Molecular characterization of a two-domain form of the neuronal voltage-gated P/Q-type calcium channel alpha(1)2.1 subunit

We characterized the neuronal two-domain (95kD-alpha(1)2.1) form of the alpha(1)2.1 subunit of the voltage-gated calcium channels using genetic and molecular analysis. The 95kD-alpha(1)2.1 is absent in neuronal preparations from CACNA1A null mouse demonstrating that alpha(1)2.1 and 95kD-alpha(1)2.1 arise from the same gene. A recombinant two-domain form (alpha(1AI-II)) of alpha(1)2.1 associates with the beta subunit and is trafficked to the plasma membrane. Translocation of the alpha(1AI-II) to the plasma membrane requires association with the beta subunit, since a mutation in the alpha(1AI-II) that inhibits beta subunit association reduces membrane trafficking. Though the alpha(1AI-II) protein does not conduct any voltage-gated currents, we have previously shown that it generates a high density of non-linear charge movements [Ahern et al., Proc. Natl. Acad. Sci. USA 98 (2001) 6935-6940]. In this study, we demonstrate that co-expression of the alpha(1AI-II) significantly reduces the current amplitude of alpha(1)2.1/beta(1a)/alpha(2)delta channels, via competition for the beta subunit. Taken together, our results demonstrate a dual functional role for the alpha(1AI-II) protein, both as a voltage sensor and modulator of P/Q-type currents in recombinant systems. These studies suggest an in vivo role for the 95kD-alpha(1)2.1 in altering synaptic activity via protein-protein interactions and/or regulation of P/Q-type currents.     

Genetic and developmental characterization of Dmca1D,a calcium channel alpha1 subunit gene in Drosophila melanogaster.

To begin unraveling the functional significance of calcium channel diversity, we identified mutations in Dmca1D, a Drosophila calcium channel alpha1 subunit cDNA that we recently cloned. These mutations constitute the l(2)35Fa lethal locus, which we rename Dmca1D. A severe allele, Dmca1D(X10), truncates the channel after the IV-S4 transmembrane domain. These mutants die as late embryos because they lack vigorous hatching movements. In the weaker allele, Dmca1D(AR66), a cysteine in transmembrane domain I-S1 is changed to tyrosine. Dmca1D(AR66) embryos hatch but pharate adults have difficulty eclosing. Those that do eclose have difficulty in fluid-filling of the wings. These studies show that this member of the calcium channel alpha1 subunit gene family plays a nonredundant, vital role in larvae and adults.     

Ca2+]i regulates trafficking of Cav1.3 (alpha1D Ca2+ channel) in insulin-secreting cells

Chronic exposure of pancreatic -cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of -cell basal intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We have investigated the relationship between the basal intracellular free Ca²⁺ and the L-type (Ca_v1.3) Ca²⁺ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca²⁺ influx, which was attenuated when the basal [Ca²⁺]_i was elevated. Using voltage-clamp techniques, we found that changing [Ca²⁺]_i could regulate the amplitude of the Ca²⁺ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Ca_v1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca²⁺]_i conditions by deconvolution microscopy. The [Ca²⁺]_i-dependent translocation of Ca_v1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca²⁺]_i. These results suggest that Ca_v1.3 channel trafficking is involved in glucose desensitization of pancreatic -cells. internalization; intracellular free calcium; glucose desensitization     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Functional properties of a new voltage-dependent calcium channel alpha(2)delta auxiliary subunit gene (CACNA2D2)

We have positionally cloned and characterized a new calcium channel auxiliary subunit, alpha(2)delta-2 (CACNA2D2), which shares 56% amino acid identity with the known alpha(2)delta-1 subunit. The gene maps to the critical human tumor suppressor gene region in chromosome 3p21.3, showing very frequent allele loss and occasional homozygous deletions in lung, breast, and other cancers. The tissue distribution of alpha(2)delta-2 expression is different from alpha(2)delta-1, and alpha(2)delta-2 mRNA is most abundantly expressed in lung and testis and well expressed in brain, heart, and pancreas. In contrast, alpha(2)delta-1 is expressed predominantly in brain, heart, and skeletal muscle. When co-expressed (via cRNA injections) with alpha(1B) and beta(3) subunits in Xenopus oocytes, alpha(2)delta-2 increased peak size of the N-type Ca(2+) currents 9-fold, and when co-expressed with alpha(1C) or alpha(1G) subunits in Xenopus oocytes increased peak size of L-type channels 2-fold and T-type channels 1.8-fold, respectively. Anti-peptide antibodies detect the expression of a 129-kDa alpha(2)delta-2 polypeptide in some but not all lung tumor cells. We conclude that the alpha(2)delta-2 gene encodes a functional auxiliary subunit of voltage-gated Ca(2+) channels. Because of its chromosomal location and expression patterns, CACNA2D2 needs to be explored as a potential tumor suppressor gene linking Ca(2+) signaling and lung, breast, and other cancer pathogenesis. The homologous location on mouse chromosome 9 is also the site of the mouse neurologic mutant ducky (du), and thus, CACNA2D2 is also a candidate gene for this inherited idiopathic generalized epilepsy syndrome.     

straightjacket is required for the synaptic stabilization of cacophony, a voltage-gated calcium channel alpha1 subunit

In a screen to identify genes involved in synaptic function, we isolated mutations in Drosophila melanogaster straightjacket (stj), an alpha(2)delta subunit of the voltage-gated calcium channel. stj mutant photoreceptors develop normal synaptic connections but display reduced "on-off" transients in electroretinogram recordings, indicating a failure to evoke postsynaptic responses and, thus, a defect in neurotransmission. stj is expressed in neurons but excluded from glia. Mutants exhibit endogenous seizure-like activity, indicating altered neuronal excitability. However, at the synaptic level, stj larval neuromuscular junctions exhibit approximately fourfold reduction in synaptic release compared with controls stemming from a reduced release probability at these synapses. These defects likely stem from destabilization of Cacophony (Cac), the primary presynaptic alpha(1) subunit in D. melanogaster. Interestingly, neuronal overexpression of cac partially rescues the viability and physiological defects in stj mutants, indicating a role for the alpha(2)delta Ca(2+) channel subunit in mediating the proper localization of an alpha(1) subunit at synapses.     

Biochemical properties and subcellular distribution of an N-type calcium channel alpha 1 subunit.

A site-directed anti-peptide antibody, CNB-1, that recognizes the alpha 1 subunit of rat brain class B calcium channels (rbB) immunoprecipitated 43% of the N-type calcium channels labeled by [125I]omega-conotoxin. CNB-1 recognized proteins of 240 and 210 kd, suggesting the presence of two size forms of this alpha 1 subunit. Calcium channels recognized by CNB-1 were localized predominantly in dendrites; both dendritic shafts and punctate synaptic structures upon the dendrites were labeled. The large terminals of the mossy fibers of the dentate gyrus granule neurons were heavily labeled, suggesting that the punctate labeling pattern represents calcium channels in nerve terminals. The pattern of immunostaining was cell specific. The cell bodies of some pyramidal cells in layers II, III, and V of the dorsal cortex, Purkinje cells, and scattered cell bodies elsewhere in the brain were also labeled at a low level. The results define complementary distributions of N- and L-type calcium channels in dendrites, nerve terminals, and cell bodies of most central neurons and support distinct functional roles in calcium-dependent electrical activity, intracellular calcium regulation, and neurotransmitter release for these two channel types.     

Expression and modulation of an invertebrate presynaptic calcium channel alpha1 subunit homolog

Here we report the first assessment of the expression and modulation of an invertebrate alpha1 subunit homolog of mammalian presynaptic Cav2 calcium channels (N-type and P/Q-type) in mammalian cells. Our data show that molluscan channel (LCav2a) isolated from Lymnaea stagnalis is effectively membrane-targeted and electrophysiologically recordable in tsA-201 cells only when the first 44 amino acids of LCav2a are substituted for the corresponding region of rat Cav2.1. When coexpressed with rat accessory subunits, the biophysical properties of LCav2a-5'rbA resemble those of mammalian N-type calcium channels with respect to activation and inactivation, lack of pronounced calcium dependent inactivation, preferential permeation of barium ions, and cadmium block. Consistent with reports of native Lymnaea calcium currents, the LCav2a-5'rbA channel is insensitive to micromolar concentrations of omega-conotoxin GVIA and is not affected by nifedipine, thus confirming that it is not of the L-type. Interestingly, the LCav2a-5'rbA channel is almost completely and irreversibly inhibited by guanosine 5'-3-O-(thio)triphosphate but not regulated by syntaxin1, suggesting that invertebrate presynaptic calcium channels are differently modulated from their vertebrate counterparts.     

C-terminal fragments of the alpha 1C (CaV1.2) subunit associate with and regulate L-type calcium channels containing C-terminal-truncated alpha 1C subunits

L-type Ca(2+) channels in native tissues have been found to contain a pore-forming alpha(1) subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminal-truncated alpha(1C) (Ca(V)1.2) subunits, we performed electrophysiological and biochemical experiments. In tsA201 cells expressing either wild type or C-terminal-truncated alpha(1C) subunits in combination with a beta(2a) subunit, truncation of the alpha(1C) subunit by as little as 147 amino acids led to a 10-15-fold increase in currents compared with those obtained from control, full-length alpha(1C) subunits. Dialysis of cells expressing the truncated alpha(1C) subunits with C-terminal fragments applied through the patch pipette reconstituted the inhibition of the channels seen with full-length alpha(1C) subunits. In addition, C-terminal deletion mutants containing a tethered C terminus also exhibited the C-terminal-induced inhibition. Immunoprecipitation assays demonstrated the association of the C-terminal fragments with truncated alpha(1C) subunits. In addition, glutathione S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the C- terminal fragments of the alpha(1C) subunit can associate with C-terminal-truncated alpha(1C) subunits and inhibit the currents through L-type Ca(2+) channels.     

Cloning of a calcium channel alpha1 subunit from the reef-building coral, Stylophora pistillata

While the mechanisms of cellular Ca2+ entry associated with cell activation are well characterized, the pathway of continuous uptake of the large amount of Ca2+ needed in the biomineralization process remains largely unknown. Scleractinian corals are one of the major calcifying groups of organisms. Recent studies have suggested that a voltage-dependent Ca2+ channel is involved in the transepithelial transport of Ca2+ used for coral calcification. We report here the cloning and sequencing of a cDNA coding a coral alpha1 subunit Ca2+ channel. This channel is closely related to the L-type family found in vertebrates and invertebrates. Immunohistochemical analysis shows that this channel is present within the calicoblastic ectoderm, the site involved in calcium carbonate precipitation. These data and previous results provide molecular evidence that voltage-dependent Ca2+ channels are involved in calcification. Cnidarians are the most primitive organisms in which a Ca2+ channel has been cloned up to now; evolutionary perspectives on Ca2+ channel diversity are discussed.     

Functional properties and modulation of extracellular epitope - tagged CaV2.1 voltage-gated calcium channels

Depolarisation-induced Ca²⁺ influx into electrically excitable cells is determined by the density of voltage-gated Ca²⁺ channels at the cell surface. Surface expression is modulated by physiological stimuli as well as by drugs and can be altered under pathological conditions. Extracellular epitope tagging of channel subunits allows to quantify their surface expression and to distinguish surface channels from those in intracellular compartments. Here we report the first systematic characterisation of extracellularly epitope tagged Ca_V2.1 channels. We identified a permissive region in the pore-loop of repeat IV within the Ca_V2.1 α1 subunit which allowed integration of several different tags (hemagluttinine [HA], double HA; 6-histidine tag [His], 9-His, bungarotoxin-binding site) without compromising α1 subunit protein expression (in transfected tsA-201 cells) and function (after expression in X. laevis oocytes). Immunofluorescent studies revealed that the double-HA tagged construct (1722-HAGHA) was targeted to presynaptic sites in transfected cultured hippocampal neurons as expected for Ca_V2.1 channels. We also demonstrate that introduction of tags into this permissive position creates artifical sites for channel modulation. This was demonstrated by partial inhibition of 1722-HA channel currents with anti-HA antibodies and the concentration-dependent stimulation or partial inhibition by Ni-nitrilo triacetic acid (NTA) and novel bulkier derivatives (Ni-trisNTA, Ni-tetrakisNTA, Ni-nitro-o-phenyl-bisNTA, Ni-nitro-p-phenyl-bisNTA). Therefore our data also provide evidence for the concept that artificial modulatory sites for small ligands can be introduced into voltage-gated Ca²⁺ channel for their selective modulation.     

Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca(v)alpha1

Alternative splicing is a critical mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes. This review focuses on recent studies of voltage-gated calcium (Ca) channel Ca_vα₁ subunit splice isoforms in neurons. Voltage-gated Ca channels couple changes in neuronal activity to rapid changes in intracellular Ca levels that in turn regulate an astounding range of cellular processes. Only ten genes have been identified that encode Ca_vα₁ subunits, an insufficient number to account for the level of functional diversity among voltage-gated Ca channels. The consequences of regulated alternative splicing among the genes that comprise voltage-gated Ca channels permits specialization of channel function, optimizing Ca signaling in different regions of the brain and in different cellular compartments. Although the full extent of alternative splicing is not yet known for any of the major subtypes of voltage-gated Ca channels, it is already clear that it adds a rich layer of structural and functional diversity”.     

Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line

The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.     

Cloning and expression study of the mouse tetrodotoxin-resistant voltage-gated sodium channel alpha subunit NaT/Scn11a

We have cloned a tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel alpha subunit from a mouse cDNA library and designated it as NaT. It encodes 1765 amino acid residues and is virtually identical to that of Scn11a, which has been reported recently, except for 40 nt and 14 aa substitutions. The amino acid identity of NaT/Scn11a with rat NaN/SNS2 is 88%. NaT/Scn11a was mapped to mouse chromosome 9F3-F4 by fluorescence in situ hybridization (FISH). While rat NaN/SNS2 has been reported to be expressed specifically in the peripheral sensory neurons, NaT/Scn11a is expressed not only in the peripheral sensory neurons but also in the spinal cord, uterus, testis, ovary, placenta, and small intestine. NaT is detectable in mouse embryos 15 days postcoitus (p.c.), around the phase of organogenesis and gonadal differentiation. These findings demonstrate a unique distribution of NaT/Scn11a and suggest some of its roles in the above-mentioned processes.     

Cloning of the beta(2a) subunit of the voltage-dependent calcium channel from human heart: cooperative effect of alpha(2)/delta and beta(2a) on the membrane expression of the alpha(1C) subunit

Expression and membrane localization of an epitope-tagged human Ca(2+) channel alpha(1C) subunit were monitored in Xenopus oocytes by confocal microscopy and electrophysiological recording. When alpha(2)/delta and beta(2a) were separately coexpressed with the alpha(1C) subunit, assessment by confocal microscopy showed an 86 and 225% increase of the channel density, respectively. Simultaneous coexpression of alpha(2)/delta and beta(2a) subunits resulted in a cooperative (470%) increase. Electrophysiological measurements performed in parallel revealed that the current augmentation by the alpha(2)/delta subunit is totally attributable to an increase in channel density, whereas the beta(2a) subunit, in addition to increasing channel density, also facilitates channel opening.