Current Literature In Basic Science
When Inhibiting Repetitive Firing is Pro-Epileptic
De Novo KCNB1 Mutations in Infantile Epilepsy Inhibit Repetitive Neuronal Firing. Saitsu H, Akita T, Tohyama J, Goldberg-Stern H, Kobayashi Y, Cohen R, Kato M, Ohba C, Miyatake S, Tsurusaki Y, Nakashima M, Miyake N, Fukuda A, Matsumoto N. Sci Rep 2015;5:15199. doi:10.1038/srep15199.
The voltage-gated Kv2.1 potassium channel encoded by KCNB1 produces the major delayed rectifier potassium current in pyramidal neurons. Recently, de novo heterozygous missense KCNB1 mutations have been identified in three patients with epileptic encephalopathy and a patient with neurodevelopmental disorder. However, the frequency of KCNB1 mutations in infantile epileptic patients and their effects on neuronal activity are yet unknown. We searched whole exome sequencing data of a total of 437 patients with infantile epilepsy, and found novel de novo heterozygous missense KCNB1 mutations in two patients showing psychomotor developmental delay and severe infantile generalized seizures with high-amplitude spike-and-wave electroencephalogram discharges. The mutation located in the channel voltage sensor (p.R306C) disrupted sensitivity and cooperativity of the sensor, while the mutation in the channel pore domain (p.G401R) selectively abolished endogenous Kv2 currents in transfected pyramidal neurons, indicating a dominant negative effect. Both mutants inhibited repetitive neuronal firing through preventing production of deep interspike voltages. Thus KCNB1 mutations can be a rare genetic cause of infantile epilepsy, and insufficient firing of pyramidal neurons would disturb both development and stability of neuronal circuits, leading to the disease phenotypes.
Commentary Mutations in KCNB1 were first reported in patients with early infantile epileptic encephalopathy type 26 (EIEE26) in 2014 (1). The clinical features included infant-onset refractory seizures of multiple types, diffuse slow-wave EEG with multifocal spikes, global developmental delay, and hypotonia. Subsequently, KCNB1 mutations have been reported in additional patients with neurodevelopmental delay and overlapping epilepsy phenotypes (2–4). KCNB1 encodes the Kv2.1 voltage-gated potassium channel α subunit, which is a major contributor to delayed rectifier potassium current (5). Kv2.1 channels are localized on the soma, proximal dendrites, and axon initial segment of neurons, where they play an important role in modulating neuronal excitability (5, 6). Voltage-gated potassium channels comprise four α subunits that co-assemble with four-fold symmetry around a central ion conduction pore. Each subunit contains six transmembrane segments. Segments S1 to S4 comprise the voltage-sensing domain and segments S5 and S6 comprise the ion-selective pore domain. The S4 segment has a repeating motif of two neutral amino acids and one positively charged amino acid (usually arginine) that is critical for voltage-sensitive gating. Changes in membrane potential are transduced from the voltage-sensor domain to the pore domain, which Epilepsy Currents, Vol. 16, No. 2 (March/April) 2016 pp. 114–115 © American Epilepsy Society
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then conducts potassium ions in response to that change. Of interest, the reported KCNB1 mutations were all clustered within the pore domain of the channel. Functional characterization of the mutant channels in Chinese hamster ovary (CHO) K1 cells demonstrated a loss of ion selectivity and voltage-dependence for three of the mutations (S347R, T374I, G379R) (1) and loss of ion selectivity (V378A) (3). Additionally, the V378A mutation showed disruption of channel trafficking to the cell surface in COS-7 cells (3). These studies suggested a nascent genotype–phenotype correlation in which pore domain mutations result in similar functional defects and clinical features. In the current report, Saitsu and colleagues report two novel de novo mutations of KCNB1 in patients with infantile epilepsy and neurodevelopmental delay. These were discovered as part of a large-scale whole exome sequencing in patients with intractable genetic disorders. Among 437 patients with infantile epilepsy, they identified de novo KCNB1 mutations in two unrelated patients. The patients initially presented with developmental delay, and subsequently developed epilepsy at 12 to 18 months of age. Multiple seizure types were reported, including spasms, focal, clonic, tonic–clonic, and myoclonic. Seizures were refractory to therapy. On EEG, patients showed high-amplitude diffuse spike-and-wave discharges, which was also a common feature in previously reported patients (1, 3, 4). This finding provided further evidence that infantile onset seizures with spike-and-wave discharges are a key feature of KCNB1-associated epilepsy. The newly described KCNB1 mutations, R306C and G401R, are located in different functional domains of the Kv2.1
KCNB1 Mutations Inhibit Repetitive Firing
channel protein. Arginine 306 is one of the critical positively charged arginines that comprise the voltage sensor, while glycine 401 is located in the pore domain. In order to examine the effects of these mutations on Kv2.1 channel function, the authors expressed Kv2.1 mutants in Neuro2A cells and performed voltage-clamp recordings. Neuro2A is a mouse-derived neuroblastoma cell line that endogenously expresses Kv2.1 currents (7). Transfection of Neuro2A cells with wild-type Kv2.1 resulted in currents that were two orders of magnitude larger than the endogenous currents. Thus, in their system, wild-type Kv2.1 is endogenously present, while the transfected Kv2.1 is overexpressed. Transfection with the R306C mutant resulted in currents that were approximately two times larger than the endogenous current, similar to the magnitude seen with transfected wildtype. However, voltagedependence and activation kinetics differed from wildtype, with delayed channel opening and inactivation. In contrast, transfection with the G401R mutant resulted in elimination of endogenous currents, suggesting a dominant negative effect on wild-type channels. Previously characterized KCNB1 pore mutations (S347R, T374I, G379R) exhibited similar dominant negative effects when co-expressed with wild-type channels (1). However, loss of ion selectivity, a common feature observed with other KCNB1 pore mutations (S347R, T374I, G379R, V378A) (1, 3), was not observed for G401R. As the first reported voltage-sensor mutation, R306C has unique properties that cannot be directly compared with previous studies. However, it does share some loss-of-function characteristics with the other mutations, including reduced current density and reduced conductance in voltage ranges where wild-type Kv2.1 is usually most active. To further investigate how these functional defects would influence neuronal excitability, Saitsu and colleagues tested mutation effects on total potassium currents and firing of cultured cortical neurons. Because cortical neurons contain a variety of endogenous potassium currents, they used a stimulation protocol that attempts to isolate Kv2-mediated currents and confirmed this by blocking the residual current with a Kv2-selective toxin. When transfected with R306C, the observed currents were similar in amplitude to endogenous in the positive voltage range, but larger in the negative voltage range. Additionally, the time course of activation was slower, particularly in the positive voltage range. Transfection with G401R resulted in loss of Kv2-mediated current, consistent with a dominant negative effect. Under current clamp recording, R306C and G401R transfected neurons exhibited decreased input resistance and increased minimum current required to fire an action potential (rheobase). Of most interest, R306C- and G401R-transfected neurons exhibited deficiencies in repetitive action potential firing in response to long current injections. Facilitation and maintenance of repetitive firing is a prominent function of Kv2.1 (5) and loss of this hallmark
feature is likely an important defect. Precisely how inability to facilitate repetitive firing translates to human epilepsy is currently unknown and will require further studies to understand the network effects. The current study and other reports support mutation of KCNB1 as pathogenic, resulting in epileptic encephalopathy characterized by global developmental delay, infantile onset refractory seizures, diffuse slowing and spike-and-wave discharges on EEG, and hypotonia. The R306C mutation, located in the voltage sensor, expands the range of mutation sites and functional defects that can result in EIEE26. Functional studies demonstrate loss of Kv2.1 function as a putative shared molecular defect, suggesting that restoration of Kv2.1 function may be a useful therapy. by Jennifer A. Kearney, PhD References 1. Torkamani A, Bersell K, Jorge BS, Bjork RL Jr, Friedman JR, Bloss CS, Cohen J, Gupta S, Naidu S, Vanoye CG, George AL Jr, Kearney JA. De novo KCNB1 mutations in epileptic encephalopathy [published online ahead of print September 19, 2014]. Ann Neurol 2014;76:529–540. doi:10.1002/ana.24263. 2. Soden SE, Saunders CJ, Willig LK, Farrow EG, Smith LD, Petrikin JE, LePichon JB, Miller NA, Thiffault I, Dinwiddie DL, Twist G, Noll A, Heese BA, Zellmer L, Atherton AM, Abdelmoity AT, Safina N, Nyp SS, Zuccarelli B, Larson IA, Modrcin A, Herd S, Creed M, Ye Z, Yuan X, Brodsky RA, Kingsmore SF. Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders. Sci Transl Med 2014;6:265ra168. doi:10.1126/scitranslmed.3010076. 3. Thiffault I, Speca DJ, Austin DC, Cobb MM, Eum KS, Safina NP, Grote L, Farrow EG, Miller N, Soden S, Kingsmore SF, Trimmer JS, Saunders CJ, Sack JT. A novel epileptic encephalopathy mutation in KCNB1 disrupts Kv2.1 ion selectivity, expression, and localization. J Gen Physiol 2015;146:399–410. 4. Allen NM, Conroy J, Shahwan A, Lynch B, Correa RG, Pena SD, McCreary D, Magalhães TR, Ennis S, Lynch SA, King MD. Unexplained early onset epileptic encephalopathy: Exome screening and phenotype expansion [published online ahead of print December 9, 2015]. Epilepsia 2016;57:e12–e17. doi:10.1111/epi.13250. 5. Misonou H, Mohapatra DP, Trimmer JS. Kv2.1: A voltage-gated k+ channel critical to dynamic control of neuronal excitability. Neurotoxicology 2005;26:743–752. 6. Sarmiere PD, Weigle CM, Tamkun MM. The Kv2.1 K+ channel targets to the axon initial segment of hippocampal and cortical neurons in culture and in situ. BMC Neurosci 2008;9:112. doi: 10.1186/1471-22029-112. 7. Leung YM, Huang CF, Chao CC, Lu DY, Kuo CS, Cheng TH, Chang LY, Chou CH. Voltage-gated K+ channels play a role in cAMP-stimulated neuritogenesis in mouse neuroblastoma N2A cells. J Cell Physiol 2011;226:1090–1098.
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When Inhibiting Repetitive Firing is Pro-Epileptic
De Novo KCNB1 Mutations in Infantile Epilepsy Inhibit Repetitive Neuronal Firing. Saitsu H, Akita T, Tohyama J, Goldberg-Stern H, Kobayashi Y, Cohen R, Kato M, Ohba C, Miyatake S, Tsurusaki Y, Nakashima M, Miyake N, Fukuda A, Matsumoto N. Sci Rep 2015;5:15199. doi:10.1038/srep15199.
The voltage-gated Kv2.1 potassium channel encoded by KCNB1 produces the major delayed rectifier potassium current in pyramidal neurons. Recently, de novo heterozygous missense KCNB1 mutations have been identified in three patients with epileptic encephalopathy and a patient with neurodevelopmental disorder. However, the frequency of KCNB1 mutations in infantile epileptic patients and their effects on neuronal activity are yet unknown. We searched whole exome sequencing data of a total of 437 patients with infantile epilepsy, and found novel de novo heterozygous missense KCNB1 mutations in two patients showing psychomotor developmental delay and severe infantile generalized seizures with high-amplitude spike-and-wave electroencephalogram discharges. The mutation located in the channel voltage sensor (p.R306C) disrupted sensitivity and cooperativity of the sensor, while the mutation in the channel pore domain (p.G401R) selectively abolished endogenous Kv2 currents in transfected pyramidal neurons, indicating a dominant negative effect. Both mutants inhibited repetitive neuronal firing through preventing production of deep interspike voltages. Thus KCNB1 mutations can be a rare genetic cause of infantile epilepsy, and insufficient firing of pyramidal neurons would disturb both development and stability of neuronal circuits, leading to the disease phenotypes.
Commentary Mutations in KCNB1 were first reported in patients with early infantile epileptic encephalopathy type 26 (EIEE26) in 2014 (1). The clinical features included infant-onset refractory seizures of multiple types, diffuse slow-wave EEG with multifocal spikes, global developmental delay, and hypotonia. Subsequently, KCNB1 mutations have been reported in additional patients with neurodevelopmental delay and overlapping epilepsy phenotypes (2–4). KCNB1 encodes the Kv2.1 voltage-gated potassium channel α subunit, which is a major contributor to delayed rectifier potassium current (5). Kv2.1 channels are localized on the soma, proximal dendrites, and axon initial segment of neurons, where they play an important role in modulating neuronal excitability (5, 6). Voltage-gated potassium channels comprise four α subunits that co-assemble with four-fold symmetry around a central ion conduction pore. Each subunit contains six transmembrane segments. Segments S1 to S4 comprise the voltage-sensing domain and segments S5 and S6 comprise the ion-selective pore domain. The S4 segment has a repeating motif of two neutral amino acids and one positively charged amino acid (usually arginine) that is critical for voltage-sensitive gating. Changes in membrane potential are transduced from the voltage-sensor domain to the pore domain, which Epilepsy Currents, Vol. 16, No. 2 (March/April) 2016 pp. 114–115 © American Epilepsy Society
114
then conducts potassium ions in response to that change. Of interest, the reported KCNB1 mutations were all clustered within the pore domain of the channel. Functional characterization of the mutant channels in Chinese hamster ovary (CHO) K1 cells demonstrated a loss of ion selectivity and voltage-dependence for three of the mutations (S347R, T374I, G379R) (1) and loss of ion selectivity (V378A) (3). Additionally, the V378A mutation showed disruption of channel trafficking to the cell surface in COS-7 cells (3). These studies suggested a nascent genotype–phenotype correlation in which pore domain mutations result in similar functional defects and clinical features. In the current report, Saitsu and colleagues report two novel de novo mutations of KCNB1 in patients with infantile epilepsy and neurodevelopmental delay. These were discovered as part of a large-scale whole exome sequencing in patients with intractable genetic disorders. Among 437 patients with infantile epilepsy, they identified de novo KCNB1 mutations in two unrelated patients. The patients initially presented with developmental delay, and subsequently developed epilepsy at 12 to 18 months of age. Multiple seizure types were reported, including spasms, focal, clonic, tonic–clonic, and myoclonic. Seizures were refractory to therapy. On EEG, patients showed high-amplitude diffuse spike-and-wave discharges, which was also a common feature in previously reported patients (1, 3, 4). This finding provided further evidence that infantile onset seizures with spike-and-wave discharges are a key feature of KCNB1-associated epilepsy. The newly described KCNB1 mutations, R306C and G401R, are located in different functional domains of the Kv2.1
KCNB1 Mutations Inhibit Repetitive Firing
channel protein. Arginine 306 is one of the critical positively charged arginines that comprise the voltage sensor, while glycine 401 is located in the pore domain. In order to examine the effects of these mutations on Kv2.1 channel function, the authors expressed Kv2.1 mutants in Neuro2A cells and performed voltage-clamp recordings. Neuro2A is a mouse-derived neuroblastoma cell line that endogenously expresses Kv2.1 currents (7). Transfection of Neuro2A cells with wild-type Kv2.1 resulted in currents that were two orders of magnitude larger than the endogenous currents. Thus, in their system, wild-type Kv2.1 is endogenously present, while the transfected Kv2.1 is overexpressed. Transfection with the R306C mutant resulted in currents that were approximately two times larger than the endogenous current, similar to the magnitude seen with transfected wildtype. However, voltagedependence and activation kinetics differed from wildtype, with delayed channel opening and inactivation. In contrast, transfection with the G401R mutant resulted in elimination of endogenous currents, suggesting a dominant negative effect on wild-type channels. Previously characterized KCNB1 pore mutations (S347R, T374I, G379R) exhibited similar dominant negative effects when co-expressed with wild-type channels (1). However, loss of ion selectivity, a common feature observed with other KCNB1 pore mutations (S347R, T374I, G379R, V378A) (1, 3), was not observed for G401R. As the first reported voltage-sensor mutation, R306C has unique properties that cannot be directly compared with previous studies. However, it does share some loss-of-function characteristics with the other mutations, including reduced current density and reduced conductance in voltage ranges where wild-type Kv2.1 is usually most active. To further investigate how these functional defects would influence neuronal excitability, Saitsu and colleagues tested mutation effects on total potassium currents and firing of cultured cortical neurons. Because cortical neurons contain a variety of endogenous potassium currents, they used a stimulation protocol that attempts to isolate Kv2-mediated currents and confirmed this by blocking the residual current with a Kv2-selective toxin. When transfected with R306C, the observed currents were similar in amplitude to endogenous in the positive voltage range, but larger in the negative voltage range. Additionally, the time course of activation was slower, particularly in the positive voltage range. Transfection with G401R resulted in loss of Kv2-mediated current, consistent with a dominant negative effect. Under current clamp recording, R306C and G401R transfected neurons exhibited decreased input resistance and increased minimum current required to fire an action potential (rheobase). Of most interest, R306C- and G401R-transfected neurons exhibited deficiencies in repetitive action potential firing in response to long current injections. Facilitation and maintenance of repetitive firing is a prominent function of Kv2.1 (5) and loss of this hallmark
feature is likely an important defect. Precisely how inability to facilitate repetitive firing translates to human epilepsy is currently unknown and will require further studies to understand the network effects. The current study and other reports support mutation of KCNB1 as pathogenic, resulting in epileptic encephalopathy characterized by global developmental delay, infantile onset refractory seizures, diffuse slowing and spike-and-wave discharges on EEG, and hypotonia. The R306C mutation, located in the voltage sensor, expands the range of mutation sites and functional defects that can result in EIEE26. Functional studies demonstrate loss of Kv2.1 function as a putative shared molecular defect, suggesting that restoration of Kv2.1 function may be a useful therapy. by Jennifer A. Kearney, PhD References 1. Torkamani A, Bersell K, Jorge BS, Bjork RL Jr, Friedman JR, Bloss CS, Cohen J, Gupta S, Naidu S, Vanoye CG, George AL Jr, Kearney JA. De novo KCNB1 mutations in epileptic encephalopathy [published online ahead of print September 19, 2014]. Ann Neurol 2014;76:529–540. doi:10.1002/ana.24263. 2. Soden SE, Saunders CJ, Willig LK, Farrow EG, Smith LD, Petrikin JE, LePichon JB, Miller NA, Thiffault I, Dinwiddie DL, Twist G, Noll A, Heese BA, Zellmer L, Atherton AM, Abdelmoity AT, Safina N, Nyp SS, Zuccarelli B, Larson IA, Modrcin A, Herd S, Creed M, Ye Z, Yuan X, Brodsky RA, Kingsmore SF. Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders. Sci Transl Med 2014;6:265ra168. doi:10.1126/scitranslmed.3010076. 3. Thiffault I, Speca DJ, Austin DC, Cobb MM, Eum KS, Safina NP, Grote L, Farrow EG, Miller N, Soden S, Kingsmore SF, Trimmer JS, Saunders CJ, Sack JT. A novel epileptic encephalopathy mutation in KCNB1 disrupts Kv2.1 ion selectivity, expression, and localization. J Gen Physiol 2015;146:399–410. 4. Allen NM, Conroy J, Shahwan A, Lynch B, Correa RG, Pena SD, McCreary D, Magalhães TR, Ennis S, Lynch SA, King MD. Unexplained early onset epileptic encephalopathy: Exome screening and phenotype expansion [published online ahead of print December 9, 2015]. Epilepsia 2016;57:e12–e17. doi:10.1111/epi.13250. 5. Misonou H, Mohapatra DP, Trimmer JS. Kv2.1: A voltage-gated k+ channel critical to dynamic control of neuronal excitability. Neurotoxicology 2005;26:743–752. 6. Sarmiere PD, Weigle CM, Tamkun MM. The Kv2.1 K+ channel targets to the axon initial segment of hippocampal and cortical neurons in culture and in situ. BMC Neurosci 2008;9:112. doi: 10.1186/1471-22029-112. 7. Leung YM, Huang CF, Chao CC, Lu DY, Kuo CS, Cheng TH, Chang LY, Chou CH. Voltage-gated K+ channels play a role in cAMP-stimulated neuritogenesis in mouse neuroblastoma N2A cells. J Cell Physiol 2011;226:1090–1098.
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