Native American ataxia medicines rescue ataxia-linked mutant potassium channel activity via binding to the voltage sensing domain.

There are currently no drugs known to rescue the function of Kv1.1 voltage-gated potassium channels carrying loss-of-function sequence variants underlying the inherited movement disorder, Episodic Ataxia 1 (EA1). The Kwakwaka'wakw First Nations of the Pacific Northwest Coast used Fucus gardneri (bladderwrack kelp), Physocarpus capitatus (Pacific ninebark) and Urtica dioica (common nettle) to treat locomotor ataxia. Here, we show that extracts of these plants enhance wild-type Kv1.1 current, especially at subthreshold potentials. Screening of their constituents revealed that gallic acid and tannic acid similarly augment wild-type Kv1.1 current, with submicromolar potency. Crucially, the extracts and their constituents also enhance activity of Kv1.1 channels containing EA1-linked sequence variants. Molecular dynamics simulations reveal that gallic acid augments Kv1.1 activity via a small-molecule binding site in the extracellular S1-S2 linker. Thus, traditional Native American ataxia treatments utilize a molecular mechanistic foundation that can inform small-molecule approaches to therapeutically correcting EA1 and potentially other Kv1.1-linked channelopathies.

Polysaccharide extracts of Astragalus membranaceus and Atractylodes macrocephala promote intestinal epithelial cell migration by activating the polyamine-mediated K+ channel.

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 µmol·L-1, SPD), alpha-difluoromethylornithine (2.5 mmol·L-1, DFMO), 4-Aminopyridine (40 µmol·L-1, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L-1), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca2+ ([Ca2+]cyt) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca2+]cyt and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca2+]cyt, but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.

A novel metabolism-based phenotypic drug discovery platform in zebrafish uncovers HDACs 1 and 3 as a potential combined anti-seizure drug target.

Despite the development of newer anti-seizure medications over the past 50 years, 30-40% of patients with epilepsy remain refractory to treatment. One explanation for this lack of progress is that the current screening process is largely biased towards transmembrane channels and receptors, and ignores intracellular proteins and enzymes that might serve as efficacious molecular targets. Here, we report the development of a novel drug screening platform that harnesses the power of zebrafish genetics and combines it with in vivo bioenergetics screening assays to uncover therapeutic agents that improve mitochondrial health in diseased animals. By screening commercially available chemical libraries of approved drugs, for which the molecular targets and pathways are well characterized, we were able to reverse-identify the proteins targeted by efficacious compounds and confirm the physiological roles that they play by utilizing other pharmacological ligands. Indeed, using an 870-compound screen in kcna1-morpholino epileptic zebrafish larvae, we uncovered vorinostat (Zolinza™; suberanilohydroxamic acid, SAHA) as a potent anti-seizure agent. We further demonstrated that vorinostat decreased average daily seizures by ∼60% in epileptic Kcna1-null mice using video-EEG recordings. Given that vorinostat is a broad histone deacetylase (HDAC) inhibitor, we then delineated a specific subset of HDACs, namely HDACs 1 and 3, as potential drug targets for future screening. In summary, we have developed a novel phenotypic, metabolism-based experimental therapeutics platform that can be used to identify new molecular targets for future drug discovery in epilepsy.

Input-dependent regulation of excitability controls dendritic maturation in somatosensory thalamocortical neurons.

Input from the sensory organs is required to pattern neurons into topographical maps during development. Dendritic complexity critically determines this patterning process; yet, how signals from the periphery act to control dendritic maturation is unclear. Here, using genetic and surgical manipulations of sensory input in mouse somatosensory thalamocortical neurons, we show that membrane excitability is a critical component of dendritic development. Using a combination of genetic approaches, we find that ablation of N-methyl-D-aspartate (NMDA) receptors during postnatal development leads to epigenetic repression of Kv1.1-type potassium channels, increased excitability, and impaired dendritic maturation. Lesions to whisker input pathways had similar effects. Overexpression of Kv1.1 was sufficient to enable dendritic maturation in the absence of sensory input. Thus, Kv1.1 acts to tune neuronal excitability and maintain it within a physiological range, allowing dendritic maturation to proceed. Together, these results reveal an input-dependent control over neuronal excitability and dendritic complexity in the development and plasticity of sensory pathways.

Functional reconstitution of cell-free synthesized purified Kv channels.

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

Heterogeneous calretinin expression in the avian cochlear nucleus angularis.

Multiple calcium-binding proteins (CaBPs) are expressed at high levels and in complementary patterns in the auditory pathways of birds, mammals, and other vertebrates, but whether specific members of the CaBP family can be used to identify neuronal subpopulations is unclear. We used double immunofluorescence labeling of calretinin (CR) in combination with neuronal markers to investigate the distribution of CR-expressing neurons in brainstem sections of the cochlear nucleus in the chicken (Gallus gallus domesticus). While CR was homogeneously expressed in cochlear nucleus magnocellularis, CR expression was highly heterogeneous in cochlear nucleus angularis (NA), a nucleus with diverse cell types analogous in function to neurons in the mammalian ventral cochlear nucleus. To quantify the distribution of CR in the total NA cell population, we used antibodies against neuronal nuclear protein (NeuN), a postmitotic neuron-specific nuclear marker. In NA neurons, NeuN label was variably localized to the cell nucleus and the cytoplasm, and the intensity of NeuN immunoreactivity was inversely correlated with the intensity of CR immunoreactivity. The percentage of CR + neurons in NA increased from 31 % in embryonic (E)17/18 chicks, to 44 % around hatching (E21), to 51 % in postnatal day (P) 8 chicks. By P8, the distribution of CR + neurons was uniform, both rostrocaudal and in the tonotopic (dorsoventral) axis. Immunoreactivity for the voltage-gated potassium ion channel Kv1.1, used as a marker for physiological type, showed broad and heterogeneous postsynaptic expression in NA, but did not correlate with CR expression. These results suggest that CR may define a subpopulation of neurons within nucleus angularis.

Atractylodes macrocephala Koidz promotes intestinal epithelial restitution via the polyamine--voltage-gated K+ channel pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes macrocephala Koidz (AMK) has been used widely as a digestive and tonic in traditional Chinese medicine. AMK has shown noteworthy promoting effect on intestinal epithelial cell migration, which might represent a promising candidate for the treatment of intestinal mucosa injury. The aim of this study was to investigate the efficacy of AMK on intestinal mucosal restitution and the underlying mechanisms via IEC-6 cell migration model. MATERIALS AND METHODS: A wounding model of IEC-6 cells was induced by a single-edge razor blade along the diameter of six-well polystyrene plates. The cells were grown in control cultures and in cultures containing spermidine (5 µmol/L, SPD, reference drug), alpha-difluoromethylornithine (2.5 mmol/L, DFMO, polyamine inhibitor), AMK (50, 100, and 200 µg/mL), DFMO plus SPD and DFMO plus AMK for 24h. The membrane potential (MP) and cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) were detected by flow cytometry, and polyamines content was determined via high-performance liquid chromatography (HPLC). The expression of Kv1.1 mRNA and protein levels were assessed by RT-qPCR and Western blot analysis, respectively. Cell migration assay was carried out using the Image-Pro Plus software. All of these indexes were used to evaluate the effectiveness of AMK. RESULTS: (1) Treatment with AMK caused significant increases in cellular polyamines content, membrane hyperpolarization, an elevation of [Ca(2+)]cyt and an acceleration of cell migration in IEC-6 cells, as compared to control group. (2) AMK not only reversed the inhibitory effects of DFMO on the polyamines content, MP, and [Ca(2+)]cyt but also restored IEC-6 cell migration to control levels. (3) The Kv1.1 mRNA and protein expression were significantly increased by AMK treatment in control and polyamine-deficient IEC-6 cells. CONCLUSIONS: The results of our current studies revealed that treatment with AMK significantly stimulates the migration of intestinal epithelial cells through polyamine-Kv1.1 channel signaling pathway, which could promote the healing of intestinal injury. These results suggest the potential usefulness of AMK to cure intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.

Acoustic startle hypersensitivity in Mceph mice and its effect on hippocampal excitability.

Current therapies and research for epilepsy concentrate mainly on controlling the disease, but not on prevention of its development and progression. This is partly due to the under-appreciated heterogeneity of the different epileptic syndromes, and a lack of knowledge about the underlying mechanisms of hypersensitivity and hypersynchrony in epilepsy development and spread. In this study we investigate mechanisms underlying the increased susceptibility to acoustic startle in a mouse model homozygous for the spontaneous megencephaly (mceph) mutation, which results in a lack of the functional potassium channel Kv1.1. Mceph mice are hypersensitive to acoustic startle, a response that is not seen in the wild-type (WT) littermates. After acoustic startle, a strong activation of astrocytes, as indicated by glial fibrillary acidic protein, occurred in the inferior colliculus and hippocampus. Both the hypersensitivity of acoustic startle as well as activation of astrocytes could be maintained at WT levels by pre-treating the Mceph mice with the anti-epileptic drug valproate. Furthermore, we utilized the Mceph mouse model to investigate whether acoustic startle-induced hypersensitivity has negative consequences for synchronous neuronal activity in other, non-auditory, systems and networks in the brain, such as the hippocampus. Our findings show that acoustic startle-induced hypersensitivity primes hippocampal networks by increasing their excitability, which results in increased strength of rhythmic network activity. Our results provide novel insights into the mechanisms that underlie the spread of hypersensitivity and hypersynchrony across functionally different parts of the brain.

Low-voltage activated Kv1.1 subunits are crucial for the processing of sound source location in the lateral superior olive in mice.

Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.

Some effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes.

A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes. 1 microg/ml of the venom made the resting plasma membrane potential more negative in cells voltage-clamped at -60 mV. The effect was potentially due to the closure of one or several conductances that were investigated further. Thus, we determined the effects of the venom on the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-activated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest that the venom exerts its action mainly on a transient outward potassium-current that is probably mediated by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected the expression of the mRNA coding for xKv1.1 in the oocytes.

Expression and biophysical properties of Kv1 channels in supragranular neocortical pyramidal neurones.

Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-cell RT-PCR, immunocytochemistry, and whole cell recordings with specific peptide toxins revealed that individual pyramidal cells express multiple Kv1 alpha-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as axonal cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these cells. The alpha-dendrotoxin (alpha-DTX)-sensitive current activated more rapidly and at more negative potentials than the alpha-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The alpha-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from -70 mV. From -50 mV, this percentage increased to approximately 20%. All cells expressed an alpha-DTX-sensitive current with slow inactivation kinetics. In some cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the alpha-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.

Involvement of Scn1b and Kcna1 ion channels in audiogenic seizures and PTZ-induced epilepsy.

We have undertaken chemical genetic approach using Qingyangshenylycosides (QYS), a natural product compound, to explore the molecular mechanisms underlying different types of epilepsy models. Two animal models were used for these studies, i.e., audiogenic seizure (AGS) and pentylenetetrazol (PTZ)-induced generalized epilepsy in DBA/2J mice. We show that the latency of AGS is prolonged and the severity of seizures (the percentages of the tonus, Tonus_%) is reduced in the QYS-treated animals. These results indicate that QYS has anticonvulsant effect on the AGS model. However, we find that administration of QYS has an opposite effects on PTZ-induced generalized epilepsy. Both the latency of the generalized epilepsy and the latency of death are decreased after QYS treatment in PTZ-induced epilepsy. We examine the molecular basis of the distinct roles of QYS in these two epilepsy models by using gene expression data. Our results show that a voltage-gated sodium channel (Scn1b) and a voltage-gated potassium channel (Kcna1) are differentially expressed in AGS and PTZ-induced epilepsy models as well as in QYS-treated animals. Our results demonstrate that a chemical genetic approach may help to reveal both the molecular mechanisms of different epilepsies and the mechanism of action of the antiepileptic drugs.