Tsutomu Tanabe

Nomenclature of voltage-gated sodium channels

Voltage-gated Ca2+ channels mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission, and gene expression. They are members of a gene superfamily of transmembrane ion channel proteins that includes voltage-gated K+ and Na+ channels. The Ca2+ channels that have been characterized biochemically are complex proteins composed of four or five distinct subunits, which are encoded by multiple genes. The α1 subunit of 190–250 kDa is the largest subunit, and it incorporates the conduction pore, the voltage sensor and gating apparatus, and the known sites of channel regulation by second messengers, drugs, and toxins. An intracellular β subunit and a transmembrane, disulfide-linked α2δ subunit complex are components of most types of Ca2+ channels. A γ subunit has also been found in skeletal muscle Ca2+ channels, and related subunits are expressed in heart and brain. Although these auxiliary subunits modulate the properties of the channel complex, the pharmacological and electrophysiological diversity of Ca2+ channels arises primarily from the existence of multiple forms of α1 subunits. Mammalian α1 subunits are encoded by at least ten distinct genes. Historically, various names have been given to the corresponding gene products, giving rise to distinct and sometimes confusing nomenclatures. In 1994, some of us proposed a unified nomenclature based on the most widely accepted system at the time: α1 subunits were referred to as α1S for the original skeletal muscle isoform and α1A through α1E for those discovered subsequently (Birnbaumer et al. 1994xBirnbaumer, L., Campbell, K.P., Catterall, W.A., Harpold, M.M., Hofmann, F., Horne, W.A., Mori, Y., Schwartz, A., Snutch, T.P., Tanabe, T. et al. Neuron. 1994; 13: 505–506Abstract | Full Text PDF | PubMed | Scopus (264)See all ReferencesBirnbaumer et al. 1994). Since then, four new α1 subunits have been identified, which were named α1F through α1I.Ca2+ currents recorded in different cell types have diverse physiological and pharmacological properties, and an alphabetical nomenclature has also evolved for the distinct classes of Ca2+ currents. L-type Ca2+ currents require a strong depolarization for activation, are long lasting, and are blocked by the organic L-type Ca2+ channel antagonists, including dihydropyridines, phenylalkylamines, and benzothiazepines. They are the main Ca2+ currents recorded in muscle and endocrine cells, where they initiate contraction and secretion. N-type, P/Q-type, and R-type Ca2+ currents also require strong depolarization for activation. They are unaffected by L-type Ca2+ antagonist drugs but are blocked by specific polypeptide toxins from snail and spider venoms. They are expressed primarily in neurons, where they initiate neurotransmission at most fast synapses. T-type Ca2+ currents are activated by weak depolarizations and are transient. They are resistant to both organic antagonists and to the snake and spider toxins used to define the N- and P/Q-type Ca2+ currents. They are expressed in a wide variety of cell types, where they are involved in shaping the action potential and controlling patterns of repetitive firing.As new Ca2+ channel genes are cloned, it is apparent that these two alphabetical nomenclatures will overlap at α1L, which may not mediate an L-type Ca2+ current and therefore may create confusion. Moreover, the present alphabetical nomenclature does not reveal the structural relationships among the α1 subunits, which can be grouped into three families: (1) α1S, α1C, α1D, and α1F; (2) α1A, α1B, and α1E; and (3) α1G, α1H, and α1I. The complete amino acid sequences of these α1 subunits are more than 70% identical within a family but less than 40% identical among families. These family relationships are illustrated for the more conserved transmembrane and pore domains in Figure 1Figure 1. Division of calcium channels into these three families is phylogenetically ancient, as representatives of each are found in the C. elegans genome. Ideally, a nomenclature for Ca2+ channel α1 subunits should provide a systematic organization based on their structural relationships and should be coordinated with nomenclatures for the other families of voltage-gated ion channels of different ionic selectivities (ie., K+ and Na+).Figure 1Phylogeny of Voltage-Gated Ca2+ Channel α1 SubunitsOnly the membrane-spanning segments and the pore loops (∼350 amino acids) are compared. First, all sequence pairs were compared, which clearly defines three families with intrafamily sequence identities above 80% (CaV1.m, CaV2.m, CaV3.m). Then, a consensus sequence was defined for each family, and these three sequences were compared to one another, with interfamily sequence identities of ∼52% (CaV1.m versus CaV2.m) and 28% (CaV3.m versus CaV1.m or CaV2.m).View Large Image | View Hi-Res Image | Download PowerPoint SlideFor these reasons, we wish to propose a new nomenclature of voltage-gated Ca2+ channels (Table 1Table 1), which is more systematic and mimics the well-defined K+ channel nomenclature (Chandy et al., 1991xChandy, K.G. Nature. 1991; 352: 26Crossref | PubMedSee all ReferencesChandy et al., 1991). This nomenclature uses a numerical system (KV1.1, KV2.1, KV3.1, etc.) to define families and subfamilies of K+ channels based on similarities in amino acid sequences. In a similar manner, we propose that Ca2+ channels should be renamed using the chemical symbol of the principal permeating ion (Ca) with the principal physiological regulator (voltage) indicated as a subscript (CaV). The numerical identifier would correspond to the CaV channel α1 subunit gene family (1 through 3 at present) and the order of discovery of the α1 subunit within that family (1 through m). According to this nomenclature, the CaV1 family (CaV1.1 through CaV1.4) includes channels containing α1S, α1C, α1D, and α1F, which mediate L-type Ca2+ currents (Table 1Table 1). The CaV2 family (CaV2.1 through CaV2.3) includes channels containing α1A, α1B, and α1E, which mediate P/Q-type, N-type, and R-type Ca2+ currents, respectively (Table 1Table 1). The CaV3 family (CaV3.1 through CaV3.3) includes channels containing α1G, α1H, and α1I, which mediate T-type Ca2+ currents (Table 1Table 1). When specific reference to the α1 subunit within the Ca2+ channel complex is intended, the designation α11.m, α12.m, or α13.m may be used, where the numeral m represents the individual gene/protein within the family. Where applicable, lowercase letters are used to distinguish alternatively spliced variants (e.g., CaV1.2a corresponds to channels containing the cardiac variant of the former α1C). Such a systematic nomenclature has proved successful for the KV channel proteins. Its strength resides in the rational basis derived from the structural relationships among the channel proteins and the ease and precision with which new channels can be added.Table 1Proposed Nomenclature for Cloned Voltage-Gated Ca2+ Channel α1 SubunitsNameFormer NamesAccession NumberGene Name and Human ChromosomeSplice TypesFormer NamesPrimary TissuesCav1.1 α11.1α1S, α1Skm, CaCh1X05921CACNA1S; 1q31-32skeletal muscleCav1.2α1C, rbC, CaCh2CaCh2, X15539CACNA1C; 12p13.3Cav1.2aα1C-aheartα11.2Cav1.2bα1C-bsmooth musclerbC-I, M67516; rbC-II, M67515Cav1.2cα1C-bbrain, heart, pituitary, adrenalCav1.3 α11.3α1D, rbD, CaCh3M76558CACNA1D; 3p14.3brain, pancreas, kidney, ovary, cochleaCav1.4α1FAJ224874CACNA1F; Xp11.23retinaα11.4Cav2.1α1A, rbA, CaCh4, BIrbA, M64373; BI-1, X57476CACNA1A; 19p13Cav2.1aBI1brain, cochlea, pituitaryα12.1BI-2, X57477Cav2.1bBI2brain, cochlea, pituitaryCav2.2α1B, rbB, CaCh5, BIIIrbB, M92905; BIII, D14157;CACNA1B; 9q34Cav2.2aα1B-1brain, nervous systemα12.2human α1B, M94172Cav2.2bα1B-2brain, nervous systemCav2.3α1E, rbE, CaCh6, BIIrbE, L15453, BII-1, X67855;CACNA1E; 1q25-31Cav2.3aBIIbrain, cochlea, retina, heart,α12.3human α1E, L29384pituitaryCav2.3bBII2brain, cochlea, retinaCav3.1α1GAF027984; AF029228CACNA1G; 17q22Cav3.1abrain, nervous systemα13.1Cav3.2α1HAF051946; AF073931CACNA1H; 16p13.3Cav3.2abrain, heart, kidney, liverα13.2Cav3.3α1IAF086827CACNA1I; 22q12.3-13-2Cav3.3abrainα13.3The cloned voltage-gated Ca2+ channels and most widely studied alternate splice forms are presented together with the proposed nomenclature and previous nomenclatures.The nomenclature of the auxiliary subunits is not modified, since it already includes numbers for the gene family and lowercase letters for the splice variants. Thus, the subunit compositions of the voltage-dependent Ca2+ channels CaVn.mx may be described as α1n.mx/βm′x′/γm′′x′′/α2δm′′′x′′′ complexes, where the number n defines a main family, the numbers m, m′, m′′, and m′′′ refer to the individual genes/proteins within the families, and the letters x, x′, x′′, and x′′′ identify the splice variants. Standard prefixes can be placed in front of the channel name to identify the species of origin. In this notation, the skeletal muscle calcium channel would be written α11.1a/β1a/γ1a/α2δ1a. With this new nomenclature, the CaV designation may also be used to identify calcium channel auxiliary subunits such as CaVβ or CaVγ independent of their presence in a calcium channel complex.We hope that this new nomenclature for α1 subunits will be a stimulus to further research on voltage-gated Ca2+ channels by providing a common, easily accessible standard of reference for scientists working in this field. A full-length review article** is planned to present a more detailed proposal for nomenclature of the many alternate splice forms of the α1 subunits and the auxiliary subunits of Ca2+ channels that have been described in cDNA cloning experiments.*This nomenclature has been approved by the Nomenclature Committee of the International Union of Pharmacology, and a review article giving more details of the nomenclature for calcium channel subunits and splice variants is planned for Pharmacological Reviews.

Structural homology of Torpedo californica acetylcholine receptor subunits

The nicotinic acetylcholine receptor (AChR) from the electroplax of the ray Torpedo californica is composed of five subunits present in a molar stoichiometry of α2βγδ (refs 1–3) and contains both the binding site for the neurotransmitter and the cation gating unit (reviewed in refs 4–6). We have recently elucidated the complete primary structures of the α-, β- and δ-subunit precursors of the T. californica AChR by cloning and sequencing cDNAs for these polypeptides7,8. Here, we report the whole primary structure of the γ-subunit precursor of the AChR deduced from the nucleotide sequence of the cloned cDNA. Comparison of the amino acid sequences of the four subunits reveals marked homology among them. The close resemblance among the hydrophilicity profiles and predicted secondary structures of all the subunits suggests that these polypeptides are oriented in a pseudosymmetric fashion across the membrane. Each subunit contains four putative transmembrane segments that may be involved in the ionic channel. The transmembrane topology of the subunit molecules has also been inferred.

Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N‐type Ca2+ channel

The importance of voltage‐dependent Ca2+ channels (VDCCs) in pain transmission has been noticed gradually, as several VDCC blockers have been shown to be effective in inhibiting this process. In particular, the N‐type VDCC has attracted attention, because inhibitors of this channel are effective in various aspects of pain‐related phenomena. To understand the genuine contribution of the N‐type VDCC to the pain transmission system, we generated mice deficient in this channel by gene targeting. We report here that mice lacking N‐type VDCCs show suppressed responses to a painful stimulus that induces inflammation and show markedly reduced symptoms of neuropathic pain, which is caused by nerve injury and is known to be difficult to treat by currently available therapeutic methods. This finding clearly demonstrates that the N‐type VDCC is essential for development of neuropathic pain and, therefore, controlling the activity of this channel can be of great importance for the management of neuropathic pain.

Ataxin-3 is translocated into the nucleus for the formation of intranuclear inclusions in normal and Machado-Joseph disease brains

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is one of the dominantly inherited cerebellar ataxias. The gene responsible for the disease, a novel gene of unknown function, encodes ataxin-3 containing a polyglutamine stretch. Although it has been known that ataxin-3 is incorporated into neuronal intranuclear inclusions (NIIs) in neurons of affected regions, the relationship between NII formation and neuronal degeneration still remains uncertain. In the present study we show two different conditions in which ataxin-3 is recruited into the nucleus and suggest a process to form nuclear inclusions. In normal brains, wild-type ataxin-3 localizes within the ubiquitin-positive nuclear inclusion, the Marinesco body, indicating that ataxin-3 is recruited into the nuclear inclusion even in the absence of pathologically expanded polyglutamine. In MJD/SCA3 brains, immunohistochemical analyses with anti-ataxin-3 antibody, anti-ubiquitin antibody, and monoclonal antibody 1C2 known to recognize expanded polyglutamine revealed differences in frequency and in diameter among NIIs recognized by each antibody. These results were confirmed in the same inclusions by double immunofluorescent staining, suggesting that expanded ataxin-3 forms a core, thereby recruiting wild-type ataxin-3 into the nucleus around the core portion, and then followed by activation of the ubiquitin/ATP-dependent pathway. Recruitment of ataxin-3 into the nucleus and formation of nuclear inclusion under two different conditions suggest that ataxin-3 may be translocated into the nucleus under certain conditions stressful on neuronal cells such as aging and polyglutamine neurotoxicity.

Effects of ablation of N- and R-type Ca2+ channels on pain transmission

Recently several mutant mouse lines lacking neuronal voltage-dependent Ca(2+) channels (VDCCs) have been established by the use of gene targeting in embryonic stem cells. Pain-related behaviors in Ca(v)2.2 (alpha(1B)) and Ca(v)2.3 (alpha(1E)) knockout mice were studied to gain further insight into the mechanism of pain transmission, where VDCCs are thought to play important roles. We review here the data from these recent studies. Ca(v)2.3-/- mice showed normal responses to acute painful stimuli, and reduced responses to the somatic inflammatory pain stimuli. Ca(v)2.3+/- mice exhibited reduced symptoms of visceral inflammatory pain. Ca(v)2.3-/- mice showed abnormal behavior related to the descending antinociceptive mechanism activated by the intraperitoneal injection of acetic acid. Ca(v)2.2-/- mice showed variable acute nociceptive responses depending on the mutant lines. However, all the lines of Ca(v)2.2-/- mice exhibited reduced responses in the phase 2 of the formalin test, suggesting a suppression of inflammatory pain. Furthermore Ca(v)2.2-/- mice showed markedly reduced neuropathic pain symptoms after spinal nerve ligation. Impaired antinociception, similar to that seen in the Ca(v)2.3-/- mice, was also observed in the Ca(v)2.2-/- mice. Therefore, it is suggested that these mutant mice could provide novel models to delineate the nociceptive and antinociceptive mechanisms.

Cytoplasmic and nuclear polyglutamine aggregates in SCA6 Purkinje cells

Aggregations of the alpha1A-calcium channel protein have been previously demonstrated in spinocerebellar ataxia type 6 (SCA6). Here the authors show that small aggregates, labeled by a monoclonal antibody 1C2 that preferentially detects expanded polyglutamine larger than that in SCA6 mutation, are present mainly in the cytoplasm but also in the nucleus of Purkinje cells. Although the length of expansion is small in SCA6, the current finding might indicate that SCA6 conforms to the pathogenic mechanism(s) in other polyglutamine diseases.

Changes in Expression of Voltage-Dependent Ion Channel Subunits in Dorsal Root Ganglia of Rats with Radicular Injury and Pain

Study Design. Changes in expression of voltage-dependent ion channel subunits were examined in the radicular pain state. Furthermore, antinociceptive effects of gabapentin on radicular pain were compared with effects on peripheral neuropathic pain. Objectives. To clarify molecular substrates involved in the development of radicular pain, and to investigate the responsiveness of radicular pain to gabapentin. Summary of Background Data Peripheral nerve injuries are known to induce dynamic changes of voltage-dependent Na+ and Ca2+ channel subunits expression in dorsal root ganglion neurons. However, the expression profiles of Na+ and Ca2+ channel subunits in the radicular pain state have not been examined. Methods. Two radicular pain models and one peripheral neuropathic pain model were prepared. By using semiquantitative reverse transcriptase–polymerase chain reaction, the expression levels of several Na+ and Ca2+ channel subunits in the dorsal root ganglions of these pain model rats were investigated. The antinociceptive effects of gabapentin were examined in a behavioral study using the aforementioned pain models. Results. All three neuropathic pain operations induced comparable mechanical allodynia and thermal hyperalgesia. The upregulation of the Nav1.3 Na+ channel and Cav&agr;2&dgr; Ca2+ channel subunits was observed only in the peripheral nerve injury model. A downregulation of the Nav1.9 channel was observed in all three pain model rats. A lower dose of gabapentin was significantly more effective in alleviating the mechanical allodynia of rats with radicular pain. Conclusions. The reduction of Nav1.9 found in all three models may link to the neuropathic pain state, including radicular pain. The lower sensitivity to gabapentin in rats with peripheral neuropathic pain might be partly explained by the marked upregulation of Cav&agr;2&dgr; in the dorsal root ganglions, suggesting that gabapentin may be more effective in radicular pain treatment.

CO-EXPRESSION IN CHO CELLS OF TWO MUSCLE PROTEINS INVOLVED IN EXCITATION-CONTRACTION COUPLING

SummaryRyanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the α2, β and γ subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cells plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Properties of human Cav2.1 channel with a spinocerebellar ataxia type 6 mutation expressed in Purkinje cells

Spinocerebellar ataxia type 6 (SCA6) is caused by polyglutamine expansion in P/Q-type Ca2+ channels (Ca(v)2.1) and is characterized by predominant degeneration of cerebellar Purkinje cells. To characterize the Ca(v)2.1 channel with an SCA6 mutation in cerebellar Purkinje cells, we have generated knock-in mouse models that express human Ca(v)2.1 with 28 polyglutamine repeats (disease range) and with 13 polyglutamine repeats (normal range). Patch-clamp recordings of the Purkinje cells from homozygous control or SCA6 knock-in mice revealed a non-inactivating current that is highly sensitive to a spider toxin omega-Agatoxin IVA, indicating that the human Ca(v)2.1 expressed in Purkinje cells exhibits typical P-type properties in contrast to the previous data showing Q-type properties, when it was expressed in cultured cell lines. Furthermore, the voltage dependence of activation and inactivation and current density were not different between SCA6 and control, though these properties were altered in previous reports using non-neuronal cells as expression systems. Therefore, our results do not support the notion that the alteration of the channel properties may underlie the pathogenic mechanism of SCA6.

Cav2.3 (α1E) Ca2+ channel participates in the control of sperm function

To know the function of the Ca2+ channel containing α12.3 (α1E) subunit (Cav2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Cav2.3 channel. The averaged rising rates of Ca2+ transients induced by α‐D‐mannose–bovine serum albumin in the head region of Cav2.3−/− sperm were significantly lower than those of Cav2.3+/+ sperm. A computer‐assisted sperm motility assay revealed that straight‐line velocity and linearity were greater in Cav2.3−/− sperm than those in Cav2.3+/+ sperm. These results suggest that the Cav2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.