Michael Miller Harpold

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.

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 …

Functional Consequences of Mutations in the Human α1A Calcium Channel Subunit Linked to Familial Hemiplegic Migraine

Mutations in α1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human α1A-2subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

The use of male-specific chromosomal DNA fragments to determine the sex of bovine preimplantation embryos

The ability to determine the sex of embryos prior to transfer to recipients has commercial application in the embryo transfer industry. A number of methods of sex determination have been attempted; however, none of these has been commercially successful to date. The identification of repetitive, male-specific bovine chromosomal DNA fragments enables the use of DNA-probe technology in determining the sex of preimplantation embryos from a small embryonic sample. The identification and isolation of three repetitive, male-specific bovine chromosomal DNA fragments, and the application of these probes in a sensitive and highly accurate embryo sexing assay are reported.

Structure and Functional Characterization of a Novel Human Low‐Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage‐gated Ca2+ channel α1 subunit, α1H, from a human medullary thyroid carcinoma cell line. The α1H subunit is structurally similar to previously described α1 subunits. Northern blot analysis indicates that α1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing α1H activated at relatively hyperpolarized potentials (‐50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single‐channel conductance of ~9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 μM) and the T‐type channel antagonists mibefradil (~50% block at 1 μM) and amiloride (IC50 = 167 μM). Thus, α1H‐containing channels exhibit biophysical and pharmacological properties characteristic of low voltage‐activated, or T‐type, Ca2+ channels.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum. Within the cerebellar cortex, these pore forming proteins were found to have differential localisations when examined in adjacent sections. The alpha 1A and alpha 1B subunits broadly colocalised and were both present, though at apparently different levels, in the molecular, Purkinje and granule cell layers whilst alpha 1E was predominantly expressed in Purkinje cells. In the dentate nucleus, an area which has received little attention in previous studies, alpha 1A was highly expressed in regions in which Purkinje cell nerve terminals form synapses with deep cerebellar neurones.

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.

Comparative structure of human neuronal α2–α7 and β2–β4 nicotinic acetylcholine receptor subunits and functional expression of the α2, α3, α4, α7, β2, and β4 subunits

AbstractcDNA clones encoding human neuronal nicotinic acetylcholine receptor α2, α3, α4, α5, α6, α7, β2, β3, and β4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human α2 and α6 and full-length β3 and β4 clones have not been previously reported. Deduced amino acid sequences of the α2, α6, β3, and β4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (α2), 87 (α6), 89 (β3), and 84% (β4) identity to the corresponding rat sequences. The amino termini of the human α2 and β3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat α2 and β3. Recombinant receptors were expressed inXenopus laevis oocytes injected with in vitro transcripts encoding either α7 alone or α2, α3, or α4 in pairwise combination with β2 or β4. Inward currents were elicited by the application of acetylcholine (1–100 µM) and other agonists; these responses were blocked 65–97% by application of 10 µM d-tubocurare, confirming functional expression of human nicotinic receptors.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells

Abstract HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations α3β2 or α4β2. [ 3 H]-(±)Epibatidine ([ 3 H]-(±)EPI) bound to membranes from A3B2 (α3β2) and A4B2.2 (α4β2) cells with K d values of 7.5 and 33.4 pM and B max values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca 2+ concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-β-erythroidine (DHβE) in A3B2 cells and MEC=DHβE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in α3β2 and α4β2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca 2+ , respectively. Our results indicate that stably expressed α3β2 and α4β2 human nAChRs are pharmacologically and functionally distinct.

Distribution ofα1A, α1B andα1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

Abstract The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits,α 1A , α 1B andα 1E in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level,α 1A , α 1B andα 1E subunits were differentially localized. In general,α 1A -immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-a and pri-a cells. Theα 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. Theα 1E staining pattern shared features in common with bothα 1A andα 1B with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-α cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison ofα 1B andα 1E staining in the CA3 stratum lucidum with calbindin-immunoreactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.