1. Kv4.2 channel activity controls intrinsic firing dynamics of arcuate kisspeptin neurons
Philipe R F Mendonça, Victoria Kyle, Hugh P C Robinson, William H Colledge, Shel-Hwa Yeo J Physiol . 2018 Mar 1;596(5):885-899. doi: 10.1113/JP274474.
Key points:Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development. Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known. In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation. The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is. Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development.Abstract:Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole-cell patch-clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc), in brain slices isolated from adult male Kiss-Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A-type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular-spiking cortical interneurons, in which the A-type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arcneurons, A-type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single-cell RT-PCR. In addition, firing irregularity was correlated to the density of A-type current in the membrane. Using conductance injection, we demonstrated that adding Kv4-like potassium conductance (gKv4) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arcneurons, shaping their physiological function in gonadotropin release.
2. Elevated ocular pressure reduces voltage-gated sodium channel NaV1.2 protein expression in retinal ganglion cell axons
Nolan R McGrady, Michael L Risner, Silvia Pasini, David J Calkins, Wendi S Lambert Exp Eye Res . 2020 Jan;190:107873. doi: 10.1016/j.exer.2019.107873.
Glaucoma is an age-related neurodegenerative disease that is commonly associated with sensitivity to intraocular pressure. The disease selectively targets retinal ganglion cells (RGCs) and constituent axons. RGC axons are rich in voltage-gated sodium channels, which are essential for action potential initiation and regeneration. Here, we identified voltage-dependent sodium channel, NaV1.2, in the retina, examined how this channel contributes to RGC light responses, and monitored NaV1.2 mRNA and protein expression in the retina during progression of modeled glaucoma. We found NaV1.2 is predominately localized in ganglion cell intraretinal axons with dispersed expression in the outer and inner plexiform layers. We showed Phrixotoxin-3, a potent NaV1.2 channel blocker, significantly decreased RGC electrical activity in a dose-dependent manner with an IC50 of 40 nM. Finally, we found four weeks of raised intraocular pressure (30% above baseline) significantly increased NaV1.2 mRNA expression but reduced NaV1.2 protein level in the retina up to 57% (p < 0.001). Following prolonged intraocular pressure elevation, NaV1.2 protein expression particularly diminished at distal sections of ganglion cell intraretinal axons (p ≤ 0.01). Our results suggest NaV1.2 might be a therapeutic target during disease progression to maintain RGC excitability, preserving presynaptic connections through action potential backpropagation.
3. Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant
J Marshall Shafer, Junkai Xie, Muriel Eaton, William C Skarnes, Tiange Xiao, Zhefu Que, Anke M Tukker, Chang-Deng Hu, Kyle Wettschurack, Zhuo Huang, Layan Yunis, Maria I Olivero-Acosta, Aaron B Bowman, James A Schaber, Jean-Christophe Rochet, Jiaxiang Wu, Yang Yang, Xiaoling Chen, Darci J Trader, Jingliang Zhang, Chongli Yuan J Neurosci . 2021 Dec 8;41(49):10194-10208. doi: 10.1523/JNEUROSCI.0564-21.2021.
With the wide adoption of genomic sequencing in children having seizures, an increasing number ofSCN2Agenetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by geneSCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrentSCN2Agenetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as anin vitroplatform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENTA mounting number ofSCN2Agenetic variants have been identified from patients with epilepsy, but howSCN2Avariants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurringSCN2Avariant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform forin vitrodrug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenicSCN2Avariant.
