Genome Sequencing of Sub-Arctic Mesomycetozoean Sphaeroforma sirkka Strain B5, Performed with the Oxford Nanopore minION and Illumina HiSeq Systems.

The Mesomycetozoea branch near the animal-fungal divergence and are believed to be important to understanding the origins of multicellularity. In 2012, a free-living saprotrophic mesomycetozoean was isolated from the sub-Arctic Bering Sea. A hybrid assembly using Illumina and Nanopore sequences yielded 2,688 contigs with a total length of 125,635,304 bases.

Differential cerebellar GABAA receptor expression in mice with mutations in CaV2.1 (P/Q-type) calcium channels.

Ataxia is the predominant clinical manifestation of cerebellar dysfunction. Mutations in the human CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels, underlie several neurological disorders, including Episodic Ataxia type 2 and Familial Hemiplegic Migraine type 1 (FHM1). Several mouse mutants exist that harbor mutations in the orthologous Cacna1a gene. The spontaneous Cacna1a mutants Rolling Nagoya (tg(rol)), Tottering (tg) and Leaner (tg(ln)) mice exhibit behavioral motor phenotypes, including ataxia. Transgenic knock-in (KI) mouse strains with the human FHM1 R192Q and S218L missense mutations have been generated. R192Q KI mice are non-ataxic, whereas S218L KI mice display a complex behavioral phenotype that includes cerebellar ataxia. Given the dependence of γ-aminobutyric acid type A (GABAA) receptor subunit functioning on localized calcium currents, and the functional link between GABAergic inhibition and ataxia, we hypothesized that cerebellar GABAA receptor expression is differentially affected in Cacna1a mutants and contributes to the ataxic phenotype. Herein we quantified functional GABAA receptors and pharmacologically dissociated cerebellar GABAA receptors in several Cacna1a mutants. We did not identify differences in the expression of GABAA receptor subunits or in the number of functional GABAA receptors in the non-ataxic R192Q KI strain. In contrast, tg(rol) mice had a ∼15% decrease in the number of functional GABAA receptors, whereas S218L KI mice showed a ∼29% increase. Our data suggest that differential changes in cerebellar GABAA receptor expression profile may contribute to the neurological phenotype of cerebellar ataxia and that targeting GABAA receptors might represent a feasible complementary strategy to treat cerebellar ataxia.

The transient receptor potential channel antagonist SKF96365 is a potent blocker of low-voltage-activated T-type calcium channels.

BACKGROUND AND PURPOSE: SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents. EXPERIMENTAL APPROACH: Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed. KEY RESULTS: SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca(V)1, Ca(V)2 and Ca(V)3) at concentrations typically utilized to assay TRPC function (10 microM). Particularly, human Ca(V)3.1 T-type Ca channels were more potently inhibited by SKF (IC(50) approximately 560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca(V)3.1 T-type currents in a rat cerebellar PC slice preparation. CONCLUSIONS AND IMPLICATIONS: SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues.

Leftward shift in the voltage-dependence for Ca2+ currents activation induced by a new toxin from Phoneutria reidyi (Aranae, Ctenidae) venom.

Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an alpha-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 microg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 microM inhibited by 20% the KCl-induced increase on [Ca2+]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (-4 mV), P/Q (-8 mV) and R (-5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents.

Calcium channelopathies: voltage-gated calcium channels.

Since the initial identification of native calcium currents, significant progress has been made towards our understanding of the molecular and cellular contributions of voltage-gated calcium channels in multiple physiological processes. Moreover, we are beginning to comprehend their pathophysiological roles through both naturally occurring channelopathies in humans and mice and through targeted gene deletions. The data illustrate that small perturbations in voltage-gated calcium channel function induced by genetic alterations can affect a wide variety of mammalian developmental, physiological and behavioral functions. At least in those instances wherein the channelopathies can be attributed to gain-of-function mechanisms, the data point towards new therapeutic strategies for developing highly selective calcium channel antagonists.

Temperature dependence of T-type calcium channel gating.

T-type calcium channel isoforms are expressed in a multitude of tissues and have a key role in a variety of physiological processes. To fully appreciate the physiological role of distinct channel isoforms it is essential to determine their kinetic properties under physiologically relevant conditions. We therefore characterized the gating behavior of expressed rat voltage-dependent calcium channels (Ca(v)) 3.1, Ca(v)3.2, and Ca(v)3.3, as well as human Ca(v)3.3 at 21 degrees C and 37 degrees C in saline that approximates physiological conditions. Exposure to 37 degrees C caused significant increases in the rates of activation, inactivation, and recovery from inactivation, increased the current amplitudes, and induced a hyperpolarizing shift of half-activation for Ca(v)3.1 and Ca(v)3.2. At 37 degrees C the half-inactivation showed a hyperpolarizing shift for Ca(v)3.1 and Ca(v)3.2 and human Ca(v)3.3, but not rat Ca(v)3.3. The observed changes in the kinetics were significant but not identical for the three isoforms, showing that the ability of T-type channels to conduct calcium varies with both channel isoform and temperature.

Genetic heterogeneity in paroxysmal nonkinesigenic dyskinesia.

Paroxysmal nonkinesigenic dyskinesia (PNKD) is characterized by attacks of dystonia or chorea lasting minutes to hours. Recently, mutations in the myofibrillogenesis regulator 1 gene (MR-1) have been identified in 10 unrelated PNKD kindreds. The authors describe a Canadian PNKD family who does not have mutations in the MR-1 gene and links to a separate locus at 2q31. This indicates that there are at least two different genes responsible for PNKD.

Mutation analysis of the sodium/hydrogen exchanger gene (NHE5) in familial paroxysmal kinesigenic dyskinesia.

Familial Paroxysmal Kinesigenic Dyskinesia (PKD) is an autosomal dominant condition characterized by attacks of dystonia or chorea triggered by sudden movements. Recently two separate loci for PKD, Episodic Kinesigenic Dyskinesia 1 (EKD1) and Episodic Kinesigenic Dyskinesia 2 (EKD2), have been mapped to chromosome 16 but the causative genes have not been identified. The Na(+)/H(+) exchanger gene (NHE5) involved in regulating intracellular pH lies in the EKD2 region. The coding region of the NHE5 gene in familial PKD was sequenced. We did not identify any mutations in the exons, intron/exon boundaries or the 5' and 3'UTR. This excludes mutations in the coding region of the NHE5 gene as a cause for familial PKD, but does not rule out a possible role of sequence variants in introns or regulatory regions.

Voltage-dependent calcium channels--beyond dihydropyridine antagonists.

The blockade of L-type calcium channels by dihydropyridines, phenylalkylamines and benzothiazepines has been well described and forms the basis of a multibillion dollar market for the treatment of cardiovascular disease and migraine. More recently, neuron-specific calcium channels have become the subject of intense interest regarding their potential as therapeutic targets for the treatment of chronic and neuropathic pain. A number of recently described agents that selectively target neuronal calcium channels have been described and appear promising for a variety of pain conditions.

Residue Gly1326 of the N-type calcium channel alpha 1B subunit controls reversibility of omega-conotoxin GVIA and MVIIA block.

We recently reported that amino acid residues contained within a putative EF hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region partially overlaps with residues previously implicated in block of the channel by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF hand domain on channel block by omega-conotoxin GVIA and the structurally related omega-conotoxin MVIIA. Both of the toxins irreversibly block the activity of wild type alpha(1B) N-type channels. We find that in addition to previously identified amino acid residues, residues in positions 1326 and 1332 are important determinants of omega-conotoxin GVIA blockade. Substitution of residue Glu(1332) to arginine slows the time course of development of block. Point mutations in position Gly(1326) to either arginine, glutamic acid, or proline dramatically decrease the time constant for development of the block. Additionally, in the G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with omega-conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly(1326) may form a barrier, which controls the access of peptide toxins to their blocking site within the outer vestibule of the channel pore and also stabilizes the toxin-channel interaction.

Amino acid residues outside of the pore region contribute to N-type calcium channel permeation.

It is widely believed that the selectivity of voltage-dependent calcium channels is mainly controlled by amino acid residues contained within four p-loop motifs forming the pore of the channel. An examination of the amino acid sequences of high voltage-activated calcium channels reveals that their domain III S5-H5 regions contain a highly conserved motif with homology to known EF hand calcium binding proteins, hinting that this region may contribute to channel permeation. To test this hypothesis, we used site-directed mutagenesis to replace three conserved negatively charged residues in the N-type calcium channel alpha1B subunit (Glu-1321, Asp-1323, and Glu-1332) with positively charged amino acids (lysine and arginine) and studied their effect on ion selectivity using whole cell and single channel patch clamp recordings. Whereas the wild type channels conducted barium much more effectively than calcium, the mutant displayed nearly equal permeabilities for these two ions. Individual replacement of residue 1332 or a double substitution of residues 1321 and 1323 with lysine and arginine, respectively, were equally effective. Disruption of the putative EF hand motif through replacement of the central glycine residue (1326) with proline resulted in a similar effect, indicating that the responses observed with the triple mutant were not due to changes in the net charge of the channel. Overall, our data indicate that residues outside of the narrow region of the pore have the propensity to contribute to calcium channel permeation. They also raise the possibility that interactions of calcium ions with a putative calcium binding domain at the extracellular side of the channel may underlie the differential permeabilities of the channel for barium and calcium ions.

Molecular and functional characterization of a family of rat brain T-type calcium channels.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (alpha(1G), alpha(1H), and alpha(1I)). Northern blot and reverse transcriptase-polymerase chain reaction analyses show alpha(1G), alpha(1H), and alpha(1I) to be expressed throughout the newborn and juvenile rat brain. In contrast, while alpha(1G) and alpha(1H) mRNA are expressed in all regions in adult rat brain, alpha(1I) mRNA expression is restricted to the striatum. Expression of alpha(1G), alpha(1H), and alpha(1I) subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of alpha(1G), alpha(1H), and alpha(1I) under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of alpha(1G), alpha(1H), and alpha(1I) T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

Voltage-gated calcium channels direct neuronal migration in Caenorhabditis elegans.

Calcium signaling is known to be important for regulating the guidance of migrating neurons, yet the molecular mechanisms underlying this process are not well understood. We have found that two different voltage-gated calcium channels are important for the accurate guidance of postembryonic neuronal migrations in the nematode Caenorhabditis elegans. In mutants carrying loss-of-function alleles of the calcium channel gene unc-2, the touch receptor neuron AVM and the interneuron SDQR often migrated inappropriately, leading to misplacement of their cell bodies. However, the AVM neurons in unc-2 mutant animals extended axons in a wild-type pattern, suggesting that the UNC-2 calcium channel specifically directs migration of the neuronal cell body and is not required for axonal pathfinding. In contrast, mutations in egl-19, which affect a different voltage-gated calcium channel, affected the migration of the AVM and SDQR bodies, as well as the guidance of the AVM axon. Thus, cell migration and axonal pathfinding in the AVM neurons appear to involve distinct calcium channel subtypes. Mutants defective in the unc-43/CaM kinase gene showed a defect in SDQR and AVM positioning that resembled that of unc-2 mutants; thus, CaM kinase may function as an effector of the UNC-2-mediated calcium influx in guiding cell migration.

Determinants of voltage-dependent inactivation affect Mibefradil block of calcium channels.

The voltage gated calcium channel family is a major target for a range of therapeutic drugs. Mibefradil (Ro 40-5967) belongs to a new chemical class of these molecules which differs from other Ca2+ antagonists by its ability to potently block T-type Ca2+ channels. However, this molecule has also been shown to inhibit other Ca2+ channel subtypes. To further analyze the mechanism governing the Ca2+ channel-Mibefradil interaction, we examined the effect of Mibefradil on various recombinant Ca2+ channels expressed in mammalian cells from their cloned cDNAs, using Ca2+ as the permeant ion at physiological concentration. Expression of alpha1A, alpha1C, and alpha1E in tsA 201 cells resulted in Ca2+ currents with functional characteristics closely related to those of their native counterparts. Mibefradil blocked alpha1A and alpha1E with a Kd comparable to that reported for T-type channels, but had a lower affinity (approximately 30-fold) for alpha1C. For each channel, inhibition by Mibefradil was consistent with high-affinity binding to the inactivated state. Modulation of the voltage-dependent inactivation properties by the nature of the coexpressed beta subunit or the alpha1 splice variant altered block at the Mibefradil receptor site. Therefore, we conclude that the tissue and sub-cellular localization of calcium channel subunits as well as their specific associations are essential parameters to understand the in vivo effects of Mibefradil.

P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A.

Spatial and temporal changes in intracellular calcium concentrations are critical for controlling gene expression in neurons. In many neurons, activity-dependent calcium influx through L-type channels stimulates transcription that depends on the transcription factor CREB by activating a calmodulin-dependent pathway. Here we show that selective influx of calcium through P/Q-type channels is responsible for activating expression of syntaxin-1A, a presynaptic protein that mediates vesicle docking, fusion and neurotransmitter release. The initial P/Q-type calcium signal is amplified by release of calcium from intracellular stores and acts through phosphorylation that is dependent on the calmodulin-dependent kinase CaM K II/IV, protein kinase A and mitogen-activated protein kinase kinase. Initiation of syntaxin-1A expression is rapid and short-lived, with syntaxin-1A ultimately interacting with the P/Q-type calcium channel to decrease channel availability. Our results define an activity-dependent feedback pathway that may regulate synaptic efficacy and function in the nervous system.

alpha 1B N-type calcium channel isoforms with distinct biophysical properties.

N-type calcium channels both generate the initial calcium signal to trigger neurotransmitter release and also interact with synaptic release proteins at many mammalian central nervous system synapses. Two isoforms of the alpha 1B N-type channel from rat brain (alpha 1B-I and alpha 1B-II) were found to differ in four regions: (1) a glutamate (Glu) to glycine (Gly) substitution in domain I S3; (2) a Gly to Glu substitution in the domain I-II linker; (3) the insertion or deletion of an alanine (Ala) in the domain I-II linker; and (4) the presence or absence of serine/phenylalanine/methionine/glycine (SFMG) in the linker between domain III S3-S4. Comparison of the electrophysiological properties of the alpha 1B-I and alpha 1B-II N-type channels shows that they exhibit distinct kinetics as well as altered current-voltage relations. Utilizing chimeric alpha 1B-I and alpha 1B-II cDNAs, we show that: (1) the Glu 177 to Gly substitution in domain I S3 increases the rate of activation by approximately 15-fold; (2) the presence or absence of Ala 415 in the domain I-II linker alters current-voltage relations by approximately 10 mV but does not affect channel kinetics; (3) the substitution of Gly 387 to Glu in the domain I-II linker also has no effect on kinetics; and (4) the presence or absence of SFMG (1236-1239) in domain III S3-S4 did not significantly affect channel current-voltage relations, kinetics, or steady state inactivation. We conclude that molecularly distinct alpha 1B isoforms are expressed in rat brain and may account for some of the functional diversity of N-type currents in native cells.

Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

A new beta subtype-specific interaction in alpha1A subunit controls P/Q-type Ca2+ channel activation.

The cytoplasmic beta subunit of voltage-dependent calcium channels modulates channel properties in a subtype-specific manner and is important in channel targeting. A high affinity interaction site between the alpha1 interaction domain (AID) in the I-II cytoplasmic loop of alpha1 and the beta interaction domain (BID) of the beta subunit is highly conserved among subunit subtypes. We describe a new subtype-specific interaction (Ss1) between the amino-terminal cytoplasmic domain of alpha1A (BI-2) and the carboxyl terminus of beta4. Like the interaction identified previously () between the carboxyl termini of alpha1A and beta4 (Ss2), the affinity of this interaction is lower than AID-BID, suggesting that these are secondary interactions. Ss1 and Ss2 involve overlapping sites on beta4 and are competitive, but neither inhibits the interaction with AID. The interaction with the amino terminus of alpha1 is isoform-dependent, suggesting a role in the specificity of alpha1-beta pairing. Coexpression of beta4 in Xenopus oocytes produces a reduced hyperpolarizing shift in the I-V curve of the alpha1A channel compared with beta3 (not exhibiting this interaction). Replacing the amino terminus of alpha1A with that of alpha1C abolishes this difference. Our data contribute to our understanding of the molecular organization of calcium channels, providing a functional basis for variation in subunit composition of native P/Q-type channels.

Identification of an integration center for cross-talk between protein kinase C and G protein modulation of N-type calcium channels.

The modulation of presynaptic calcium channel activity by second messengers provides a fine tuning mechanism for neurotransmitter release. In neurons, the activation of certain G protein-coupled receptors reduces N-type channel activity by approximately 60%. In contrast, activation of protein kinase C (PKC) results in an approximately 50% increase in N-type channel activity, and subsequent G protein inhibition is antagonized. Here, we describe the molecular determinants that control the dual effects of PKC-dependent phosphorylation. The double substitution of two adjacent PKC consensus sites in the calcium channel domain I-II linker (Thr422, Ser425) to alanines abolished both PKC-dependent up-regulation and the PKC-G protein cross-talk. The single substitution of Ser425 to glutamic acid abolished PKC up-regulation but had no effect on G protein modulation. Replacement of Thr422 with glutamic acid eliminated PKC-dependent up-regulation and mimicked the effects of PKC phosphorylation on G protein inhibition. Our data suggest that Thr422 mediates the antagonistic effect of PKC on G protein modulation, while phosphorylation of either Thr422 or Ser425 are sufficient to increase N-type channel activity. Thus, Thr422 serves as a molecular switch by which PKC is able to simultaneously trigger the up-regulation of channel activity and antagonize G protein inhibition.