca2+ channel protein stability or an upregulation of the Cav1.2 gene expression. Further experimentation on this model should shed light on Ca2+ channel expression patterns. http://tips.trends.com
PAA, BZT
DHP α1-subunit
b Beguin, P. et al. (2001) Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/GEM. Nature 411, 701–706 c Zamponi, G.W. et al. (1997) Crosstalk between G proteins and protein kinase C mediated by the Ca2+ channel α1-subunit. Nature 385, 442–446 d De Waard, M. et al. (1997) Direct binding of G-protein β/γ complex to voltage-dependent Ca2+ channels. Nature 385, 446–450 e Peterson, B.Z. et al. (1999) Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type Ca2+ channels. Neuron 22, 549–558 f DeMaria, C.D. et al. (2001) Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels. Nature 411, 484–489 g Dzhura, I. et al. (2000) Calmodulin kinase determines Ca2+-dependent facilitation of L-type Ca2+ channels. Nat. Cell Biol. 2, 173–177 h De Jongh, K.S. et al. (1996) Specific phosphorylation of a site in the full-length of the α1-subunit of the cardiac L-type Ca2+ channel by adenosine 3′,5′-cyclic monophosphatedependent protein kinase. Biochemistry 35, 10392–10402
α1C) overexpression Cav1.2 (α
Opening of cardiac Ca2+ channels increases the local intracellular Ca2+ concentration, initiating the release of internal stores of Ca2+ through the ryanodine RY2 receptor, a Ca2+ channel closely associated with the sarcoplasmic reticulum (SR). Maintenance of normal Ca2+ handling is essential to maintain cardiac function and retain myocardial integrity. A loss of Ca2+ homeostasis is common in cardiovascular disease; in fact, Ca2+ overload is one documented hallmark of terminal heart failure. The role of the L-VDCC either in the initiation of, in the progression of, or as a source
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Table 1. Ca2+ channel transgenic mice Channela
Gene
Tissue distribution
Phenotype
Cav1.2 (OE) CACNA1C
Heart
Hypertrophy and death in 1 year, 11,12 gene transcriptional changes
Cav1.2 (KO) CACNA1C
Heart, smooth muscle, brain
Embryonic lethal <14.5, unidentified Ca2+ channel
10
Cav1.3 (KO) CACNA1D
Endocrine, smooth muscle, heart
Deaf, sinus arrhythmia, bradycardia
13
Cav2.1 (KO) CACNA1A
Brain, cochlea, pituitary
Ataxia, dystonia, increased L- and N-type current, death in <4 weeks
21
Cav2.2 (KO) CACNA1B
Brain, neuronal cells
Decreased sympathetic nervous system response, increased heart rate, increased blood pressure
23
Cav2.3 (KO) CACNA1E
Brain, cochlea, retina, heart, pituitary
Increased somatic inflammatory pain response
25
β1 (KO)
CACNB1
Skeletal muscle, heart, brain
34
β3 (KO)
CACNB3
Brain, heart, aorta
Decreased L-type Ca2+ current, skeletal muscle dysfunction, death at birth (asphyxia) Decreased L- and N-type Ca2+ current, no identifiable pathology
γ1 (KO)
CACNG1
Skeletal muscle
Increased L-type Ca2+ current, no identifiable pathology
40
aAbbreviations:
Refs
37
KO, knockout; OE, overexpression.
of Ca2+ imbalance in heart disease remains unclear. To clarify the role of the L-VDCC in heart failure, we overexpressed the channel in a tissue-specific manner to determine if a small, sustained increased influx of Ca2+ would recapitulate the characteristics of human heart disease. Overexpression of the α1-subunit (Cav1.2) in mice increased Ca2+ influx into cardiomyocytes, which resulted in an increase in cardiac contractile force. Interestingly, expression of the accessory subunits was unchanged compared with nontransgenic animals. Because no differences were observed in the activation or inactivation kinetics of the channel, and no change was noted in single channel conductance (A. Schwartz et al., unpublished), it is believed that the core stoichiometry remains intact in both transgenic and nontransgenic animals11. Two major findings in this transgenic model include an early blunting of the β-adrenoceptor signaling pathway and a slowprogressing development of hypertrophy and eventual heart failure, both common characteristics of human pathology. Additionally, enhanced activation of PKC-α was an early event, which might serve as the common link between disease development and the defect in β-adrenoceptor signaling12. Numerous signaling pathways have been implicated in cardiac hypertrophy with no clear common component other than Ca2+. Clearly, in this model, the increased entry of Ca2+ initiates the hypertrophic program with associated fibrosis, alterations in gene transcription, and apoptosis, in addition to an elevation of PKC-α (Ref. 12). Thus, the Ca2+ channel overexpression mouse model http://tips.trends.com
provides a useful ‘tool’ to define the prominent intracellular signaling pathways influenced by aberrant Ca2+ signaling and to emphasize a possible central role for Ca2+ in hypertrophy and failure. α1D) knockout mice Cav1.3 (α
An additional L-type VDCC (Cav1.3, α1D) is expressed in neurons and neuroendocrine cells with its primary localization in the brain, pancreas, kidney, ovary, cochlea tissues and heart atria. Genetic ablation of the gene encoding Cav1.3 revealed the contribution of high-voltage-activated L-type Ca2+ currents from the α1D channel and identified their physiological role. Although homozygous mice displayed normal reproductive abilities and were only slightly underrepresented in litters, α1D–/– mice were deaf and experienced sinoatrial node dysfunction resulting in bradycardia and arrhythmias13. Ca2+ entry through VDCCs is required for neurotransmitter release in auditory hair cells14,15, and deafness was a result of this loss in Ca2+ influx. In addition, Ca2+ entry also appears to be important in maintaining normal cochlear morphology. Both inner and outer hair cell populations were degenerated in α1D–/– mice by postnatal day 35, although complete deafness was already present, which suggests that deafness is a result of loss of Ca2+ current rather than a loss of hair cells13. α1A) knockout mice Cav2.1 (α
Cav2.1 (α1A) is the most abundant VDCC in the vertebrate brain with the cell body layers in the cerebellum and hippocampus the most predominant expressors16. The α1A-subunit encodes the P/Q-type Ca2+ channel and mediates the Ca2+ influx responsible for neurotransmitter release from presynaptic and somatodendritic membranes17. Spontaneous mutations of Cav2.1 create defective channels with varying phenotypic characteristics in both mice and humans18–20 (Box 2). To identify the consequence of a complete loss of α1A-dependent channel activity, the gene encoding Cav2.1 was disrupted21. Heterozygous mice for the gene encoding Cav2.1 reproduced normally and displayed no gross abnormalities. Although homozygous animals were not embryonic lethal, at approximately day 10, knockout mice displayed signs of ataxia whereby they would lose their balance while walking and would roll on their backs. The ataxia became worse with age, and dystonia, absent seizures, stunted growth and death occurred at four weeks. Histological assessment identified a smaller cerebellum in mutant mice, but the Purkinje cell monolayer and granule cell layers developed normally. The Cav2.1 knockout animal provides direct evidence that the gene encodes the P/Q-type Ca2+ currents measured in brain. Interestingly, disruption of the Ca2+ influx mediated by the gene encoding Cav2.1 alters the expression and functional properties of other Ca2+ channels in neurons. The R-type (α1E, Cav2.3) current was reduced, suggesting either a modulation at the expression level or a direct effect on functional activity,
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Box 2. Mutations identified in Ca2++ channel subunits Several spontaneous mutations have been identified in α1-subunits, which comprise four homologous motifs (I–IV), each of which have six transmembrane domains, and β-subunits of voltagedependent Ca2+ channels (VDCCs). These mutations, the location of which are indicated in Fig. Ia, result in defects ranging from slightly aberrant Ca2+ channel function to disease. Fig. Ib summarizes the known channelopathies that result from mutations in VDCC subunits and the diseases that appear to be associated with these defectsa–j. References a Ptacek, L. et al. (1994) Dihydropyridine receptor mutations cause hypokalaemic periodic paralysis. Cell 77, 863–868 b Monnier, N. et al. (1997) Malignant hyperthermia susceptibility is associated with a mutation of the α1-subunit of the human dihydropyridine-sensitive L-type voltagedependent calcium channel receptor in skeletal muscle. Am. J. Hum. Genet. 60, 1316–1325 c Chaudhari, N. (1992) A single nucleotide deletion in the skeletal muscle-specific calcium channel transcript of muscular dysgenesis (mdg) mice. J. Biol. Chem. 267, 25636–25639 d Strom, T. et al. (1998) An L-type calcium channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 260–263 e Bech-Hansen, N. et al. (1998) Loss-of-function mutations in a calcium channel α1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 264–267 f Ophoff, R.A. et al. (1996) Familial hemiplegic migrane and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87, 543–552 g Zhuchenko, O. et al. (1997) Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel. Nat. Genet. 15, 62–68 h Fletcher, C.F. et al. (1996) Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 87, 607–617
Fig. I (a)
+ 1 2 3 4 5 +
6
NH2 COOH NH2
COOH
(b) Name
Disease
Location of mutation
Refs
Cav1.1 (human)
Hypokalemic periodic paralysis Hypokalemic periodic paralysis Malignant hyperthermia Muscular dysgenesis
IV S4 II S4 II S3–S4 loop IV S6 deletion
a a b c
Cav1.4 (human)
Congenital stationary night blindness
Loop I–II Loop II–III I S6 III S1–S2 loop III S4 III S5–S6 loop IV S2 IV S4 IV S5–S6 loop C-terminal cytoplasmic tail
d e d e d d,e e e d d,e
Cav2.1 (human)
Familial hemiplegic migraine
I S4 II S5–S6 loop II S6 IV S6 III S1 III S2 C-terminal cytoplasmic tail
f f f f f f g
Cav1.1 (mouse)
Episodic ataxia type 2 Spinocerebellar ataxia type 6
Cav2.1 (mouse)
Tottering Leaner
II S5–S6 loop C-terminal cytoplasmic tail
h i
β4 (mouse)
Lethargic
β-subunit interaction domain
j
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i Doyle, J. et al. (1997) Mutations in the CACNL1A4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice. Mamm. Genome 8, 113–120
which has been reported previously22. In addition to a decrease in R-type current, elimination of the P/Q-type channel activity resulted in an enhanced functional Land N-type Ca2+ channel activity21. Thus, there appears to be an overlap in neurotransmitter release whereby a whole family of Ca2+ channels can serve as triggers. α1B) knockout mice Cav 2.2 (α
The N-type VDCCs are predominantly localized in the nervous system and have been considered to play a vital role in many neuronal functions particularly transmitter release at sympathetic postsynaptic http://tips.trends.com
IV
III
II
I
j Burgess, D.L. et al. (1997) Mutation of the Ca2+ channel beta subunit gene CCHB4 is associated with ataxia and seizures in the lethargic (lh) mouse. Cell 88, 385–392
terminals. To examine this directly, a very recent and exciting addition to our transgenic armamentaria is the generation of mice genetically deficient in the α1B-subunit23. Surprisingly, these mice have a normal life span and do not exhibit any significant behavioral aberrations. The N-type currents, of course, were completely eliminated without any change in the activity of other VDCC types in neuronal preparations. The baroreceptor reflex response was markedly reduced after bilateral carotid occlusion. In addition, isolated left atria from these mice hearts did not demonstrate positive inotropic responses to electrical sympathetic
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Fig. 1. Gene regulation by Ca2+ signals. Function of voltage-, ligand- and store-operated Ca2+ channels transiently enhances cytosolic Ca2+. The generation, duration and waning of the Ca2+ signal are regulated by several intertwining cellular mechanisms resulting in an initiation of gene transcription. Sustained Ca2+ signals can influence gene transcription by diverse cellular mechanisms. These include, but are not limited to: (1) activation of calcineurin, a Ca2+–calmodulin (CaM)activated phosphatase that dephosphorylates members of the NF-ATc transcription factors, conferring nuclear translocation and transcription initiation; and (2) activation of the Ca2+–CaM complex and translocation to the nucleus and subsequent cAMP response elementbinding protein (CREB) phosphorylation and/or activation of Ca2+–CaMdependent protein kinase (CaMK) enzymes. Other cellular signal transduction pathways might interfere with the above processes. These include cytosolic Ca2+ itself, and trimeric G-protein-coupled receptors, either directly or indirectly, might activate protein kinase C (PKC) or other transduction pathways (Ras, Rac and mitogenactivated kinase family). These signals can all funnel into initiation of the transcription processes. It is likely that the intracellular Ca2+ signaling is integrated into a canonical organization of numerous other signal transduction pathways. Abbreviations: CRAC, Ca2+ releaseactivated Ca2+ channel; PP2B, protein phosphatase 2B; Trp, transient receptor potential; VDCC, voltagedependent Ca2+ channel. Adapted, with permission, from Ref. 47.
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Receptor
Trp, CRAC Ca2+
NMDA receptor Ca2+
Furthermore, as previously mentioned, an 80% decrease in R-type current was observed in the Cav2.1 (α1A) knockout mice22. Thus, it appears that the contribution of the α1E Ca2+ channel component of the R-type current is dependent on the neuronal cell population, with additional VDCC α1-subunits contributing to the total R-type current.
VDCC Ca2+
P Ras, Rac, PKC
Ca2+ +
β1 knockout mice
NF-ATc
+ Ca2+–CaM
+
PP2B P NF-ATc
Ca2+–CaM
NF-ATn
CREB
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neuronal stimulation. Although, the parasympathetic system seemed intact, both blood pressure and heart rate were elevated. These mice should be of considerable value in further assessing the role of the adrenergic arm of the autonomic nervous system. α1E) knockout mice Cav2.3 (α
Of the known VDCC channels in the brain, the R-type is the least understood because of its resistance to antagonists of L-, N- and P/Q-type channels. Cloning of the gene encoding Cav2.3 (α1E) and heterologous expression studies suggested that this channel was responsible for R-type current, but the channel also possessed properties that resembled a T-type current. However, a specific inhibitor of class E Ca2+ channels [SNX482 (a synthetic version of a novel 41-amino-acid peptide isolated from the venom of the West African tarantula Hysterocrates gigas)] was insufficient in blocking all R-type currents24. Thus, the in vivo function of the α1E channel remained controversial until knockout studies of the gene encoding Cav2.3 were performed. Transgenic mice displayed a reduced response to somatic inflammatory pain and an altered response to visceral inflammatory pain. Further investigation identified expression of the gene encoding Cav2.3 in the spinal cord and dorsal root ganglion25. Taken together, the data indicate that the α1E Ca2+ channel controls pain behaviors by both spinal and supraspinal mechanisms making it an ideal target for blocking nociception. In addition, the α1E Ca2+ channel also appears to play a role in formation of accurate spatial memory but not fear memory26. So, is the α1E Ca2+ channel responsible for the R-type current in neurons? Experiments using antisense oligonucleotides to the α1E-subunits in neurons support the idea that the R-type current is a result of the α1E Ca2+ channel27,28. However, Wilson et al.29 identified substantial R-type currents in all α1E knockout neurons measured. http://tips.trends.com
The β1 knockout mouse was the first Ca2+ channel transgenic animal created. The β1-subunit is predominantly found in skeletal muscle and brain with lower expression levels in the spleen and heart30–33. Coexpression of the β1-subunit in heterologous expression studies enhances Ca2+ channel current amplitude and density (Box 1)2–9. Ca2+ influx across the skeletal muscle membrane through the α1S-subunit (Cav1.1) is essential in coupling the electrical depolarizing signal to the release of Ca2+ from the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum and eventual muscle contraction [i.e. excitation contraction (EC) coupling]. Homozygous transgenic fetuses were immobile with disorganization of thick and thin filaments and died at birth from asphyxiation34. Consistent with the heterologous expression systems, the L-type Ca2+ current was decreased by 10–20-fold with not only a loss of β1-subunit membrane localization, but also a loss of α1S-subunit membrane localization, which suggests a trafficking role for the β1-subunit34. Radioligand binding {e.g. using [3H]PN200110} revealed a 3.9-fold decrease in Bmax for the L-type channel and a 2.8-fold decrease in charge movement35. Early death of the homozygous animals made it impossible to assess the role of the β1-subunit in the brain, spleen and heart. Interestingly, heterozygous animals were indistinguishable from wild-type littermates. This would suggest that an excess of β1-subunit is present and is able to compensate for ablating one copy of the gene. β3 knockout mice
The β3-subunit is primarily found in the brain, heart and aorta, and is associated with ~56% of the N-type Ca2+ channels in the brain36. Although β3-deficient mice were indistinguishable from wild-type animals, L- and N-type currents were significantly reduced in the superior cervical ganglion37. Possible upregulation of other β-subunits might account for the minimal change in phenotype. γ knockout mice
The γ1-subunit is exclusively expressed in skeletal muscle, but its function has been illusive. Heterologous coexpression studies have failed to identify a role of the γ1-subunit when expressed with its native complex partners (α1S, β1 and α2–δ1)31. However, coexpression with α1C identified a shift in the steady-state inactivation and acceleration of both current activation and current inactivation38,39. γ1-Subunit-deficient mice were indistinguishable from wild-type mice, but they
Review
Note added in proof A recent article [Rottbauer, W. et al. (2001) Developmental Cell 1, 265–275] describes an embryonic lethal mutation [‘island beat (isl)’] in the α1C-subunit of the L-VDCC in zebrafish. The atria exhibit fibrillation and the ventricle is silent and fails to acquire the normal number of myocytes. This provides evidence that the L-VDCC can regulate growth independently of contraction.
TRENDS in Pharmacological Sciences Vol.22 No.10 October 2001
displayed altered Ca2+ channel activity. Increased peak Ca2+ current amplitude and shifts in inactivation kinetics were present without changes in current density or activation kinetics40. Stargazer mice, which exhibit phenotypes that are characteristic of absence epilepsy, were thought to represent a natural γ-subunit mutation. Cloning the cDNA encoding the 36-kDa protein from these mice revealed a weak homology to the skeletal muscle γ-subunit. Therefore, the gene was termed γ2 or stargazin41. In vitro coexpression of stargazin (γ2) showed an increase of steady-state inactivation of α1A. Thus, it was concluded that aberrant P/Q-type channel function might contribute to inappropriate Ca2+ entry in neuronal cells of this mutant mice. However, a recent study by Chen et al.42 clearly identified the stargazer protein as an essential component to target AMPA receptors to synapses. When mutated stargazin is present (stargazer mice), no functional AMPA receptors can be detected in granule cell synapses. Concluding remarks
Transgenic animals have proven tremendously useful in clarifying and confirming heterologous expression studies and defining in vivo functions of the Ca2+ channel family. The numerous substrates for Ca2+ signaling create great difficulties in identifying specific roles for the channel. In recent years, it has become increasingly evident that alterations in Ca2+ signaling
References 1 Ertel, E.A. et al. (2000) Nomenclature of voltagedependent calcium channels. Neuron 25, 533–535 2 Mori, Y. et al. (1996) Molecular pharmacology of voltage-dependent calcium channels. Jpn. J. Pharmacol. 72, 83–109 3 Gurnett, C.A. and Campbell, K.P. (1996) Transmembrane auxiliary subunits of voltagedependent ion channels. J. Biol. Chem. 271, 27975–27978 4 Varadi, G. et al. (1999) Molecular elements of ion permeation and selectivity within calcium channels. Crit. Rev. Biochem. Mol. Biol. 34, 181–214 5 Hoffman, F. et al. (1999) Voltage-dependent Ca2+ channels: from structure to function. Rev. Physiol. Biochem. Pharmacol. 139, 33–87 6 Catterall, W.A. (1998) Stucture and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24, 307–323 7 Pragnell, M. et al. (1994) Calcium channel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit. Nature 368, 67–70 8 Witcher, D.R. et al. (1995) Association of native Ca2+ channel β-subunits with the α1-subunit interaction domain. J. Biol. Chem. 270, 18088–18093 9 Gurnett, C.A. et al. (1996) Dual function of the voltage-dependent Ca2+ channel α2/δ-subunit in current stimulation and subunit interaction. Neuron 16, 431–440 10 Seisenberger, C. et al. (2000) Functional embryonic cardiomyocytes after disruption of the L-type α1C (Cav1.2) calcium channel gene in mouse. J. Biol. Chem. 275, 39193–39199 http://tips.trends.com
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pathways have a drastic impact on the gene expression program in excitable and also in non-excitable cells. In terms of evolution, Ca2+ is the key cation, and is also probably important early on in development in almost all biological systems that link excitation to numerous physiological end states. How a generic cation such as Ca2+ results in very specific outcomes at the level of gene expression will be one of the greatest puzzles of research in the post-genomic era. The stimulus– transcription coupling has brought into focus several other signaling pathways whose activation correlates with the activation of Ca2+ signaling. These include stimulation of cAMP response-element binding protein (CREB), phosphorylation and dephosphorylation by a Ca2+–calmodulin signal and in turn activation of CRE-regulated genes43,44. The involvement of a Ca2+–calmodulin-dependent kinase cascade in this process has also been demonstrated43. Another very important Ca2+-stimulated signaling pathway is signal transduction by calcineurin and activation of NF-ATc transcription factors45–47 (Fig. 1). These are only a few of a possible many mechanisms, and, most probably, a combinatorial and hierarchical integration of signaling pathways as observed in other examples48–50 will determine the final outcome of a Ca2+-stimulated gene expression. The recent advent of transgenic animals with specific VDCC subunits will aid immensely in fine-tuning our understanding of cellular regulation.
11 Muth, J.N. et al. (1999) Cardiac-specific overexpression of the α1-subunit of the L-type voltage-dependent Ca2+ channel in transgenic mice. J. Biol. Chem. 274, 21503–21506 12 Muth, J.N. et al. (2001) A Ca2+-dependent transgenic model of cardiac hypertrophy. A role for protein kinase Cα. Circulation 103, 140–147 13 Platzer, J. et al. (2000) Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell 102, 89–97 14 Martinez-Dunst, C. et al. (1997) Release sites and calcium channels in hair cells of the chick’s cochlea. J. Neurosci. 17, 9133–9144 15 Moser, T. and Beutner, D. (2000) Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse. Proc. Natl. Acad. Sci. U. S. A. 97, 883–888 16 Dunlap, K. et al. (1995) Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci. 18, 89–98 17 Westenbroek, R.E. et al. (1995) Immunochemical identification and subcellular distribution of the α1A-subunits of brain calcium channels. J. Neurosci. 15, 6403–6418 18 Rhyu, I.J. et al. (1999) Morphologic investigation of rolling mouse Nagoya (tg(rol)/tg(rol)) cerebellar Purkinje cells: an ataxic mutant, revisited. Neurosci. Lett. 266, 49–52 19 Ophoff, R.A. et al. (1996) Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+channel gene CACNL1A4. Cell 87, 543–552 20 Zhuchenko, O. et al. (1997) Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltagedependent calcium channel. Nat. Genet. 15, 62–69
21 Jun, K. et al. (1999) Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α1Asubunit. Proc. Natl. Acad. Sci. U. S. A. 96, 15245–15250 22 Sutton, K.G. et al. (1999) P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A. Nature 401, 800–804 23 Ino, M. et al. (2001) Functional disorders of sympathetic nervous system in mice lacking the α1B-subunit (Cav2.2) of N-type calcium channel. Proc. Natl. Acad. Sci. U. S. A. 98, 5323–5328 24 Newcomb, R. et al. (1998) Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry. 37, 15353–15362 25 Saegusa, H. et al. (2000) Altered pain responses in mice lacking α1E-subunit of the voltage-dependent Ca2+ channel. Proc. Natl. Acad. Sci. U. S. A. 97, 6132–6137 26 Kubota, M. et al. (2001) Intact LTP and fear memory but impaired spatial memory in mice lacking Cav2.3 (α1E) channel. Biochem. Biophys. Res. Commun. 282, 242–248 27 Piedras-Renteria, E.S. and Tsien, R.W. (1998) Antisense oligonucleotides against α1E reduce Rtype calcium currents in cerebellar granule cells. Proc. Natl. Acad. Sci. U. S. A. 95, 7760–7765 28 Tottene, A. et al. (2000) α1E-subunits form the pore of three cerebellar R-type Ca2+ channels with different pharmacological and permeation properties. J. Neurosci. 20, 171–178 29 Wilson, S.M. et al. (2000) The status of voltagedependent calcium channels in α1E knockout mice. J. Neurosci. 20, 8566–8571
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30 Ruth, P. et al. (1989) Primary structure of the β-subunit of the DHP-sensitive calcium channel from skeletal muscle. Science 245, 1115–1118 31 Varadi, G. et al. (1991) Acceleration of activation and inactivation by the β-subunit of the skeletal muscle calcium channel. Nature 11, 159–162 32 Pragnell, M. et al. (1991) Cloning and tissuespecific expression of the brain calcium channel β-subunit. FEBS Lett. 291, 253–258 33 Powers, P.A. et al. (1992) Skeletal muscle and brain isoforms of a β-subunit of human voltagedependent calcium channels are encoded by a single gene. J. Biol. Chem. 267, 22967–22972 34 Gregg, R.G. et al. (1996) Absence of the β-subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1-subunit and eliminates excitation–contraction coupling. Proc. Natl. Acad. Sci. U. S. A. 93, 13961–13966 35 Strube, C. et al. (1996) Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor β1-subunit. Biophys. J. 71, 2531–2543 36 Scott, V.E. et al. (1996) β-subunit heterogeneity in N-type Ca2+ channels. J. Biol. Chem. 271, 3207–3212
37 Namkung, Y. et al. (1998) Targeted disruption of the Ca2+ channel β3-subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons. Proc. Natl. Acad. Sci. U. S. A. 95, 12010–12015 38 Singer, D. et al. (1991) The roles of the subunits in the function of the calcium channel. Science 253, 553–557 39 Wei, X.Y. et al. (1991) Heterologous regulation of the cardiac Ca2+ channel α1-subunit by skeletal muscle β and γ-subunits. Implications for the structure of cardiac L-type Ca2+ channels. J. Biol. Chem. 266, 21943–21947 40 Freise, D. et al. (2000) Absence of the γ-subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties. J. Biol. Chem. 275, 14476–14481 41 Letts, V.A. et al. (1998) The mouse stargazer gene encodes a neuronal Ca2+ channel γ-subunit. Nat. Genet. 19, 340–347 42 Chen, L. et al. (2000) Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943 43 Bito, H. et al. (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214
44 Deisseroth, K. et al. (1998) Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 392, 198–202 45 Molkentin, J.D. et al. (1998) A calcineurindependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215–228 46 Graef, I.A. et al. (1999) L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401, 703–708 47 Crabtree, G.R. (1999) Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96, 611–614 48 Halfon, M.S. et al. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissuerestricted transcription factors. Cell 103, 63–74 49 Flores, G.V. et al. (2000) Combinatorial signaling in the specification of unique cell fates. Cell 103, 75–85 50 Xu, C. et al. (2000) Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye. Cell 103, 87–97
Heterodimerization of G-proteincoupled receptors: pharmacology, signaling and trafficking Lakshmi A. Devi Although classical models predict that G-protein-coupled receptors (GPCRs) function as monomers, several recent studies acknowledge that GPCRs exist as dimeric or oligomeric complexes. In addition to homodimers, heterodimers between members of the GPCR family (both closely and distantly related) have been reported. In some cases heterodimerization is required for efficient agonist binding and signaling, and in others heterodimerization appears to lead to the generation of novel binding sites. In this article, the techniques used to study GPCR heterodimers, and the ‘novel pharmacology’ and functional implications resulting from heterodimerization will be discussed.
Lakshmi A. Devi Dept of Pharmacology, New York University School of Medicine, New York, NY, USA. e-mail: lakshmi.devi@ med.nyu.edu
The function of most, if not all, cells in the body is regulated by plasma membrane receptors. The vast majority of these receptors belong to the superfamily of G-protein-coupled receptors (GPCRs), which at current estimates account for ~1% of the genes present in a mammalian genome. Agonists or antagonists of GPCRs, in addition to agents that interfere with cellular pathways regulated by these receptors, are widely used in drug therapy. All GPCRs share a common tertiary structure consisting of seven transmembrane (TM) helices http://tips.trends.com
linked by three alternating intracellular and extracellular domains, with an extracellular N-terminus and a cytoplasmic C-terminus. GPCRs interact with heterotrimeric guanine-nucleotidebinding regulatory proteins (G proteins), which then interact with effector systems and regulate various intracellular processes (for an animation of agonistinduced activation of GPCRs, see http://archive. bmn.com/supp/tips/tips2210a.html). Based on sequence similarity, GPCRs can be classified into three major receptor families1. Family A (rhodopsin, β2-adrenoceptor-like) is the largest family of GPCRs. These receptors are characterized by several conserved residues in their TM helices and a palmitoylated cysteine in the C-terminal tail. Family B [glucagon, vasoactive intestinal peptide (VIP), calcitonin receptorlike] is a relatively small group that is characterized by the presence of a large N-terminal domain that contains several well-conserved cysteine residues. Family C (metabotropic neurotransmitter, calcium-sensing receptor-like) is characterized by a very long N-terminal domain that appears to be sufficient for ligand binding.
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Use of transgenic mice to study voltage-dependent Ca2++ channels James N. Muth, Gyula Varadi and Arnold Schwartz During the past decade a great number of genes encoding high- and lowvoltage-dependent Ca2++ channels and their accessory subunits have been cloned. Studies of Ca2++ channel structure–function relationships and channel regulation using cDNA expression in heterologous expression systems have revealed intricate details of subunit interaction, regulation of channels by protein kinase A (PKA) and protein kinase C (PKC), drug binding sites, mechanisms of drug action, the ion conduction pathway and other aspects of channel function. In recent years, however, we have arrived at the brink of an entirely new strategy to study Ca2++ channels by overexpressing or knocking out genes encoding these channels in transgenic mice. In this article, various models of gene knockout or gene overexpression will be discussed. This new approach will reveal many secrets regarding Ca2++ channel regulation and the control of Ca2++-dependent cellular processes.
James N. Muth Gyula Varadi Institute of Molecular Pharmacology and Biophysics and the Dept of Cell Biology, Neurobiology and Anatomy Arnold Schwartz* Institute of Molecular Pharmacology and Biophysics, University of Cincinnati, College of Medicine, PO Box 670828, 231 Albert Sabin Way, Cincinnati, OH 45267-0828, USA. *e-mail: schwara@ email.uc.edu
Voltage-dependent Ca2+ channels (VDCCs) are present in all excitable tissues and in some nonexcitable cell types such as fibroblasts. VDCCs mediate the influx of Ca2+ in response to membrane depolarization and regulate numerous intracellular functions including contraction, secretion, neurotransmitter release and gene transcription. The present nomenclature classifies the VDCC α1-subunit families into three main groups (Cav1.X, Cav2.X and Cav3.X) on the basis of their physiological properties and sequence similarities1. Although a large body of knowledge about VDCCs has accumulated, physiological characteristics of these channels in native tissue and the contribution of the channels to broad scope function are only just being considered. Ca2+ is the most ubiquitous signaling molecule, yet it elicits the most exquisitely specific responses of all signaling molecules. Therefore, proper regulation and control of Ca2+ mobilization is essential to prevent aberrant signaling. In this review, transgenic mice that have been developed to investigate VDCCs will be discussed. Accessory subunits
Ca2+ channels are multi-subunit protein complexes composed of at least four subunits, designated α1, α2–δ and β (Refs 2–6). Depending on the tissue, a γ-subunit might also be present. The α1-subunit harbors the ion-conducting pore, voltage sensor, gating machinery and drug binding sites for molecules such as Ca2+ channel modulators [e.g. dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BZTs)] and toxins, and is also a downstream target for intracellular signaling pathways. The α2–δ-, β- and γ-subunits are accessory subunits that modulate the function of the α1-subunit2–6. http://tips.trends.com
Since the initial discovery of the multi-subunit nature of Ca2+ channels and the cloning of the channel subunits, numerous studies have been published on possible roles that the subunits might possess in channel function. The interaction of the subunits is ‘tight’ but not covalently bound, with Kds in the nanomolar range7–9. Most current knowledge concerning the role of the accessory subunits has been obtained in coexpression studies performed in Xenopus oocytes and in various mammalian cell lines. The data suggest that coexpression of accessory subunits in general enhances current amplitude and density, sometimes as high as 2–20-fold2–9, and accelerates activation and inactivation rates (Box 1). The precise interpretation of these data is rendered somewhat cloudy by the fact that heterologous expression systems might or might not possess endogenous accessory subunits. In addition, such systems might be inadequate for studying the processing and trafficking of such a large membrane protein complex. Furthermore, it is difficult to extrapolate data obtained in cells to the whole organism. In attempts to identify the native role of the subunits, a variety of transgenic animals have recently been produced (Table 1). Similarly, several spontaneous Ca2+ channel mutations have been identified that result in defects ranging from slightly aberrant Ca2+ channel function to disease (Box 2). α1C) knockout mice Cav1.2 (α
Gene ablation of the L-type VDCC (Cav1.2, α1C) addressed the ‘flip-side’ of the regulation of Ca2+ cycling in the heart whereby a decrease in Ca2+ entry was created. Not surprisingly, homozygous knockout of these channels was embryonic lethal. However, contraction of transgenic and wild-type embryonic hearts was indistinguishable until day 12.5 postcoitum (p.c.), which suggests the presence of a novel, L-type-like Ca2+ channel10. This channel displayed lower affinity to DHPs and was shown to be different from the Cav1.3. The contribution of this unidentified Ca2+ channel to adult heart contraction or, more importantly, to pathology is unclear. A reversion to fetal gene expression is a phenomenon that occurs when the hypertrophic gene program is initiated. It is tempting to speculate that expression of this unidentified Ca2+ channel might also be elevated in hypertrophy and could contribute to Ca2+ overload.
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Box 1. Ca2++ channel subunit interaction and association with cellular signaling pathways The subunits of Ca2+ channels (α1, α2–δ and β, and sometimes γ) interact tightly with each other (Fig. I). Coexpression studies have provided insights into the roles of Ca2+ channel subunits in channel function, and their interactions with other molecules. Binding sites for dihydropyridines (DHPs), phenylalkylamines (PAAs) and benzothiazepines (BZTs) are shown in Fig. I. (1) Ca2+ channel α1-subunits [which each contain four homologous motifs (I–IV), each of which have six transmembrane domains] associate with β-subunits at their intracellular connecting loop between motifs I and II. The association manifests in a broad scope of functional modulation such as enhanced translocation of α1 to the cell membrane, accelerating activation and inactivation kinetics and altering channel voltagedependencea. Recently, a small G protein (Kir/GEM) was shown to interact with the α1-subunit and interfere with its plasma membrane expressionb. (2) P/Q-, N- and R-type Ca2+ channels (Cav2.1, 2.2, 2.3) are modulated by G-protein βγ-subunit complexes. Apparently, the Gβγ interaction site is partly shared with the β-subunit interaction site. The βγ interaction is also modulated by protein kinase C (PKC) phosphorylationc,d. (3) Inactivation of high-voltage-activated Ca2+ channels depends on intracellular Ca2+. Ca2+–calmodulin (CaM) associates with an IQ domain on the large intracellular C-terminal tail and enhances channel inactivation. This interaction is also necessary for facilitation of L- and P/Q-type channels, a positive feedback mechanism that augments Ca2+ currents in response to increased intracellular Ca2+ concentrationse. In addition, CaM bifurcates the local Ca2+ signal and
Fig. I
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6
I NH2
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III
IV
1
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2
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Ca
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COOH
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mediates the Ca2+-dependent inactivation (negative feedback) and facilitation of the channel (positive feedback)f. (4) Phosphorylation of L-type Ca2+ channels by Ca2+–CaM kinase II (CaMKII) is another factor that determines Ca2+ channel current facilitation. Phosphorylation by CaMKII causes frequent and long openings at the single channel levelg. (5) Phosphorylation of cardiac Ca2+ channels at the C-terminal consensus site is one downstream target of protein kinase A (PKA) signalingh. Increases in PKA activation arise from β-adrenoceptor signaling and result in an enhanced Ca2+ influx. References a Bichet, D. et al. (2000) The I-II loop of the Ca2+ channel α1-subunit contains an endoplasmic reticulum retention signal antagonized by the β-subunit. Neuron 25, 177–190
It is also worth mentioning that Cav1.2 heterozygote knockout mice were indistinguishable from wild-type mice with respect to shape, development and behavior10. Interestingly, cardiomyocytes isolated from 12.5 p.c. heterozygotes displayed a significant reduction of Ca2+ channel currents. However, by age 14.5 p.c., Ca2+ channel currents increased to levels equivalent to wild-type animals10. Thus, the ablation of Cav1.2 (heterozygous only) can lead to either an increase in Ca2+ channel protein stability or an upregulation of the Cav1.2 gene expression. Further experimentation on this model should shed light on Ca2+ channel expression patterns. http://tips.trends.com
PAA, BZT
DHP α1-subunit
b Beguin, P. et al. (2001) Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/GEM. Nature 411, 701–706 c Zamponi, G.W. et al. (1997) Crosstalk between G proteins and protein kinase C mediated by the Ca2+ channel α1-subunit. Nature 385, 442–446 d De Waard, M. et al. (1997) Direct binding of G-protein β/γ complex to voltage-dependent Ca2+ channels. Nature 385, 446–450 e Peterson, B.Z. et al. (1999) Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type Ca2+ channels. Neuron 22, 549–558 f DeMaria, C.D. et al. (2001) Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels. Nature 411, 484–489 g Dzhura, I. et al. (2000) Calmodulin kinase determines Ca2+-dependent facilitation of L-type Ca2+ channels. Nat. Cell Biol. 2, 173–177 h De Jongh, K.S. et al. (1996) Specific phosphorylation of a site in the full-length of the α1-subunit of the cardiac L-type Ca2+ channel by adenosine 3′,5′-cyclic monophosphatedependent protein kinase. Biochemistry 35, 10392–10402
α1C) overexpression Cav1.2 (α
Opening of cardiac Ca2+ channels increases the local intracellular Ca2+ concentration, initiating the release of internal stores of Ca2+ through the ryanodine RY2 receptor, a Ca2+ channel closely associated with the sarcoplasmic reticulum (SR). Maintenance of normal Ca2+ handling is essential to maintain cardiac function and retain myocardial integrity. A loss of Ca2+ homeostasis is common in cardiovascular disease; in fact, Ca2+ overload is one documented hallmark of terminal heart failure. The role of the L-VDCC either in the initiation of, in the progression of, or as a source
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Table 1. Ca2+ channel transgenic mice Channela
Gene
Tissue distribution
Phenotype
Cav1.2 (OE) CACNA1C
Heart
Hypertrophy and death in 1 year, 11,12 gene transcriptional changes
Cav1.2 (KO) CACNA1C
Heart, smooth muscle, brain
Embryonic lethal <14.5, unidentified Ca2+ channel
10
Cav1.3 (KO) CACNA1D
Endocrine, smooth muscle, heart
Deaf, sinus arrhythmia, bradycardia
13
Cav2.1 (KO) CACNA1A
Brain, cochlea, pituitary
Ataxia, dystonia, increased L- and N-type current, death in <4 weeks
21
Cav2.2 (KO) CACNA1B
Brain, neuronal cells
Decreased sympathetic nervous system response, increased heart rate, increased blood pressure
23
Cav2.3 (KO) CACNA1E
Brain, cochlea, retina, heart, pituitary
Increased somatic inflammatory pain response
25
β1 (KO)
CACNB1
Skeletal muscle, heart, brain
34
β3 (KO)
CACNB3
Brain, heart, aorta
Decreased L-type Ca2+ current, skeletal muscle dysfunction, death at birth (asphyxia) Decreased L- and N-type Ca2+ current, no identifiable pathology
γ1 (KO)
CACNG1
Skeletal muscle
Increased L-type Ca2+ current, no identifiable pathology
40
aAbbreviations:
Refs
37
KO, knockout; OE, overexpression.
of Ca2+ imbalance in heart disease remains unclear. To clarify the role of the L-VDCC in heart failure, we overexpressed the channel in a tissue-specific manner to determine if a small, sustained increased influx of Ca2+ would recapitulate the characteristics of human heart disease. Overexpression of the α1-subunit (Cav1.2) in mice increased Ca2+ influx into cardiomyocytes, which resulted in an increase in cardiac contractile force. Interestingly, expression of the accessory subunits was unchanged compared with nontransgenic animals. Because no differences were observed in the activation or inactivation kinetics of the channel, and no change was noted in single channel conductance (A. Schwartz et al., unpublished), it is believed that the core stoichiometry remains intact in both transgenic and nontransgenic animals11. Two major findings in this transgenic model include an early blunting of the β-adrenoceptor signaling pathway and a slowprogressing development of hypertrophy and eventual heart failure, both common characteristics of human pathology. Additionally, enhanced activation of PKC-α was an early event, which might serve as the common link between disease development and the defect in β-adrenoceptor signaling12. Numerous signaling pathways have been implicated in cardiac hypertrophy with no clear common component other than Ca2+. Clearly, in this model, the increased entry of Ca2+ initiates the hypertrophic program with associated fibrosis, alterations in gene transcription, and apoptosis, in addition to an elevation of PKC-α (Ref. 12). Thus, the Ca2+ channel overexpression mouse model http://tips.trends.com
provides a useful ‘tool’ to define the prominent intracellular signaling pathways influenced by aberrant Ca2+ signaling and to emphasize a possible central role for Ca2+ in hypertrophy and failure. α1D) knockout mice Cav1.3 (α
An additional L-type VDCC (Cav1.3, α1D) is expressed in neurons and neuroendocrine cells with its primary localization in the brain, pancreas, kidney, ovary, cochlea tissues and heart atria. Genetic ablation of the gene encoding Cav1.3 revealed the contribution of high-voltage-activated L-type Ca2+ currents from the α1D channel and identified their physiological role. Although homozygous mice displayed normal reproductive abilities and were only slightly underrepresented in litters, α1D–/– mice were deaf and experienced sinoatrial node dysfunction resulting in bradycardia and arrhythmias13. Ca2+ entry through VDCCs is required for neurotransmitter release in auditory hair cells14,15, and deafness was a result of this loss in Ca2+ influx. In addition, Ca2+ entry also appears to be important in maintaining normal cochlear morphology. Both inner and outer hair cell populations were degenerated in α1D–/– mice by postnatal day 35, although complete deafness was already present, which suggests that deafness is a result of loss of Ca2+ current rather than a loss of hair cells13. α1A) knockout mice Cav2.1 (α
Cav2.1 (α1A) is the most abundant VDCC in the vertebrate brain with the cell body layers in the cerebellum and hippocampus the most predominant expressors16. The α1A-subunit encodes the P/Q-type Ca2+ channel and mediates the Ca2+ influx responsible for neurotransmitter release from presynaptic and somatodendritic membranes17. Spontaneous mutations of Cav2.1 create defective channels with varying phenotypic characteristics in both mice and humans18–20 (Box 2). To identify the consequence of a complete loss of α1A-dependent channel activity, the gene encoding Cav2.1 was disrupted21. Heterozygous mice for the gene encoding Cav2.1 reproduced normally and displayed no gross abnormalities. Although homozygous animals were not embryonic lethal, at approximately day 10, knockout mice displayed signs of ataxia whereby they would lose their balance while walking and would roll on their backs. The ataxia became worse with age, and dystonia, absent seizures, stunted growth and death occurred at four weeks. Histological assessment identified a smaller cerebellum in mutant mice, but the Purkinje cell monolayer and granule cell layers developed normally. The Cav2.1 knockout animal provides direct evidence that the gene encodes the P/Q-type Ca2+ currents measured in brain. Interestingly, disruption of the Ca2+ influx mediated by the gene encoding Cav2.1 alters the expression and functional properties of other Ca2+ channels in neurons. The R-type (α1E, Cav2.3) current was reduced, suggesting either a modulation at the expression level or a direct effect on functional activity,
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Box 2. Mutations identified in Ca2++ channel subunits Several spontaneous mutations have been identified in α1-subunits, which comprise four homologous motifs (I–IV), each of which have six transmembrane domains, and β-subunits of voltagedependent Ca2+ channels (VDCCs). These mutations, the location of which are indicated in Fig. Ia, result in defects ranging from slightly aberrant Ca2+ channel function to disease. Fig. Ib summarizes the known channelopathies that result from mutations in VDCC subunits and the diseases that appear to be associated with these defectsa–j. References a Ptacek, L. et al. (1994) Dihydropyridine receptor mutations cause hypokalaemic periodic paralysis. Cell 77, 863–868 b Monnier, N. et al. (1997) Malignant hyperthermia susceptibility is associated with a mutation of the α1-subunit of the human dihydropyridine-sensitive L-type voltagedependent calcium channel receptor in skeletal muscle. Am. J. Hum. Genet. 60, 1316–1325 c Chaudhari, N. (1992) A single nucleotide deletion in the skeletal muscle-specific calcium channel transcript of muscular dysgenesis (mdg) mice. J. Biol. Chem. 267, 25636–25639 d Strom, T. et al. (1998) An L-type calcium channel gene mutated in incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 260–263 e Bech-Hansen, N. et al. (1998) Loss-of-function mutations in a calcium channel α1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness. Nat. Genet. 19, 264–267 f Ophoff, R.A. et al. (1996) Familial hemiplegic migrane and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 87, 543–552 g Zhuchenko, O. et al. (1997) Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel. Nat. Genet. 15, 62–68 h Fletcher, C.F. et al. (1996) Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 87, 607–617
Fig. I (a)
+ 1 2 3 4 5 +
6
NH2 COOH NH2
COOH
(b) Name
Disease
Location of mutation
Refs
Cav1.1 (human)
Hypokalemic periodic paralysis Hypokalemic periodic paralysis Malignant hyperthermia Muscular dysgenesis
IV S4 II S4 II S3–S4 loop IV S6 deletion
a a b c
Cav1.4 (human)
Congenital stationary night blindness
Loop I–II Loop II–III I S6 III S1–S2 loop III S4 III S5–S6 loop IV S2 IV S4 IV S5–S6 loop C-terminal cytoplasmic tail
d e d e d d,e e e d d,e
Cav2.1 (human)
Familial hemiplegic migraine
I S4 II S5–S6 loop II S6 IV S6 III S1 III S2 C-terminal cytoplasmic tail
f f f f f f g
Cav1.1 (mouse)
Episodic ataxia type 2 Spinocerebellar ataxia type 6
Cav2.1 (mouse)
Tottering Leaner
II S5–S6 loop C-terminal cytoplasmic tail
h i
β4 (mouse)
Lethargic
β-subunit interaction domain
j
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i Doyle, J. et al. (1997) Mutations in the CACNL1A4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice. Mamm. Genome 8, 113–120
which has been reported previously22. In addition to a decrease in R-type current, elimination of the P/Q-type channel activity resulted in an enhanced functional Land N-type Ca2+ channel activity21. Thus, there appears to be an overlap in neurotransmitter release whereby a whole family of Ca2+ channels can serve as triggers. α1B) knockout mice Cav 2.2 (α
The N-type VDCCs are predominantly localized in the nervous system and have been considered to play a vital role in many neuronal functions particularly transmitter release at sympathetic postsynaptic http://tips.trends.com
IV
III
II
I
j Burgess, D.L. et al. (1997) Mutation of the Ca2+ channel beta subunit gene CCHB4 is associated with ataxia and seizures in the lethargic (lh) mouse. Cell 88, 385–392
terminals. To examine this directly, a very recent and exciting addition to our transgenic armamentaria is the generation of mice genetically deficient in the α1B-subunit23. Surprisingly, these mice have a normal life span and do not exhibit any significant behavioral aberrations. The N-type currents, of course, were completely eliminated without any change in the activity of other VDCC types in neuronal preparations. The baroreceptor reflex response was markedly reduced after bilateral carotid occlusion. In addition, isolated left atria from these mice hearts did not demonstrate positive inotropic responses to electrical sympathetic
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Fig. 1. Gene regulation by Ca2+ signals. Function of voltage-, ligand- and store-operated Ca2+ channels transiently enhances cytosolic Ca2+. The generation, duration and waning of the Ca2+ signal are regulated by several intertwining cellular mechanisms resulting in an initiation of gene transcription. Sustained Ca2+ signals can influence gene transcription by diverse cellular mechanisms. These include, but are not limited to: (1) activation of calcineurin, a Ca2+–calmodulin (CaM)activated phosphatase that dephosphorylates members of the NF-ATc transcription factors, conferring nuclear translocation and transcription initiation; and (2) activation of the Ca2+–CaM complex and translocation to the nucleus and subsequent cAMP response elementbinding protein (CREB) phosphorylation and/or activation of Ca2+–CaMdependent protein kinase (CaMK) enzymes. Other cellular signal transduction pathways might interfere with the above processes. These include cytosolic Ca2+ itself, and trimeric G-protein-coupled receptors, either directly or indirectly, might activate protein kinase C (PKC) or other transduction pathways (Ras, Rac and mitogenactivated kinase family). These signals can all funnel into initiation of the transcription processes. It is likely that the intracellular Ca2+ signaling is integrated into a canonical organization of numerous other signal transduction pathways. Abbreviations: CRAC, Ca2+ releaseactivated Ca2+ channel; PP2B, protein phosphatase 2B; Trp, transient receptor potential; VDCC, voltagedependent Ca2+ channel. Adapted, with permission, from Ref. 47.
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Receptor
Trp, CRAC Ca2+
NMDA receptor Ca2+
Furthermore, as previously mentioned, an 80% decrease in R-type current was observed in the Cav2.1 (α1A) knockout mice22. Thus, it appears that the contribution of the α1E Ca2+ channel component of the R-type current is dependent on the neuronal cell population, with additional VDCC α1-subunits contributing to the total R-type current.
VDCC Ca2+
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+ Ca2+–CaM
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neuronal stimulation. Although, the parasympathetic system seemed intact, both blood pressure and heart rate were elevated. These mice should be of considerable value in further assessing the role of the adrenergic arm of the autonomic nervous system. α1E) knockout mice Cav2.3 (α
Of the known VDCC channels in the brain, the R-type is the least understood because of its resistance to antagonists of L-, N- and P/Q-type channels. Cloning of the gene encoding Cav2.3 (α1E) and heterologous expression studies suggested that this channel was responsible for R-type current, but the channel also possessed properties that resembled a T-type current. However, a specific inhibitor of class E Ca2+ channels [SNX482 (a synthetic version of a novel 41-amino-acid peptide isolated from the venom of the West African tarantula Hysterocrates gigas)] was insufficient in blocking all R-type currents24. Thus, the in vivo function of the α1E channel remained controversial until knockout studies of the gene encoding Cav2.3 were performed. Transgenic mice displayed a reduced response to somatic inflammatory pain and an altered response to visceral inflammatory pain. Further investigation identified expression of the gene encoding Cav2.3 in the spinal cord and dorsal root ganglion25. Taken together, the data indicate that the α1E Ca2+ channel controls pain behaviors by both spinal and supraspinal mechanisms making it an ideal target for blocking nociception. In addition, the α1E Ca2+ channel also appears to play a role in formation of accurate spatial memory but not fear memory26. So, is the α1E Ca2+ channel responsible for the R-type current in neurons? Experiments using antisense oligonucleotides to the α1E-subunits in neurons support the idea that the R-type current is a result of the α1E Ca2+ channel27,28. However, Wilson et al.29 identified substantial R-type currents in all α1E knockout neurons measured. http://tips.trends.com
The β1 knockout mouse was the first Ca2+ channel transgenic animal created. The β1-subunit is predominantly found in skeletal muscle and brain with lower expression levels in the spleen and heart30–33. Coexpression of the β1-subunit in heterologous expression studies enhances Ca2+ channel current amplitude and density (Box 1)2–9. Ca2+ influx across the skeletal muscle membrane through the α1S-subunit (Cav1.1) is essential in coupling the electrical depolarizing signal to the release of Ca2+ from the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum and eventual muscle contraction [i.e. excitation contraction (EC) coupling]. Homozygous transgenic fetuses were immobile with disorganization of thick and thin filaments and died at birth from asphyxiation34. Consistent with the heterologous expression systems, the L-type Ca2+ current was decreased by 10–20-fold with not only a loss of β1-subunit membrane localization, but also a loss of α1S-subunit membrane localization, which suggests a trafficking role for the β1-subunit34. Radioligand binding {e.g. using [3H]PN200110} revealed a 3.9-fold decrease in Bmax for the L-type channel and a 2.8-fold decrease in charge movement35. Early death of the homozygous animals made it impossible to assess the role of the β1-subunit in the brain, spleen and heart. Interestingly, heterozygous animals were indistinguishable from wild-type littermates. This would suggest that an excess of β1-subunit is present and is able to compensate for ablating one copy of the gene. β3 knockout mice
The β3-subunit is primarily found in the brain, heart and aorta, and is associated with ~56% of the N-type Ca2+ channels in the brain36. Although β3-deficient mice were indistinguishable from wild-type animals, L- and N-type currents were significantly reduced in the superior cervical ganglion37. Possible upregulation of other β-subunits might account for the minimal change in phenotype. γ knockout mice
The γ1-subunit is exclusively expressed in skeletal muscle, but its function has been illusive. Heterologous coexpression studies have failed to identify a role of the γ1-subunit when expressed with its native complex partners (α1S, β1 and α2–δ1)31. However, coexpression with α1C identified a shift in the steady-state inactivation and acceleration of both current activation and current inactivation38,39. γ1-Subunit-deficient mice were indistinguishable from wild-type mice, but they
Review
Note added in proof A recent article [Rottbauer, W. et al. (2001) Developmental Cell 1, 265–275] describes an embryonic lethal mutation [‘island beat (isl)’] in the α1C-subunit of the L-VDCC in zebrafish. The atria exhibit fibrillation and the ventricle is silent and fails to acquire the normal number of myocytes. This provides evidence that the L-VDCC can regulate growth independently of contraction.
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displayed altered Ca2+ channel activity. Increased peak Ca2+ current amplitude and shifts in inactivation kinetics were present without changes in current density or activation kinetics40. Stargazer mice, which exhibit phenotypes that are characteristic of absence epilepsy, were thought to represent a natural γ-subunit mutation. Cloning the cDNA encoding the 36-kDa protein from these mice revealed a weak homology to the skeletal muscle γ-subunit. Therefore, the gene was termed γ2 or stargazin41. In vitro coexpression of stargazin (γ2) showed an increase of steady-state inactivation of α1A. Thus, it was concluded that aberrant P/Q-type channel function might contribute to inappropriate Ca2+ entry in neuronal cells of this mutant mice. However, a recent study by Chen et al.42 clearly identified the stargazer protein as an essential component to target AMPA receptors to synapses. When mutated stargazin is present (stargazer mice), no functional AMPA receptors can be detected in granule cell synapses. Concluding remarks
Transgenic animals have proven tremendously useful in clarifying and confirming heterologous expression studies and defining in vivo functions of the Ca2+ channel family. The numerous substrates for Ca2+ signaling create great difficulties in identifying specific roles for the channel. In recent years, it has become increasingly evident that alterations in Ca2+ signaling
References 1 Ertel, E.A. et al. (2000) Nomenclature of voltagedependent calcium channels. Neuron 25, 533–535 2 Mori, Y. et al. (1996) Molecular pharmacology of voltage-dependent calcium channels. Jpn. J. Pharmacol. 72, 83–109 3 Gurnett, C.A. and Campbell, K.P. (1996) Transmembrane auxiliary subunits of voltagedependent ion channels. J. Biol. Chem. 271, 27975–27978 4 Varadi, G. et al. (1999) Molecular elements of ion permeation and selectivity within calcium channels. Crit. Rev. Biochem. Mol. Biol. 34, 181–214 5 Hoffman, F. et al. (1999) Voltage-dependent Ca2+ channels: from structure to function. Rev. Physiol. Biochem. Pharmacol. 139, 33–87 6 Catterall, W.A. (1998) Stucture and function of neuronal Ca2+ channels and their role in neurotransmitter release. Cell Calcium 24, 307–323 7 Pragnell, M. et al. (1994) Calcium channel β-subunit binds to a conserved motif in the I-II cytoplasmic linker of the α1-subunit. Nature 368, 67–70 8 Witcher, D.R. et al. (1995) Association of native Ca2+ channel β-subunits with the α1-subunit interaction domain. J. Biol. Chem. 270, 18088–18093 9 Gurnett, C.A. et al. (1996) Dual function of the voltage-dependent Ca2+ channel α2/δ-subunit in current stimulation and subunit interaction. Neuron 16, 431–440 10 Seisenberger, C. et al. (2000) Functional embryonic cardiomyocytes after disruption of the L-type α1C (Cav1.2) calcium channel gene in mouse. J. Biol. Chem. 275, 39193–39199 http://tips.trends.com
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pathways have a drastic impact on the gene expression program in excitable and also in non-excitable cells. In terms of evolution, Ca2+ is the key cation, and is also probably important early on in development in almost all biological systems that link excitation to numerous physiological end states. How a generic cation such as Ca2+ results in very specific outcomes at the level of gene expression will be one of the greatest puzzles of research in the post-genomic era. The stimulus– transcription coupling has brought into focus several other signaling pathways whose activation correlates with the activation of Ca2+ signaling. These include stimulation of cAMP response-element binding protein (CREB), phosphorylation and dephosphorylation by a Ca2+–calmodulin signal and in turn activation of CRE-regulated genes43,44. The involvement of a Ca2+–calmodulin-dependent kinase cascade in this process has also been demonstrated43. Another very important Ca2+-stimulated signaling pathway is signal transduction by calcineurin and activation of NF-ATc transcription factors45–47 (Fig. 1). These are only a few of a possible many mechanisms, and, most probably, a combinatorial and hierarchical integration of signaling pathways as observed in other examples48–50 will determine the final outcome of a Ca2+-stimulated gene expression. The recent advent of transgenic animals with specific VDCC subunits will aid immensely in fine-tuning our understanding of cellular regulation.
11 Muth, J.N. et al. (1999) Cardiac-specific overexpression of the α1-subunit of the L-type voltage-dependent Ca2+ channel in transgenic mice. J. Biol. Chem. 274, 21503–21506 12 Muth, J.N. et al. (2001) A Ca2+-dependent transgenic model of cardiac hypertrophy. A role for protein kinase Cα. Circulation 103, 140–147 13 Platzer, J. et al. (2000) Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell 102, 89–97 14 Martinez-Dunst, C. et al. (1997) Release sites and calcium channels in hair cells of the chick’s cochlea. J. Neurosci. 17, 9133–9144 15 Moser, T. and Beutner, D. (2000) Kinetics of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse of the mouse. Proc. Natl. Acad. Sci. U. S. A. 97, 883–888 16 Dunlap, K. et al. (1995) Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci. 18, 89–98 17 Westenbroek, R.E. et al. (1995) Immunochemical identification and subcellular distribution of the α1A-subunits of brain calcium channels. J. Neurosci. 15, 6403–6418 18 Rhyu, I.J. et al. (1999) Morphologic investigation of rolling mouse Nagoya (tg(rol)/tg(rol)) cerebellar Purkinje cells: an ataxic mutant, revisited. Neurosci. Lett. 266, 49–52 19 Ophoff, R.A. et al. (1996) Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+channel gene CACNL1A4. Cell 87, 543–552 20 Zhuchenko, O. et al. (1997) Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltagedependent calcium channel. Nat. Genet. 15, 62–69
21 Jun, K. et al. (1999) Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α1Asubunit. Proc. Natl. Acad. Sci. U. S. A. 96, 15245–15250 22 Sutton, K.G. et al. (1999) P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A. Nature 401, 800–804 23 Ino, M. et al. (2001) Functional disorders of sympathetic nervous system in mice lacking the α1B-subunit (Cav2.2) of N-type calcium channel. Proc. Natl. Acad. Sci. U. S. A. 98, 5323–5328 24 Newcomb, R. et al. (1998) Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry. 37, 15353–15362 25 Saegusa, H. et al. (2000) Altered pain responses in mice lacking α1E-subunit of the voltage-dependent Ca2+ channel. Proc. Natl. Acad. Sci. U. S. A. 97, 6132–6137 26 Kubota, M. et al. (2001) Intact LTP and fear memory but impaired spatial memory in mice lacking Cav2.3 (α1E) channel. Biochem. Biophys. Res. Commun. 282, 242–248 27 Piedras-Renteria, E.S. and Tsien, R.W. (1998) Antisense oligonucleotides against α1E reduce Rtype calcium currents in cerebellar granule cells. Proc. Natl. Acad. Sci. U. S. A. 95, 7760–7765 28 Tottene, A. et al. (2000) α1E-subunits form the pore of three cerebellar R-type Ca2+ channels with different pharmacological and permeation properties. J. Neurosci. 20, 171–178 29 Wilson, S.M. et al. (2000) The status of voltagedependent calcium channels in α1E knockout mice. J. Neurosci. 20, 8566–8571
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30 Ruth, P. et al. (1989) Primary structure of the β-subunit of the DHP-sensitive calcium channel from skeletal muscle. Science 245, 1115–1118 31 Varadi, G. et al. (1991) Acceleration of activation and inactivation by the β-subunit of the skeletal muscle calcium channel. Nature 11, 159–162 32 Pragnell, M. et al. (1991) Cloning and tissuespecific expression of the brain calcium channel β-subunit. FEBS Lett. 291, 253–258 33 Powers, P.A. et al. (1992) Skeletal muscle and brain isoforms of a β-subunit of human voltagedependent calcium channels are encoded by a single gene. J. Biol. Chem. 267, 22967–22972 34 Gregg, R.G. et al. (1996) Absence of the β-subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1-subunit and eliminates excitation–contraction coupling. Proc. Natl. Acad. Sci. U. S. A. 93, 13961–13966 35 Strube, C. et al. (1996) Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor β1-subunit. Biophys. J. 71, 2531–2543 36 Scott, V.E. et al. (1996) β-subunit heterogeneity in N-type Ca2+ channels. J. Biol. Chem. 271, 3207–3212
37 Namkung, Y. et al. (1998) Targeted disruption of the Ca2+ channel β3-subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons. Proc. Natl. Acad. Sci. U. S. A. 95, 12010–12015 38 Singer, D. et al. (1991) The roles of the subunits in the function of the calcium channel. Science 253, 553–557 39 Wei, X.Y. et al. (1991) Heterologous regulation of the cardiac Ca2+ channel α1-subunit by skeletal muscle β and γ-subunits. Implications for the structure of cardiac L-type Ca2+ channels. J. Biol. Chem. 266, 21943–21947 40 Freise, D. et al. (2000) Absence of the γ-subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties. J. Biol. Chem. 275, 14476–14481 41 Letts, V.A. et al. (1998) The mouse stargazer gene encodes a neuronal Ca2+ channel γ-subunit. Nat. Genet. 19, 340–347 42 Chen, L. et al. (2000) Stargazing regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943 43 Bito, H. et al. (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214
44 Deisseroth, K. et al. (1998) Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 392, 198–202 45 Molkentin, J.D. et al. (1998) A calcineurindependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215–228 46 Graef, I.A. et al. (1999) L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401, 703–708 47 Crabtree, G.R. (1999) Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell 96, 611–614 48 Halfon, M.S. et al. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissuerestricted transcription factors. Cell 103, 63–74 49 Flores, G.V. et al. (2000) Combinatorial signaling in the specification of unique cell fates. Cell 103, 75–85 50 Xu, C. et al. (2000) Overlapping activators and repressors delimit transcriptional response to receptor tyrosine kinase signals in the Drosophila eye. Cell 103, 87–97
Heterodimerization of G-proteincoupled receptors: pharmacology, signaling and trafficking Lakshmi A. Devi Although classical models predict that G-protein-coupled receptors (GPCRs) function as monomers, several recent studies acknowledge that GPCRs exist as dimeric or oligomeric complexes. In addition to homodimers, heterodimers between members of the GPCR family (both closely and distantly related) have been reported. In some cases heterodimerization is required for efficient agonist binding and signaling, and in others heterodimerization appears to lead to the generation of novel binding sites. In this article, the techniques used to study GPCR heterodimers, and the ‘novel pharmacology’ and functional implications resulting from heterodimerization will be discussed.
Lakshmi A. Devi Dept of Pharmacology, New York University School of Medicine, New York, NY, USA. e-mail: lakshmi.devi@ med.nyu.edu
The function of most, if not all, cells in the body is regulated by plasma membrane receptors. The vast majority of these receptors belong to the superfamily of G-protein-coupled receptors (GPCRs), which at current estimates account for ~1% of the genes present in a mammalian genome. Agonists or antagonists of GPCRs, in addition to agents that interfere with cellular pathways regulated by these receptors, are widely used in drug therapy. All GPCRs share a common tertiary structure consisting of seven transmembrane (TM) helices http://tips.trends.com
linked by three alternating intracellular and extracellular domains, with an extracellular N-terminus and a cytoplasmic C-terminus. GPCRs interact with heterotrimeric guanine-nucleotidebinding regulatory proteins (G proteins), which then interact with effector systems and regulate various intracellular processes (for an animation of agonistinduced activation of GPCRs, see http://archive. bmn.com/supp/tips/tips2210a.html). Based on sequence similarity, GPCRs can be classified into three major receptor families1. Family A (rhodopsin, β2-adrenoceptor-like) is the largest family of GPCRs. These receptors are characterized by several conserved residues in their TM helices and a palmitoylated cysteine in the C-terminal tail. Family B [glucagon, vasoactive intestinal peptide (VIP), calcitonin receptorlike] is a relatively small group that is characterized by the presence of a large N-terminal domain that contains several well-conserved cysteine residues. Family C (metabotropic neurotransmitter, calcium-sensing receptor-like) is characterized by a very long N-terminal domain that appears to be sufficient for ligand binding.
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