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Lori L Isom - One of the best experts on this subject based on the ideXlab platform.
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Modulation of Kv1 Voltage-Gated Potassium Channels by Sodium Channel Beta Subunits
Biophysical Journal, 2012Co-Authors: Hai M. Nguyen, Lori L Isom, Jeffrey D. Calhoun, Alan L. Goldin, George K. ChandyAbstract:Sodium channel beta subunits (SCN1b-SCN4b) are integral members of voltage-gated sodium channel (VGSC)-complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, where they modulate the function of VGSCs. Mutations of these genes results in neurological (e.g. epilepsy) and cardiovascular (e.g. Brugada syndrome) diseases. Here we report that SCN1b modulates the Kv1-subfamily of K+ channels, each in a unique fashion, when co-expressed in Xenopus oocytes or mammalian cells (Table). SCN2B, but not SCN3b, has similar modulatory properties. Pull-down experiments show that SCN1b is physically coupled to Kv1 channels. Using chimeras of SCN1b and the myelin Po protein, we demonstrate that the external domain of SCN1b is essential for channel modulation. Two known epilepsy-causing mutations in the Ig-domain of SCN1b, R85C and C121W, disrupt Kv1 channel-modulation. Thus, sodium channel beta subunits may regulate action potential firing and propagation in normal and diseased conditions by modulating the function of both VGSCs and Kv channels.
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na channel β subunits overachievers of the ion channel family
Frontiers in Pharmacology, 2011Co-Authors: William J. Brackenbury, Lori L IsomAbstract:Voltage gated Na+ channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B-SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSC α subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs) and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na+ current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington’s disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independent of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.
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na channel scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo
Journal of Biological Chemistry, 2011Co-Authors: Luis F Lopezsantiago, William J. Brackenbury, Chunling Chen, Lori L IsomAbstract:Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ current (INa) mediated by voltage-gated Na+ channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by SCN2B, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S INa inactivation, reduced persistent TTX-R INa, a prolonged rate of recovery of TTX-R INa from inactivation, and reduced cell surface expression of Nav1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K+ current (Ito) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both INa and IK.
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Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo
The Journal of biological chemistry, 2011Co-Authors: Luis F. Lopez-santiago, Chunling Chen, William J. Brackenbury, Lori L IsomAbstract:Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ current (INa) mediated by voltage-gated Na+ channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by SCN2B, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S INa inactivation, reduced persistent TTX-R INa, a prolonged rate of recovery of TTX-R INa from inactivation, and reduced cell surface expression of Nav1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K+ current (Ito) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both INa and IK.
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Efficacy Loss of the Anticonvulsant Carbamazepine in Mice Lacking Sodium Channel β Subunits via Paradoxical Effects on Persistent Sodium Currents
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2010Co-Authors: Mischa Uebachs, Lori L Isom, Thoralf Opitz, Michel Royeck, Gesa Dickhof, Marie Therese Horstmann, Heinz BeckAbstract:Neuronal excitability is critically determined by the properties of voltage-gated Na + currents. Fast transient Na + currents ( I NaT ) mediate the fast upstroke of action potentials, whereas low-voltage-activated persistent Na + currents ( I NaP ) contribute to subthreshold excitation. Na + channels are composed of a pore-forming α subunit and β subunits, which modify the biophysical properties of α subunits. We have examined the idea that the presence of β subunits also modifies the pharmacological properties of the Na + channel complex using mice lacking either the β 1 ( Scn1b ) or β 2 ( SCN2B ) subunit. Classical effects of the anticonvulsant carbamazepine (CBZ), such as the use-dependent reduction of I NaT and effects on I NaT voltage dependence of inactivation, were unaltered in mice lacking β subunits. Surprisingly, CBZ induced a small but significant shift of the voltage dependence of activation of I NaT and I NaP to more hyperpolarized potentials. This novel CBZ effect on I NaP was strongly enhanced in Scn1b null mice, leading to a pronounced increase of I NaP within the subthreshold potential range, in particular at low CBZ concentrations of 10–30 μm. A combination of current-clamp and computational modeling studies revealed that this effect causes a complete loss of CBZ efficacy in reducing repetitive firing. Thus, β subunits modify not only the biophysical but also the pharmacological properties of Na + channels, in particular with respect to I NaP . Consequently, altered expression of β subunits in other neurological disorders may cause altered neuronal sensitivity to drugs targeting Na + channels.
Noriyoshi Teramoto - One of the best experts on this subject based on the ideXlab platform.
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Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na^+ channels of mouse vas deferens myocytes and recombinant Na_V1.6 channels
Naunyn-Schmiedeberg's Archives of Pharmacology, 2018Co-Authors: Kohei Takahara, Tadashi Yamamoto, Keiichiro Uchida, Hai-lei Zhu, Atsushi Shibata, Tetsuichiro Inai, Mitsuru Noguchi, Mari Yotsu-yamashita, Noriyoshi TeramotoAbstract:Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na^+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes ( SCN2B – Scn4b ). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na^+ current peak amplitude in myocytes (i.e., native I_Na) were compared with its inhibitory potency on recombinant Na_V1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native I_Na and recombinant Na_V1.6 expressed in HEK293 cells (i.e., recombinant I_Na). The current decay of native I_Na was similar to the recombinant Na_V1.6 current co-expressed with β_1-subunits. The current-voltage (I-V) relationships of native I_Na were similar to those of recombinant Na_V1.6 currents co-expressed with β_1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native I_Na ( K _ i = 510 nM), recombinant I_Na ( K _ i = 112 nM), and recombinant I_Na co-expressed with β_1-subunits ( K _ i = 92 nM). The half-maximal (V_half) activation and inactivation of native I_Na values were similar to those observed in recombinant I_Na co-expressed with β_1-subunits. These results suggest that β_1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native I_Na but also recombinant I_Na and recombinant I_Na co-expressed with β_1-subunits in a concentration-dependent manner.
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effects of 4 9 anhydrotetrodotoxin on voltage gated na channels of mouse vas deferens myocytes and recombinant nav1 6 channels
Naunyn-schmiedebergs Archives of Pharmacology, 2018Co-Authors: Kohei Takahara, Tadashi Yamamoto, Keiichiro Uchida, Hai-lei Zhu, Atsushi Shibata, Tetsuichiro Inai, Mitsuru Noguchi, Noriyoshi Teramoto, Mari YotsuyamashitaAbstract:Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (SCN2B – Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with β1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with β1-subunits in a concentration-dependent manner.
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Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.
Naunyn-Schmiedeberg's archives of pharmacology, 2018Co-Authors: Kohei Takahara, Tadashi Yamamoto, Keiichiro Uchida, Hai-lei Zhu, Atsushi Shibata, Tetsuichiro Inai, Mitsuru Noguchi, Mari Yotsu-yamashita, Noriyoshi TeramotoAbstract:Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (SCN2B – Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with β1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with β1-subunits in a concentration-dependent manner.
Chunling Chen - One of the best experts on this subject based on the ideXlab platform.
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SCN2B Deletion in Mice Results in Ventricular and Atrial Arrhythmias
Circulation. Arrhythmia and electrophysiology, 2016Co-Authors: Yangyang Bao, Chunling Chen, Luis F. Lopez-santiago, B. Cicero Willis, Chad R. Frasier, Xianming Lin, Roberto Ramos-mondragón, David S. Auerbach, Zhenxun Wang, Justus M.b. AnumonwoAbstract:Background—Mutations in SCN2B, encoding voltage-gated sodium channel β2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this,...
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na channel scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo
Journal of Biological Chemistry, 2011Co-Authors: Luis F Lopezsantiago, William J. Brackenbury, Chunling Chen, Lori L IsomAbstract:Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ current (INa) mediated by voltage-gated Na+ channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by SCN2B, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S INa inactivation, reduced persistent TTX-R INa, a prolonged rate of recovery of TTX-R INa from inactivation, and reduced cell surface expression of Nav1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K+ current (Ito) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both INa and IK.
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Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo
The Journal of biological chemistry, 2011Co-Authors: Luis F. Lopez-santiago, Chunling Chen, William J. Brackenbury, Lori L IsomAbstract:Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na+ current (INa) mediated by voltage-gated Na+ channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by SCN2B, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S INa inactivation, reduced persistent TTX-R INa, a prolonged rate of recovery of TTX-R INa from inactivation, and reduced cell surface expression of Nav1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K+ current (Ito) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both INa and IK.
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A Functional Null Mutation of SCN1B in a Patient with Dravet Syndrome
Journal of Neuroscience, 2009Co-Authors: Gustavo A. Patino, Haruko Miyazaki, Chunling Chen, Luis F. Lopez-santiago, L Claes, Emily A. Slat, Raja S R Dondeti, Heather A. O'malley, Charles B.b. Gray, Nobuyuki NukinaAbstract:Dravet syndrome (also called severe myoclonic epilepsy of infancy) is one of the most severe forms of childhood epilepsy. Most patients have heterozygous mutations in SCN1A, encoding voltage-gated sodium channel Na(v)1.1 alpha subunits. Sodium channels are modulated by beta1 subunits, encoded by SCN1B, a gene also linked to epilepsy. Here we report the first patient with Dravet syndrome associated with a recessive mutation in SCN1B (p.R125C). Biochemical characterization of p.R125C in a heterologous system demonstrated little to no cell surface expression despite normal total cellular expression. This occurred regardless of coexpression of Na(v)1.1 alpha subunits. Because the patient was homozygous for the mutation, these data suggest a functional SCN1B null phenotype. To understand the consequences of the lack of beta1 cell surface expression in vivo, hippocampal slice recordings were performed in Scn1b(-/-) versus Scn1b(+/+) mice. Scn1b(-/-) CA3 neurons fired evoked action potentials with a significantly higher peak voltage and significantly greater amplitude compared with wild type. However, in contrast to the Scn1a(+/-) model of Dravet syndrome, we found no measurable differences in sodium current density in acutely dissociated CA3 hippocampal neurons. Whereas Scn1b(-/-) mice seize spontaneously, the seizure susceptibility of Scn1b(+/-) mice was similar to wild type, suggesting that, like the parents of this patient, one functional SCN1B allele is sufficient for normal control of electrical excitability. We conclude that SCN1B p.R125C is an autosomal recessive cause of Dravet syndrome through functional gene inactivation.
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Regulation of Persistent Na Current by Interactions between β Subunits of Voltage-Gated Na Channels
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2009Co-Authors: Teresa K. Aman, Lori L Isom, Chunling Chen, Emily A. Slat, Tina M. Grieco-calub, Raffaella Rusconi, Indira M. RamanAbstract:The beta subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming alpha subunits, as well as their trafficking and localization. In heterologous expression systems, beta1, beta2, and beta3 subunits influence inactivation and persistent current in different ways. To test how the beta4 protein regulates Na channel gating, we transfected beta4 into HEK (human embryonic kidney) cells stably expressing Na(V)1.1. Unlike a free peptide with a sequence from the beta4 cytoplasmic domain, the full-length beta4 protein did not block open channels. Instead, beta4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of beta1 or chimeric subunits including the beta1 extracellular domain, however, favored inactivation. Coexpressing Na(V)1.1 and beta4 with beta1 produced tiny persistent currents, indicating that beta1 overcomes the effects of beta4 in heterotrimeric channels. In contrast, beta1(C121W), which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by beta4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with beta4, persistent current was slightly but significantly increased. Moreover, in beta4-expressing neurons from Scn1b and Scn1b/SCN2B null mice, entry into inactivated states was slowed. These data suggest that beta1 and beta4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted beta1 subunits.
Kohei Takahara - One of the best experts on this subject based on the ideXlab platform.
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Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na^+ channels of mouse vas deferens myocytes and recombinant Na_V1.6 channels
Naunyn-Schmiedeberg's Archives of Pharmacology, 2018Co-Authors: Kohei Takahara, Tadashi Yamamoto, Keiichiro Uchida, Hai-lei Zhu, Atsushi Shibata, Tetsuichiro Inai, Mitsuru Noguchi, Mari Yotsu-yamashita, Noriyoshi TeramotoAbstract:Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na^+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes ( SCN2B – Scn4b ). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na^+ current peak amplitude in myocytes (i.e., native I_Na) were compared with its inhibitory potency on recombinant Na_V1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native I_Na and recombinant Na_V1.6 expressed in HEK293 cells (i.e., recombinant I_Na). The current decay of native I_Na was similar to the recombinant Na_V1.6 current co-expressed with β_1-subunits. The current-voltage (I-V) relationships of native I_Na were similar to those of recombinant Na_V1.6 currents co-expressed with β_1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native I_Na ( K _ i = 510 nM), recombinant I_Na ( K _ i = 112 nM), and recombinant I_Na co-expressed with β_1-subunits ( K _ i = 92 nM). The half-maximal (V_half) activation and inactivation of native I_Na values were similar to those observed in recombinant I_Na co-expressed with β_1-subunits. These results suggest that β_1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native I_Na but also recombinant I_Na and recombinant I_Na co-expressed with β_1-subunits in a concentration-dependent manner.
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effects of 4 9 anhydrotetrodotoxin on voltage gated na channels of mouse vas deferens myocytes and recombinant nav1 6 channels
Naunyn-schmiedebergs Archives of Pharmacology, 2018Co-Authors: Kohei Takahara, Tadashi Yamamoto, Keiichiro Uchida, Hai-lei Zhu, Atsushi Shibata, Tetsuichiro Inai, Mitsuru Noguchi, Noriyoshi Teramoto, Mari YotsuyamashitaAbstract:Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (SCN2B – Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with β1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with β1-subunits in a concentration-dependent manner.
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Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.
Naunyn-Schmiedeberg's archives of pharmacology, 2018Co-Authors: Kohei Takahara, Tadashi Yamamoto, Keiichiro Uchida, Hai-lei Zhu, Atsushi Shibata, Tetsuichiro Inai, Mitsuru Noguchi, Mari Yotsu-yamashita, Noriyoshi TeramotoAbstract:Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other β-subunit genes (SCN2B – Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with β1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with β1-subunits in a concentration-dependent manner.
Oscar Campuzano - One of the best experts on this subject based on the ideXlab platform.
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Genetic interpretation and clinical translation of minor genes related to Brugada syndrome.
Human mutation, 2019Co-Authors: Oscar Campuzano, Monica Coll, Jesus Mates, Elena Arbelo, Georgia Sarquella-brugada, Anna Fernandez-falgueras, Sergi Cesar, Alexandra Pérez-serra, Bernat Del Olmo, Paloma JordàAbstract:Brugada syndrome (BrS) is an inherited arrhythmogenic disease associated with sudden cardiac death. The main gene is SCN5A. Additional variants in 42 other genes have been reported as deleterious, although these variants have not yet received comprehensive pathogenic analysis. Our aim was to clarify the role of all currently reported variants in minor genes associated with BrS. We performed a comprehensive analysis according to the American College of Medical Genetics and Genomics guidelines of published clinical and basic data on all genes (other than SCN5A) related to BrS. Our results identified 133 rare variants potentially associated with BrS. After applying current recommendations, only six variants (4.51%) show a conclusive pathogenic role. All definitively pathogenic variants were located in four genes encoding sodium channels or related proteins: SLMAP, SEMA3A, SCNN1A, and SCN2B. In total, 33.83% of variants in 19 additional genes were potentially pathogenic. Beyond SCN5A, we conclude definitive pathogenic variants associated with BrS in four minor genes. The current list of genes associated with BrS, therefore, should include SCN5A, SLMAP, SEMA3A, SCNN1A, and SCN2B. Comprehensive genetic interpretation and careful clinical translation should be done for all variants currently classified as potentially deleterious for BrS.
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Genetic Analysis of Arrhythmogenic Diseases in the Era of NGS: The Complexity of Clinical Decision-Making in Brugada Syndrome
PLOS ONE, 2015Co-Authors: Catarina Allegue, Monica Coll, Jesus Mates, Oscar Campuzano, Anna Iglesias, Beatriz Sobrino, Maria Brion, Jorge Amigo, Angel Carracedo, Pedro BrugadaAbstract:Background The use of next-generation sequencing enables a rapid analysis of many genes associated with sudden cardiac death in diseases like Brugada Syndrome. Genetic variation is identified and associated with 30–35% of cases of Brugada Syndrome, with nearly 20–25% attributable to variants in SCN5A, meaning many cases remain undiagnosed genetically. To evaluate the role of genetic variants in arrhythmogenic diseases and the utility of next-generation sequencing, we applied this technology to resequence 28 main genes associated with arrhythmogenic disorders. Materials and Methods A cohort of 45 clinically diagnosed Brugada Syndrome patients classified as SCN5A-negative was analyzed using next generation sequencing. Twenty-eight genes were resequenced: AKAP9, ANK2, CACNA1C, CACNB2, CASQ2, CAV3, DSC2, DSG2, DSP, GPD1L, HCN4, JUP, KCNE1, KCNE2, KCNE3, KCNH2, KCNJ2, KCNJ5, KCNQ1, NOS1AP, PKP2, RYR2, SCN1B, SCN3B, SCN4B, SCN5A, SNTA1, and TMEM43. A total of 85 clinically evaluated relatives were also genetically analyzed to ascertain familial segregation. Results and Discussion Twenty-two patients carried 30 rare genetic variants in 12 genes, only 4 of which were previously associated with Brugada Syndrome. Neither insertion/deletion nor copy number variation were detected. We identified genetic variants in novel candidate genes potentially associated to Brugada Syndrome. These include: 4 genetic variations in AKAP9 including a de novo genetic variation in 3 positive cases; 5 genetic variations in ANK2 detected in 4 cases; variations in KCNJ2 together with CASQ2 in 1 case; genetic variations in RYR2, including a de novo genetic variation and desmosomal proteins encoding genes including DSG2, DSP and JUP, detected in 3 of the cases. Larger gene panels or whole exome sequencing should be considered to identify novel genes associated to Brugada Syndrome. However, application of approaches such as whole exome sequencing would difficult the interpretation for clinical purposes due to the large amount of data generated. The identification of these genetic variants opens new perspectives on the implications of genetic background in the arrhythmogenic substrate for research purposes. Conclusions As a paradigm for other arrhythmogenic diseases and for unexplained sudden death, our data show that clinical genetic diagnosis is justified in a family perspective for confirmation of genetic causality. In the era of personalized medicine using high-throughput tools, clinical decision-making is increasingly complex.
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A missense mutation in the sodium channel β1b subunit reveals SCN1B as a susceptibility gene underlying long QT syndrome.
Heart rhythm, 2014Co-Authors: Helena Riuró, Oscar Campuzano, Anna Iglesias, Elena Arbelo, Montserrat Batlle, Félix Pérez-villa, Josep Brugada, Guillermo J. Pérez, Fabiana S. Scornik, Ramon BrugadaAbstract:BACKGROUND Long QT syndrome (LQTS) is associated with sudden cardiac death and the prolongation of the QT interval on the electrocardiogram. A comprehensive screening of all genes previously associated with this disease leaves 30% of the patients without a genetic diagnosis. Pathogenic mutations in the sodium channel β subunits have been associated with cardiac channelopathies, including SCN4B mutations in LQTS. OBJECTIVE To evaluate the role of mutations in the sodium channel β subunits in LQTS. METHODS We screened for mutations in the genes encoding the 5 sodium β subunits (SCN1B isoforms a and b, SCN2B, SCN3B, and SCN4B) from 30 nonrelated patients who were clinically diagnosed with LQTS without mutations in common LQTS-related genes. We used the patch-clamp technique to study the properties of sodium currents and the action potential duration in human embryonic kidney and HL-1 cells, respectively, in the presence of β1b subunits. RESULTS The genetic screening revealed a novel mutation in the SCN1Bb gene (β1bP213T) in an 8-year-old boy. Our electrophysiological analysis revealed that β1bP213T increases late sodium current. In addition, β1bP213T subtly altered Nav1.5 function by shifting the window current, accelerating recovery from inactivation, and decreasing the slow inactivation rate. Moreover, experiments using HL-1 cells revealed that the action potential duration significantly increases when the mutant β1b was overexpressed compared with β1bWT. CONCLUSION These data revealed SCN1Bb as a susceptibility gene responsible for LQTS, highlighting the importance of continuing the search for new genes and mechanisms to decrease the percentage of patients with LQTS remaining without genetic diagnosis.
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A missense mutation in the sodium channel β2 subunit reveals SCN2B as a new candidate gene for Brugada syndrome.
Human mutation, 2013Co-Authors: Helena Riuró, Oscar Campuzano, Anna Iglesias, Josep Brugada, Pedro Beltran-alvarez, Anna Tarradas, Elisabet Selga, Marcel Verges, Sara Pagans, Pedro BrugadaAbstract:Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa ) from cells coexpressing Nav 1.5 and wild-type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav 1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav 1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav 1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.