Richard W. Tsien

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.

Multiple types of neuronal calcium channels and their selective modulation

Abstract Voltage-gated Ca 2+ channels control the entry of Ca 2+ ions across the surface membrane of neurons and other cells and thereby influence electrical activity and diverse cellular responses. Here we summarize efforts at classifying multiple types of Ca 2+ channels according to differences in their gating, ionic conductance and pharmacology. The distribution of Ca 2+ channel types among different cells and their selective modulation by neurochemicals and drugs is briefly reviewed.

Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones.

1. Calcium currents in cultured dorsal root ganglion (d.r.g.) cells were studied with the whole‐cell patch‐clamp technique. Using experimental conditions that suppressed Na+ and K+ currents, and 3‐10 mM‐external Ca2+ or Ba2+, we distinguished three distinct types of calcium currents (L, T and N) on the basis of voltage‐dependent kinetics and pharmacology. 2. Component L activates at relatively positive test potentials (t.p. greater than ‐10 mV) and shows little inactivation during a 200 ms depolarization. It is completely reprimed at a holding potential (h.p.) of ‐60 mV, and can be isolated by using a more depolarized h.p. (‐40 mV) to inactivate the other two types of calcium currents. 3. Component T can be seen in isolation with weak test pulses. It begins activating at potentials more positive than ‐70 mV and inactivates quickly and completely during a maintained depolarization (time constant, tau approximately 20‐50 ms). The current amplitude and the rate of decay increase with stronger depolarizations until both reach a maximum at approximately ‐40 mV. Inactivation is complete at h.p. greater than ‐60 mV and is progressively removed between ‐60 and ‐95 mV. 4. Component N activates at relatively strong depolarizations (t.p. greater than ‐20 mV) and decays with time constants ranging from 50 to 110 ms. Inactivation is removed over a very broad range of holding potentials (h.p. between ‐40 and ‐110 mV). 5. With 10 mM‐EGTA in the pipette solution, substitution of Ba2+ for Ca2+ as the charge carrier does not alter the rates of activation or relaxation of any component. However, T‐type channels are approximately equally permeable to Ca2+ and Ba2+, while L‐type and N‐type channels are both much more permeable to Ba2+. 6. Component N cannot be explained by current‐dependent inactivation of L current resulting from recruitment of extra L‐type channels at negative holding potentials: raising the external Ba2+ concentration to 110 mM greatly increases the amplitude of L current evoked from h.p. = ‐30 mV but produces little inactivation. 7. Cadmium ions (20‐50 microM) virtually eliminate both N and L currents (greater than 90% block) but leave T relatively unaffected (less than 50% block). 200 microM‐Cd2+ blocks all three components. 8. Nickel ions (100 microM) strongly reduce T current but leave N and L current little changed. 9. The dihydropyridine antagonist nifedipine (10 microM) inhibits L current (approximately 60% block) at a holding potential that inactivates half the L‐type channels.(ABSTRACT TRUNCATED AT 400 WORDS)

CREB Phosphorylation and Dephosphorylation: A Ca2+- and Stimulus Duration–Dependent Switch for Hippocampal Gene Expression

While changes in gene expression are critical for many brain functions, including long-term memory, little is known about the cellular processes that mediate stimulus-transcription coupling at central synapses. In studying the signaling pathways by which synaptic inputs control the phosphorylation state of cyclic AMP-responsive element binding protein (CREB) and determine expression of CRE-regulated genes, we found two important Ca2+/calmodulin (CaM)-regulated mechanisms in hippocampal neurons: a CaM kinase cascade involving nuclear CaMKIV and a calcineurin-dependent regulation of nuclear protein phosphatase 1 activity. Prolongation of the synaptic input on the time scale of minutes, in part by an activity-induced inactivation of calcineurin, greatly extends the period over which phospho-CREB levels are elevated, thus affecting induction of downstream genes.

Calmodulin supports both inactivation and facilitation of L-type calcium channels

L-type Ca2+ channels support Ca2+ entry into cells, which triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that regulate themselves through their own activity. Among the many types of voltage-gated Ca2+ channel, L-type Ca2+ channels particularly display inactivation and facilitation, both of which are closely linked to the earlier entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensivebiophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif, and a nearby consensus calmodulin-binding isoleucine-glutamine (‘IQ’) motif, in the carboxy terminus of the α1C channel subunit have been implicated,. Here we show that calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and that the nature of the modulatory effect depends on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removed Ca2+-dependent inactivation and unmasked a strong facilitation; conversion of the same residue to glutamate eliminated both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation.

Signaling from Synapse to Nucleus: Postsynaptic CREB Phosphorylation during Multiple Forms of Hippocampal Synaptic Plasticity

Phosphorylation of the transcription factor CREB is thought to be important in processes underlying long-term memory. It is unclear whether CREB phosphorylation can carry information about the sign of changes in synaptic strength, whether CREB pathways are equally activated in neurons receiving or providing synaptic input, or how synapse-to-nucleus communication is mediated. We found that Ca(2+)-dependent nuclear CREB phosphorylation was rapidly evoked by synaptic stimuli including, but not limited to, those that induced potentiation and depression of synaptic strength. In striking contrast, high frequency action potential firing alone failed to trigger CREB phosphorylation. Activation of a submembranous Ca2+ sensor, just beneath sites of Ca2+ entry, appears critical for triggering nuclear CREB phosphorylation via calmodulin and a Ca2+/calmodulin-dependent protein kinase.

MicroRNA-mediated conversion of human fibroblasts to neurons

Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.

Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons

Activation of the transcription factor CREB is thought to be important in the formation of long-term memory in several animal species. The phosphorylation of a serine residue at position 133 of CREB is critical for activation of CREB. This phosphorylation is rapid when driven by brief synaptic activity in hippocampal neurons. It is initiated by a highly local, rise in calcium ion concentration near the cell membrane, but culminates in the activation of a specific calmodulin-dependent kinase known as CaMK IV (ref. 7), which is constitutively present in the neuronal nucleus,. It is unclear how the signal is conveyed from the synapse to the nucleus. We show here that brief bursts of activity cause a swift (∼1 min) translocation of calmodulin from the cytoplasm to the nucleus, and that this translocation is important for the rapid phosphorylation of CREB. Certain Ca2+ entry systems (L-type Ca2+ channels and NMDA receptors) are able to cause mobilization of calmodulin, whereas others (N- and P/Q-type Ca2+ channels) are not. This translocation of calmodulin provides a form of cellular communication that combines the specificity of local Ca2+ signalling with the ability to produce action at a distance.

Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons

This paper provides a brief overview of the diversity of voltage-gated Ca2+ channels and our recent work on neuronal Ca2+ channels with novel pharmacological and biophysical properties that distinguish them from L, N, P or T-type channels. The Ca2+ channel alpha 1 subunit known as alpha 1A or BI [Mori Y., Friedrich T., Kim M.-S., Mikami A., Nakai J., Ruth P., Bosse E., Hofmann F., Flockerzi V., Furuichi T., Mikoshiba K., Imoto K., Tanabe T. and Numa S. (1991) Nature 350, 398-402] is generally assumed to encode the P-type Ca2+ channel. However, we find that alpha 1A expressed in Xenopus oocytes differs from P-type channels in its kinetics of inactivation and its degree of sensitivity to block by the peptide toxins omega-Aga-IVA and omega-CTx-MVIIC [Sather W. A., Tanabe T., Zhang J.-F., Mori Y., Adams M. E. and Tsien R. W. (1993) Neuron 11, 291-303]. Thus, alpha 1A is capable of generating a Ca2+ channel with characteristics quite distinct from P-type channels. Doe-1, recently cloned from the forebrain of a marine ray, is another alpha 1 subunit which exemplifies a different branch of the Ca2+ channel family tree [Horne W. A., Ellinor P. T., Inman I., Zhou M., Tsien R. W. and Schwarz T. L. (1993) Proc. Natn. Acad. Sci. U.S.A. 90, 3787-3791]. When expressed in Xenopus oocytes, doe-1 forms a high voltage-activated (HVA) Ca2+ channel [Ellinor P. T., Zhang J.-F., Randall A. D., Zhou M., Schwarz T. L., Tsien R. W. and Horne W. (1993) Nature 363, 455-458]. It inactivates more rapidly than any previously expressed calcium channel and is not blocked by dihydropyridine antagonists or omega-Aga-IVA. Doe-1 current is reduced by omega-CTx-GVIA, but the inhibition is readily reversible and requires micromolar toxin, in contrast to this toxins potent and irreversible block of N-type channels. Doe-1 shows considerable sensitivity to block by Ni2+ or Cd2+. We have identified components of Ca2+ channel current in rat cerebellar granule neurons with kinetic and pharmacological features similar to alpha 1A and doe-1 in oocytes [Randall A. D., Wendland B., Schweizer F., Miljanich G., Adams M. E. and Tsien R. W. (1993) Soc. Neurosci. Abstr. 19, 1478]. The doe-1-like component (R-type current) inactivates much more quickly than L, N or P-type channels, and also differs significantly in its pharmacology.(ABSTRACT TRUNCATED AT 400 WORDS)

Letter to the EditorNomenclature of Voltage-Gated Calcium Channels

As new Ca 2ϩ channel genes are cloned, it is apparent that these two alphabetical nomenclatures will overlap at ␣ 1L , which may not mediate an L-type Ca 2ϩ current and Voltage-gated Ca 2ϩ channels mediate calcium influx in therefore may create confusion. Moreover, the present response to membrane depolarization and regulate in-alphabetical nomenclature does not reveal the structural tracellular processes such as contraction, secretion, relationships among the ␣ 1 subunits, which can be neurotransmission, and gene expression. They are mem-grouped into three families: (1) ␣ 1S , ␣ 1C , ␣ 1D , and ␣ 1F ; (2) bers of a gene superfamily of transmembrane ion chan-The complete nel proteins that includes voltage-gated K ϩ and Na ϩ amino acid sequences of these ␣ 1 subunits are more channels. The Ca 2ϩ channels that have been character-than 70% identical within a family but less than 40% ized biochemically are complex proteins composed of identical among families. These family relationships are four or five distinct subunits, which are encoded by illustrated for the more conserved transmembrane and multiple genes. The ␣ 1 subunit of 190–250 kDa is the pore domains in Figure 1. Division of calcium channels largest subunit, and it incorporates the conduction pore, into these three families is phylogenetically ancient, as the voltage sensor and gating apparatus, and the known representatives of each are found in the C. elegans ge-sites of channel regulation by second messengers, nome. Ideally, a nomenclature for Ca 2ϩ channel ␣ 1 sub-drugs, and toxins. An intracellular ␤ subunit and a trans-units should provide a systematic organization based on membrane, disulfide-linked ␣ 2 ␦ subunit complex are their structural relationships and should be coordinated components of most types of Ca 2ϩ channels. A ␥ subunit with nomenclatures for the other families of voltage-has also been found in skeletal muscle Ca 2ϩ channels, gated ion channels of different ionic selectivities (ie., K ϩ and related subunits are expressed in heart and brain. and Na ϩ). Although these auxiliary subunits modulate the proper-For these reasons, we wish to propose a new nomen-ties of the channel complex, the pharmacological and clature of voltage-gated Ca 2ϩ channels (Table 1), which electrophysiological diversity of Ca 2ϩ channels arises is more systematic and mimics the well-defined K ϩ primarily from the existence of multiple forms of ␣ 1 sub-channel nomenclature (Chandy et al., 1991). This no-units. Mammalian ␣ 1 …