scholarly journals Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel

2020 ◽  
Vol 295 (38) ◽  
pp. 13277-13286
Author(s):  
Mark J. Burton ◽  
Joel Cresser-Brown ◽  
Morgan Thomas ◽  
Nicola Portolano ◽  
Jaswir Basran ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Normal Function ◽  
Pas Domain ◽  
Heme Binding ◽  
Channel Activity ◽  
Action Potential Firing ◽  
Voltage Gated ◽  
Conserved Domain ◽  
Potential Firing ◽  
Eag Channels

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10–12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per–ARNT–Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

scholarly journals Enhanced excitability of cortical neurons in low-divalent solutions is primarily mediated by altered voltage-dependence of voltage-gated sodium channels

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Briana J Martiszus ◽  
Timur Tsintsadze ◽  
Wenhan Chang ◽  
Stephen M Smith
Keyword(s):  
Cortical Neurons ◽  
Voltage Dependence ◽  
Wild Type ◽  
Calcium Sensing Receptor ◽  
Action Potential Firing ◽  
Voltage Gated ◽  
Calcium Sensing ◽  
Neocortical Neurons ◽  
Voltage Gated Sodium Channels ◽  
Potential Firing

Increasing extracellular [Ca2+] ([Ca2+]o) strongly decreases intrinsic excitability in neurons but the mechanism is unclear. By one hypothesis, [Ca2+]o screens surface charge, reducing voltage-gated sodium channel (VGSC) activation and by another [Ca2+]o activates Calcium-sensing receptor (CaSR) closing the sodium-leak channel (NALCN). Here we report that neocortical neurons from CaSR-deficient (Casr-/-) mice had more negative resting potentials and did not fire spontaneously in reduced divalent-containing solution (T0.2) compared to wild-type (WT). However, after setting membrane potential to -70 mV, T0.2 application similarly depolarized and increased action potential firing in Casr-/- and WT neurons. Enhanced activation of VGSCs was the dominant contributor to the depolarization and increase in excitability by T0.2 and occurred due to hyperpolarizing shifts in VGSC window currents. CaSR deletion depolarized VGSC window currents but did not affect NALCN activation. Regulation of VGSC gating by external divalents is the key mechanism mediating divalent-dependent changes in neocortical neuron excitability.

scholarly journals Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity

2020 ◽  
Vol 295 (13) ◽  
pp. 4114-4123 ◽  
Author(s):  
Ze-Jun Wang ◽  
Stephanie M. Soohoo ◽  
Purushottam B. Tiwari ◽  
Grzegorz Piszczek ◽  
Tinatin I. Brelidze
Keyword(s):  
Ligand Binding ◽  
Cancer Progression ◽  
Neurological Disorders ◽  
Neuronal Excitability ◽  
Tryptophan Fluorescence ◽  
Structural Similarity ◽  
Pas Domain ◽  
Channel Activity ◽  
Binding Domains ◽  
Eag Channels

Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per–Arnt–Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.

scholarly journals Nedd4-2 (NEDD4L) controls intracellular Na+-mediated activity of voltage-gated sodium channels in primary cortical neurons

2013 ◽  
Vol 457 (1) ◽  
pp. 27-31 ◽  
Author(s):  
Jenny A. Ekberg ◽  
Natasha A. Boase ◽  
Grigori Rychkov ◽  
Jantina Manning ◽  
Philip Poronnik ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Ubiquitin Ligase ◽  
Cortical Neurons ◽  
Channel Activity ◽  
Primary Cortical Neurons ◽  
Voltage Gated Sodium Channel ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
First Time

The study shows for the first time that the ubiquitin ligase Nedd4-2 regulates voltage-gated sodium channel activity in cortical neurons, specifically in response to elevated intracellular Na+ concentration.

Sensory renal innervation: a kidney-specific firing activity due to a unique expression pattern of voltage-gated sodium channels?

2013 ◽  
Vol 304 (5) ◽  
pp. F491-F497 ◽  
Author(s):  
Wolfgang Freisinger ◽  
Johannes Schatz ◽  
Tilmann Ditting ◽  
Angelika Lampert ◽  
Sonja Heinlein ◽  
...  
Keyword(s):  
Action Potential ◽  
Sodium Channels ◽  
Sensory Neurons ◽  
Firing Pattern ◽  
Drg Neurons ◽  
Action Potential Firing ◽  
Tonic Firing ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Potential Firing

Sensory neurons with afferent axons from the kidney are extraordinary in their response to electrical stimulation. More than 50% exhibit a tonic firing pattern, i.e., sustained action potential firing throughout depolarizing, pointing to an increased excitability, whereas nonrenal neurons show mainly a phasic response, i.e., less than five action potentials. Here we investigated whether these peculiar firing characteristics of renal afferent neurons are due to differences in the expression of voltage-gated sodium channels (Navs). Dorsal root ganglion (DRG) neurons from rats (Th11-L2) were recorded by the current-clamp technique and distinguished as “tonic” or “phasic.” In voltage-clamp recordings, Navs were characterized by their tetrodotoxoxin (TTX) sensitivity, and their molecular identity was revealed by RT-PCR. The firing pattern of 66 DRG neurons (41 renal and 25 nonrenal) was investigated. Renal neurons exhibited more often a tonic firing pattern (56.1 vs. 12%). Tonic neurons showed a more positive threshold (−21.75 ± 1.43 vs.−29.33 ± 1.63 mV; P < 0.05), a higher overshoot (56.74 [53.6–60.96] vs. 46.79 mV [38.63–54.75]; P < 0.05) and longer action potential duration (4.61 [4.15–5.85] vs. 3.35 ms [2.12–5.67]; P < 0.05). These findings point to an increased presence of the TTX-resistant Navs 1.8 and 1.9. Furthermore, tonic neurons exhibited a relatively higher portion of TTX-resistant sodium currents. Interestingly, mRNA expression of TTX-resistant sodium channels was significantly increased in renal, predominantly tonic, DRG neurons. Hence, under physiological conditions, renal sensory neurons exhibit predominantly a firing pattern associated with higher excitability. Our findings support that this is due to an increased expression and activation of TTX-resistant Navs.

Development of Ionic Currents Underlying Changes in Action Potential Waveforms in Rat Spinal Motoneurons

1998 ◽  
Vol 80 (6) ◽  
pp. 3047-3061 ◽  
Author(s):  
Bao-Xi Gao ◽  
Lea Ziskind-Conhaim
Keyword(s):  
Action Potential ◽  
Cell Voltage ◽  
Ionic Currents ◽  
Repetitive Firing ◽  
Delayed Rectifier ◽  
Ionic Mechanism ◽  
Spinal Motoneurons ◽  
Action Potential Firing ◽  
Voltage Gated ◽  
Potential Firing

Gao, Bao-Xi and Lea Ziskind-Conhaim. Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons. J. Neurophysiol. 80: 3047–3061, 1998. Differentiation of the ionic mechanism underlying changes in action potential properties was investigated in spinal motoneurons of embryonic and postnatal rats using whole cell voltage- and current-clamp recordings. Relatively slow-rising, prolonged, largely Na+-dependent action potentials were recorded in embryonic motoneurons, and afterdepolarizing potentials were elicited in response to prolonged intracellular injections of depolarizing currents. Action potential amplitude, as well as its rates of rise and repolarization significantly increased, and an afterhyperpolarizing potential (AHP) became apparent immediately after birth. Concurrently, repetitive action potential firing was elicited in response to a prolonged current injection. To determine the ionic mechanism underlying these changes, the properties of voltage-gated macroscopic Na+, Ca2+, and K+ currents were examined. Fast-rising Na+ currents ( I Na) and slow-rising Ca2+ currents ( I Ca) were expressed early in embryonic development, but only I Na was necessary and sufficient to trigger an action potential. I Na and I Ca densities significantly increased while the time to peak I Na and I Ca decreased after birth. The postnatal increase in I Na resulted in overshooting action potential with significantly faster rate of rise than that recorded before birth. Properties of three types of outward K+ currents were examined: transient type-A current ( I A), noninactivating delayed rectifier-type current ( I K), and Ca2+-dependent K+ current ( I K(Ca)). The twofold postnatal increase in I K and I K(Ca) densities resulted in shorter duration action potential and the generation of AHP. Relatively large I A was expressed early in neuronal development, but unlike I K and I K(Ca) its density did not increase after birth. The three types of K+ channels had opposite modulatory actions on action potential firing behavior: I K and I A increased the firing rate, whereas I K(Ca) decreased it. Our findings demonstrated that the developmental changes in action potential waveforms and the onset of repetitive firing were correlated with large increases in the densities of existing voltage-gated ion channels rather than the expression of new channel types.

Mitochondrial transport in processes of cortical neurons is independent of intracellular calcium

2006 ◽  
Vol 291 (6) ◽  
pp. C1193-C1197 ◽  
Author(s):  
Luis Beltran-Parrazal ◽  
Héctor E. López-Valdés ◽  
K. C. Brennan ◽  
Mauricio Díaz-Muñoz ◽  
Jean de Vellis ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Action Potentials ◽  
Synaptic Activity ◽  
Action Potential Firing ◽  
Mitochondrial Movement ◽  
Neuronal Processes ◽  
Potential Firing ◽  
Intracellular Ca ◽  
Rat Cortical Neurons ◽  
And Function

Mitochondria show extensive movement along neuronal processes, but the mechanisms and function of this movement are not clearly understood. We have used high-resolution confocal microscopy to simultaneously monitor movement of mitochondria and changes in intracellular [Ca2+] ([Ca2+]i) in rat cortical neurons. A significant percentage (27%) of the total mitochondria in cortical neuronal processes showed movement over distances of >2 μM. The average velocity was 0.52 μm/s. The velocity, direction, and pattern of mitochondrial movement were not affected by transient increases in [Ca2+]i associated with spontaneous firing of action potentials. Stimulation of Ca2+ transients with forskolin (10 μM) or bicuculline (10 μM), or sustained elevations of [Ca2+]i evoked by glutamate (10 μM) also had no effect on mitochondrial transit. Neither removal of extracellular Ca2+, depletion of intracellular Ca2+ stores with thapsigargin, or inhibition of synaptic activity with TTX (1 μM) or a cocktail of CNQX (10 μM) and MK801 (10 μM) affected mitochondrial movement. These results indicate that movement of mitochondria along processes is a fundamental activity in neurons that occurs independently of physiological changes in [Ca2+]i associated with action potential firing, synaptic activity, or release of Ca2+ from intracellular stores.

Carisbamate, a novel neuromodulator, inhibits voltage-gated sodium channels and action potential firing of rat hippocampal neurons

2009 ◽  
Vol 83 (1) ◽  
pp. 66-72 ◽  
Author(s):  
Yi Liu ◽  
George J. Yohrling ◽  
Yan Wang ◽  
Tasha L. Hutchinson ◽  
Douglas E. Brenneman ◽  
...  
Keyword(s):  
Action Potential ◽  
Sodium Channels ◽  
Hippocampal Neurons ◽  
Action Potential Firing ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Potential Firing

scholarly journals Electrophysiological properties of human β-cell lines EndoC-βH1 and -βH2 conform with human β-cells

2017 ◽  
Author(s):  
Benoît Hastoy ◽  
Mahdieh Godazgar ◽  
Anne Clark ◽  
Vibe Nylander ◽  
Ioannis Spiliotis ◽  
...  
Keyword(s):  
Action Potential ◽  
Cell Lines ◽  
Small Contribution ◽  
Channel Activity ◽  
Β Cells ◽  
Β Cell ◽  
Action Potential Firing ◽  
Electrophysiological Properties ◽  
Potential Firing ◽  
Fold Stimulation

AbstractThe electrophysiological and secretory properties of the human β-cell lines EndoC-βH1 and EndoC-βH2 were investigated. Both cell lines respond to glucose (6-20mM) with 2-to 3-fold stimulation of insulin secretion, an effect that was mimicked by tolbutamide (0.2mM) and reversed by diazoxide (0.5mM). Glucose-induced insulin release correlated with an elevation of [Ca2+]i, membrane depolarization and increased action potential firing. KATP channel activity at 1mM glucose is low and increasing glucose to 6 or 20mM reduced KATP channel activity to the same extent as application of the KATP channel blocker tolbutamide (0.2mM). The upstroke of the action potentials in EndoC-βH1 and −βH2 cells observed at high glucose principally reflects activation of L- and P/Q-type Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. Action potential repolarization involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis (measured by measurements of membrane capacitance) was triggered by membrane depolarizations >10ms to membrane potentials above -30mV. Both cell lines were well-granulated (6,000-15,000 granules/cell) and granules consisted of a central insulin core surrounded by a clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and that they represent a useful experimental model.

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