Pharmacological Dissection of the Crosstalk between NaV and CaV Channels in GH3b6 Cells

Thanks to the crosstalk between Na+ and Ca2+ channels, Na+ and Ca2+ homeostasis interplay in so-called excitable cells enables the generation of action potential in response to electrical stimulation. Here, we investigated the impact of persistent activation of voltage-gated Na+ (NaV) channels by neurotoxins, such as veratridine (VTD), on intracellular Ca2+ concentration ([Ca2+]i) in a model of excitable cells, the rat pituitary GH3b6 cells, in order to identify the molecular actors involved in Na+-Ca2+ homeostasis crosstalk. By combining RT-qPCR, immunoblotting, immunocytochemistry, and patch-clamp techniques, we showed that GH3b6 cells predominantly express the NaV1.3 channel subtype, which likely endorses their voltage-activated Na+ currents. Notably, these Na+ currents were blocked by ICA-121431 and activated by the β-scorpion toxin Tf2, two selective NaV1.3 channel ligands. Using Fura-2, we showed that VTD induced a [Ca2+]i increase. This effect was suppressed by the selective NaV channel blocker tetrodotoxin, as well by the selective L-type CaV channel (LTCC) blocker nifedipine. We also evidenced that crobenetine, a NaV channel blocker, abolished VTD-induced [Ca2+]i elevation, while it had no effects on LTCC. Altogether, our findings highlight a crosstalk between NaV and LTCC in GH3b6 cells, providing a new insight into the mode of action of neurotoxins.

Distinct dendritic Ca2+ spike forms produce opposing input-output transformations in rat CA3 pyramidal cells

Proper integration of different inputs targeting the dendritic tree of CA3 pyramidal cells (CA3PCs) is critical for associative learning and recall. Dendritic Ca2+ spikes have been proposed to perform associative computations in other PC types, by detecting conjunctive activation of different afferent input pathways, initiating afterdepolarization (ADP) and triggering burst firing. Implementation of such operations fundamentally depends on the actual biophysical properties of dendritic Ca2+ spikes; yet little is known about these properties in dendrites of CA3PCs. Using dendritic patch-clamp recordings and two-photon Ca2+ imaging in acute slices from male rats we report that, unlike CA1PCs, distal apical trunk dendrites of CA3PCs exhibit distinct forms of dendritic Ca2+ spikes. Besides ADP-type global Ca2+ spikes, a majority of dendrites expresses a novel, fast Ca2+ spike type that is initiated locally without backpropagating action potentials, can recruit additional Na+ currents, and is compartmentalized to the activated dendritic subtree. Occurrence of the different Ca2+ spike types correlates with dendritic structure, indicating morpho-functional heterogeneity among CA3PCs. Importantly, ADPs and dendritically initiated spikes produce opposing somatic output: bursts versus strictly single action potentials, respectively. The uncovered variability of dendritic Ca2+ spikes may underlie heterogeneous input-output transformation and bursting properties of CA3PCs, and might specifically contribute to key associative and non-associative computations performed by the CA3 network.

Regulation of Nav1.6-mediated sodium currents underlie the homeostatic control of neuronal intrinsic excitability in the optic tectum of the developing Xenopus laevis tadpole

For individual neurons to function appropriately within a network that is undergoing synaptic reorganization and refinement due to developmental or experience-dependent changes in circuit activity, they must homeostatically adapt their intrinsic excitability to maintain a consistent output despite the changing levels of synaptic input. This homeostatic plasticity of excitability is particularly important for the development of sensory circuits, where subtle deficits in neuronal and circuit function cause developmental disorders including autism spectrum disorder and epilepsy. Despite the critical importance of this process for normal circuit development, the molecular mechanism by which this homeostatic control of intrinsic excitability is regulated is not fully understood. Here, we demonstrate that Xenopus optic tectal neurons express distinct fast, persistent and resurgent Na+ currents. Here, we demonstrate that Xenopus optic tectal neurons express distinct fast, persistent and resurgent Na+ currents. These are regulated with developmental changes in synaptic input, and homeostatically in response to changes in visual input. We show that expression of the voltage-gated Na+ channel subtype Nav1.6 is regulated with changes in intrinsic excitability, that blocking Nav1.6 channels is sufficient to decrease intrinsic excitability. Furthermore, that upregulation of Nav1.6 expression is necessary for experience-dependent increases in Na+ currents and intrinsic excitability. Finally, by examining behaviors that rely on visual and multisensory integration, we extend these findings to show that tight regulation of Na+ channel gene expression during a critical period of tectal circuit development is required for the normal functional development of the tectal circuitry.

Identification and Characterization of Rice OsHKT1;3 Variants

In rice, the high-affinity K+ transporter, OsHKT1;3, functions as a Na+-selective transporter. mRNA variants of OsHKT1;3 have been reported previously, but their functions remain unknown. In this study, five OsHKT1;3 variants (V1-V5) were identified from japonica rice (Nipponbare) in addition to OsHKT1;3_FL. Absolute quantification qPCR analyses revealed that the transcript level of OsHKT1;3_FL was significantly higher than other variants in both the roots and shoots. Expression levels of OsHKT1;3_FL, and some variants, increased after 24 h of salt stress. Two electrode voltage clamp experiments in a heterologous expression system using Xenopus laevis oocytes revealed that oocytes expressing OsHKT1;3_FL and all of its variants exhibited smaller Na+ currents. The presented data, together with previous data, provide insights to understanding how OsHKT family members are involved in the mechanisms of ion homeostasis and salt tolerance in rice.

The Specific Effects of OD-1, a Peptide Activator, on Voltage-Gated Sodium Current and Seizure Susceptibility

OD-1, a scorpion toxin, has been previously recognized as an activator of voltage-gated Na+ currents. To what extent this agent can alter hippocampal neuronal Na+ currents and network excitability and how it can be applied to neuronal hyperexcitability research remains unclear. With the aid of patch-clamp technology, it was revealed that, in mHippoE-14 hippocampal neurons, OD-1 produced a concentration-, time-, and state-dependent rise in the peak amplitude of INa. It shifted the INa inactivation curve to a less negative potential and increased the frequency of spontaneous action currents. Further characterization of neuronal excitability revealed higher excitability in the hippocampal slices treated with OD-1 as compared with the control slices. A stereotaxic intrahippocampal injection of OD-1 generated a significantly higher frequency of spontaneous seizures and epileptiform discharges compared with intraperitoneal injection of lithium-pilocarpine- or kainic acid-induced epilepsy, with comparable pathological changes. Carbamazepine significantly attenuated OD-1 induced seizures and epileptiform discharges. The OD-1-mediated modifications of INa altered the electrical activity of neurons in vivo and OD-1 could potentially serve as a novel seizure and excitotoxicity model.

Persistent and resurgent Na+ currents in vestibular calyx afferents

Action potential firing patterns differ between groups of afferent neurons innervating vestibular epithelia. We investigated the biophysical properties of Na+ currents in specialized vestibular calyx afferent terminals during postnatal development. Mature calyces express Na+ currents with transient, persistent, and resurgent components. Nav1.6 channels contribute to resurgent Na+ currents and may enhance firing in peripheral calyx afferents. Understanding Na+ channels that contribute to vestibular nerve responses has implications for developing new treatments for vestibular dysfunction.