Research Interests: Algorithms, Chemistry, Calcium, Medicine, Quality Control, and 15 moreCell Culture, Cell line, Humans, High throughput screening, Temperature, Reproducibility of Results, Potassium Channels, Membrane Potential, Fluorometry, Extracellular, Patch Clamp, Calcium Channel Blockers, Intracellular, Pharmacology and pharmaceutical sciences, and coloring agents
Research Interests: Biophysics, Electrophysiology, Biology, Biological Chemistry, Medicine, and 14 morePatch-clamp and imaging techniques, Biological Sciences, Cell line, Humans, Sequence alignment, Mutation, Biological, CHEMICAL SCIENCES, Protein Secondary Structure Prediction, Gating, Amino Acid Sequence, Molecular Sequence Data, conserved sequence , and Medical and Health Sciences
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Research Interests: Biophysics, Chemistry, Electrophysiology, Protein Folding, Medicine, and 15 moreMultidisciplinary, Patch-clamp and imaging techniques, Humans, Mutagenesis, Complementation, Cell Polarity, Research article, Calcium Channel, PLoS one, Homeostasis, Gating, Amino Acid Profile, Amino Acid Sequence, Linker, and Protein subunits
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It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after... more
It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindling protocol is robust in that it reliably leads to epilepsy in most VGAT-Cre mice, providing a new model to test for novel antiepileptogenic drugs.
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T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca2+ channels, CACNA1H, have been found in association with various forms... more
T-type calcium channels play essential roles in regulating neuronal excitability and network oscillations in the brain. Mutations in the gene encoding Cav3.2 T-type Ca2+ channels, CACNA1H, have been found in association with various forms of idiopathic generalized epilepsy. We and others have found that these mutations may influence neuronal excitability either by altering the biophysical properties of the channels or by increasing their surface expression. The goals of the present study were to investigate the excitability of neurons expressing Cav3.2 with the epilepsy mutation, C456S, and to elucidate the mechanisms by which it influences neuronal properties. We found that expression of the recombinant C456S channels substantially increased the excitability of cultured neurons by increasing the spontaneous firing rate and reducing the threshold for rebound burst firing. Additionally, we found that molecular determinants in the I–II loop (the region in which most childhood absence epilepsy-associated mutations are found) substantially increase the surface expression of T-channels but do not alter the relative distribution of channels into dendrites of cultured hippocampal neurons. Finally, we discovered that expression of C456S channels promoted dendritic growth and arborization. These effects were reversed to normal by either the absence epilepsy drug ethosuximide or a novel T-channel blocker, TTA-P2. As Ca2+-regulated transcription factors also increase dendritic development, we tested a transactivator trap assay and found that the C456S variant can induce changes in gene transcription. Taken together, our findings suggest that gain-of-function mutations in Cav3.2 T-type Ca2+ channels increase seizure susceptibility by directly altering neuronal electrical properties and indirectly by changing gene expression.
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By means of patch-clamp technique we examined changes in volume-regulated chloride current (ICl,swell) at neuroendocrine differentiation of androgen-dependent LNCaP prostate cancer cells. In those cells with neuroendocrine differentiation... more
By means of patch-clamp technique we examined changes in volume-regulated chloride current (ICl,swell) at neuroendocrine differentiation of androgen-dependent LNCaP prostate cancer cells. In those cells with neuroendocrine differentiation resulted from an increase in the intracellular cAMP, ICl,swell became much faster in response to applying external hypotonic solution and cell swelling. Changes in final rectification and voltage-dependent inactivation were not detected, as compared to the control cells. The differentiation also diminished ICl,swell blockade by Ca2+ transported via store-operated channels (SOC). On the base of our data we suggest that potentiation of the current at neuroendocrine differentiation, at least in part, resulted from a decrease in an inhibitory effect of Ca2+, transported into a cell through SOC, on volume-sensitive chloride current. Accelerated current in those cells might be induced by cytoskeleton rearrangement at the neuron-like growth.
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By means of the patch-clamp technique we have studied the effects of intracellular applied trypsin, a known modulator of membrane channel function, on the properties of the Cl- current induced by hypotonicity-obliged cell swelling (ICl,... more
By means of the patch-clamp technique we have studied the effects of intracellular applied trypsin, a known modulator of membrane channel function, on the properties of the Cl- current induced by hypotonicity-obliged cell swelling (ICl, swell) in human prostate cancer epithelial cells, LNCaP. Intracellular infusion of 1 mg/ml of trypsin into LNCaP cells via the patch pipette shortened the delay for the onset and the time of development of ICl, swell in response to hypotonicity as well as accelerated the rate of current diminution following the return to isotonic conditions. The maximal density of ICl, swell in the presence of intracellular trypsin was 2-fold higher while the current voltage-dependent inactivation at high depolarizing potentials was virtually eliminated. Intracellular co-application of the trypsin inhibitor together with trypsin abolished all effects of trypsin. We conclude that VRACs share a great degree of functional and structural homology to voltage-gated Na+, K+...
Research Interests: Biophysics, Chemistry, Electrophysiology, Medicine, Humans, and 6 morePubmed, Male, Trypsin, LNCaP, Intracellular, and Prostatic neoplasms
Research Interests: Physiology, Biophysics, Chemistry, Neurotransmission, Medicine, and 15 moreHumans, Copper, Animals, Medical Physiology, Gating, Glycine, Amino Acid Sequence, Membrane Potential, Amino Acid Substitution Rates, Glutamic Acid, General Physiology, Glutamate Receptor, Molecular Sequence Data, binding sites, and In Vitro Techniques
revealed 12 nonsynonymous single nucleotide polymor-phisms (SNPs) that were found only in childhood absenceepilepsy (CAE) patients. One SNP, G773D, was found intwo patients. The present study reports the finding of athird patient with this... more
revealed 12 nonsynonymous single nucleotide polymor-phisms (SNPs) that were found only in childhood absenceepilepsy (CAE) patients. One SNP, G773D, was found intwo patients. The present study reports the finding of athird patient with this SNP, as well as analysis of theirparents. Because of the role of T-channels in determin-ing the intrinsic firing patterns of neurons involved in ab-sence seizures, it was suggested that these SNPs mightalterchannelfunction.Thegoalofthepresentstudywastotest this hypothesis by introducing these polymorphismsinto a human Ca
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Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Cav2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically... more
Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Cav2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Cav2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1–IS2 (H111) and IS2–IS3 (H179 and H183) loops potentiates Cav2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Cav2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the “ON” position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals...
Research Interests: Physiology, Biophysics, Chemistry, Neurotransmission, Medicine, and 15 moreHumans, Copper, Animals, Medical Physiology, Rats, Gating, Glycine, Amino Acid Sequence, Amino Acid Substitution Rates, Glutamic Acid, General Physiology, Glutamate Receptor, Molecular Sequence Data, binding sites, and In Vitro Techniques
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Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that “inhibiting inhibitory neurons” was sufficient to induce seizures, it was discovered... more
Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that “inhibiting inhibitory neurons” was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT‐Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism.
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It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after... more
It was discovered that electrical kindling of VGAT-Cre mice led to the spontaneous motor and electrographic seizures. A recent paper focused on how unique VGAT-Cre mice were used in developing spontaneous recurring seizures (SRS) after kindling and a likely mechanism - insertion of Cre into the VGAT gene - disrupted its expression and reduced GABAergic tone. The present study extends these observations to a larger cohort of mice, focusing on key issues such as how long the SRS continues after kindling and the effect of the animal's sex and age. This report describes the protocols for the following key steps: making headsets with hippocampal depth electrodes for electrical stimulation and for reading the electroencephalogram; surgery to affix the headset securely on the mouse's skull so that it does not fall off; and key details of the electrical kindling protocol such as duration of the pulse, frequency of train, duration of train, and amount of current injected. The kindlin...
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Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia (DRG) that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4... more
Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia (DRG) that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo, and that TSP4/Cavα2δ1 dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1 dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory-post-synaptic-currents in second order neurons, as well as increased VGlut2 and PSD95 positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1 dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1. Importantly, TSP4/Cavα2δ1 dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1 dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated pre-synaptic excitatory input, evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.
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Molecular diversity of T-type/Cav3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I–II linker in gating and surface expression of Cav3 channels, we... more
Molecular diversity of T-type/Cav3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I–II linker in gating and surface expression of Cav3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Δ8b variant revealed enhanced current density compared to Cav3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Δ8b channels at the plasma membrane. We conclude that alternative splicing of Cav3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased.