There are no available tools to verify whether current kinetic models correctly assume that the gating of ionotropic receptors is a stochastic process. Current models place no restrictions either on receptor conformational change rates or on the number of conformational states, making them insensitive to potential constraints imposed by deterministic principles. We formulate a new, deterministic model of receptor operation, providing a complete mathematical description and analyze the consequences of applying the model to the nicotinic receptor. In our model, the probability functions have narrowly defined forms with the number of parameters determined a priori. We redefine the origin of brief and long openings and introduce a state of partial desensitization. This model, in contrast to currently used kinetic models of nicotinic receptor, provides constant receptor affinity and enables the modulation of receptor activity without binding a modulator molecule.

The Hurst effect in the signals describing ion channels' activity has been known for many years. This effect is present in the experimental recordings of single -channel currents, but not only. The sequences of dwell times of functionally different channel states also exhibit long-range correlations. We have found that the memory effect within the dwell-time series is related to the coupling between the channel's activation gate (AG) and selectivity filter (SF), which controls the ion conduction. In this work, we analyzed both the experimental data describing the activity of potassium channels of different types (e.g., BK, mitoBK, mitoTASK-3, mitoKv1.3, TREK-2-like channels) and the series generated according to our previously proposed Hurst effect model. The obtained results suggest that the strength of the allosteric cooperation between the AG and SF determines not only the conductance of the channel - which governs how often ions in SF move or remain blocked - but also modulates the correlations present in the dwell times when sampled with a suitably high sampling rate. Moreover, we found that rectification can interfere with this process, contributing to additional changes in correlations within the channel's sojourns in subsequent states. Similarly, the correlations may be affected by processes proceeding at longer time scales, like interactions with the channel's auxiliary domains or lipid surroundings.

Mutations of the SCN1A gene, which encodes the voltage-dependent Na+ channel’s α subunit, are associated with diverse epileptic syndromes ranging in severity, even intra-family, from febrile seizures to epileptic encephalopathy. The underlying cause of this variability is unknown, suggesting the involvement of additional factors. The aim of our study was to describe the properties of mutated channels and investigate genetic causes for clinical syndromes’ variability in the family of five SCN1A gene p.Arg1596Cys mutation carriers. The analysis of additional genetic factors influencing SCN1A-associated phenotypes was conducted through exome sequencing (WES). To assess the impact of mutations, we used patch clamp analysis of mutated channels expressed in HEK cells and in vivo neural excitability studies (NESs). In cells expressing the mutant channel, sodium currents were reduced. NESs indicated increased excitability of peripheral motor neurons in mutation carriers. WES showed the absence of non-SCA1 pathogenic variants that could be causative of disease in the family. Variants of uncertain significance in three genes, as potential modifiers of the most severe phenotype, were identified. The p.Arg1596Cys substitution inhibits channel function, affecting steady-state inactivation kinetics. Its clinical manifestations involve not only epileptic symptoms but also increased excitability of peripheral motor fibers. The role of Nav1.1 in excitatory neurons cannot be ruled out as a significant factor of the clinical phenotype.

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM-NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2-GluN2A and STIM2-GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

TREK-2-like channels in the pyramidal neurons of rat prefrontal cortex are characterized by a wide range of spontaneous activity-from very low to very high-independent of the membrane potential and the stimuli that are known to activate TREK-2 channels, such as temperature or membrane stretching. The aim of this study was to discover what factors are involved in high levels of TREK-2-like channel activity in these cells. Our research focused on the PI(4,5)P2-dependent mechanism of channel activity. Single-channel patch clamp recordings were performed on freshly dissociated pyramidal neurons of rat prefrontal cortexes in both the cell-attached and inside-out configurations. To evaluate the role of endogenous stimulants, the activity of the channels was recorded in the presence of a PI(4,5)P2 analogue (PI(4,5)P2DiC8) and Ca2+. Our research revealed that calcium ions are an important factor affecting TREK-2-like channel activity and kinetics. The observation that calcium participates in the activation of TREK-2-like channels is a new finding. We showed that PI(4,5)P2-dependent TREK-2 activity occurs when the conditions for PI(4,5)P2/Ca2+ nanocluster formation are met. We present a possible model explaining the mechanism of calcium action.

Valproic acid has a long-standing reputation of effectively treating the symptoms of not only epilepsy but also psychiatric conditions. In the latter, the exact mechanism by which valproate exerts its effect remains unclear. In this study, epileptiform bursts were recorded from pyramidal neurons in the prefrontal cortex (the brain region thought to be involved in psychiatric disorders) using the patch-clamp technique. An extracellular solution with no magnesium ions and elevated potassium levels that is known to induce epileptiform activity in vitro was used. Because of their short durations, the epileptiform bursts were regarded as interictal-like epileptiform activity, which is believed to be involved in cognitive impairment. Interictal discharges occur in many neuropsychiatric disorders as well as in healthy population. Epileptic activity in prefrontal cortex pyramidal neurons was potently inhibited by two therapeutic concentrations of valproic acid (20 mu M and 200 mu M). Moreover, valproate suppressed spontaneous excitatory postsynaptic potentials. Epileptiform bursts were fully inhibited by NMDA receptor antagonist, which suggests that epileptiform activity is driven by NMDA receptors. The inhibition of excitability in prefrontal cortex pyramidal neurons by valproate was also shown. This study shows that it is possible to evoke NMDA-dependent epileptiform activity in prefrontal cortex pyramidal neurons in vitro. We suggest that the prefrontal cortex is a good region for studying the influence of drugs on interictal epileptiform activity.

BackgroundPrevious reports reported the presence of TTX-resistant Nav1.8 and Nav1.9 sodium channels in the cortex pyramidal neurons. A characteristic feature of Nav1.9 channels is activation at voltages close to - 70 mV. Therefore, they do not participate directly in the action potential, but contribute to the regulation of the resting membrane potential. Their physiological role is modulation of cell excitability. The aim of the study was to investigate, with the use of patch clamp technique, the dependence of the activation thresholds of TTX–resistant sodium currents on the concentration of extracellular calcium in medial prefrontal cortex pyramidal neurons in rats.ResultsThe recorded values of the threshold of the voltage-dependent sodium currents were in the range of -65 mV to -75 mV. This suggests that the sodium currents may result from the presence of Nav1.9 channels in the rat pyramidal neurons. The threshold for the activation of sodium currents depended on the concentration of Ca2+. Increasing the concentration of calcium ions in the extracellular solution by 5 mM caused the depolarizing shift in the activation potential by about 10 mV. The effect of calcium ion concentration on the potential of TTX-resistant currents activation suggests that Nav1.9 channels are modulated by extracellular Ca2+ concentration. ConclusionsThe study has experimentally confirmed that TTX-resistant channels are present in the cell membrane of the rat prefrontal cortex pyramidal neurons and may, therefore, take a physiological role in the conductivity of sodium currents in a manner dependent on the concentration of extracellular ions.

Background: The aim of the study was to investigate, with the use of the patch clamp technique, the dependence of the conductance of the TREK-2-like potassium leak channel in the medial prefrontal cortex pyramidal neurons on the presence of extracellular magnesium and calcium ions. It is suspected that TREK-2 channels regulate mood and may be associated with the pathophysiology of depression. Since magnesium and calcium deficiency contribute to depressive symptoms, we investigated how TREK-2- like channel pore propertie change in the absence of divalent cations. Results: Single-channel currents were recorded in a cell-attached configuration in enzymatically dispersed pyramidal neurons of the prefrontal cortex in rats. Spontaneous TREK-2-like channel activity was recorded either in the presence or absence of magnesium and calcium ions in extracellular solution. A significant increase in the inward channel conductance was observed when divalent cations were removed from the extracellular solution. Inward rectification was also increased when the bath temperature was raised to 34-37°C. Conclusions: The study confirmed that the activity of TREK-2-like channels is affected by the presence of magnesium and calcium ions in the extracellular solution. Therefore, in vivo, the TREK-2-like channel may possibly participate in the prefrontal cortex dysfunction associated with the deficiency of divalent cations.

Short-term treatment with large doses of corticosteroids can result in acute weakness of muscles in processes that have not yet been fully characterized. Corticosteroids have been shown to exert direct inhibitory action on the muscle-type nicotinic acetylcholine receptor (AChR), and therefore can promote pharmacological muscle denervation. The mechanism of hydrocortisone (HC) blockage of AChR has not been fully established yet. It is uncommon for an electrically neutral molecule, for example, HC, to induce voltage-dependent changes in AChR kinetics. Our experiments aimed to determine the source of voltage-dependency in HC action. Wild-type (WT) and αD200Q receptors were transiently expressed in HEK293 cells. Recordings were performed in either the presence or absence of HC. We showed that the D-to-Q substitution is capable of suppressing the voltage dependency in the HC-induced block. We conclude that the distance between αD200 and the agonist-binding site depends on the membrane potential. The voltage-dependent changes of the αD200 position have not been considered yet. To our knowledge, the ability to induce voltage-dependency in blocker action has not been shown previously for an amino acid located outside the transmembrane portion of the receptor. Possible mechanisms of HC block (allosteric and knocking) in WT and αD200Q receptors are discussed.

1. Kachel HS, Patel RN, Franzyk H, Mellor IR. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition. Scientific Reports. 2016; 6: 38116. CrossRef Google Scholar

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20-day-old), late adolescent (60-dayold) and adult (6-7-month-old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX-resistant, low-threshold, noninactivating and voltage-dependent Na current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats.

Valproic acid is frequently prescribed and used to treat epilepsy, bipolar disorder and other conditions. However, the mechanism of action of valproic acid has not been fully elucidated. The aim of this study was to assess the influence of valproic acid (200 mu M) on TTX-resistant sodium currents in mPFC pyramidal neurons. Valproic acid inhibited the maximal amplitude and did not change the activation parameters of TTX-resistant sodium currents. Moreover, valproic acid (2 mu M and 200 mu M) shifted the TTX-resistant sodium channel inactivation curve towards hyperpolarisation. In the presence of valproic acid, TTX-resistant sodium currents recovered from inactivation more slowly. Valproic acid did not influence the use-dependent blockade of TTX-resistant sodium currents.This study suggests that a potential new mechanism of the antiepileptic action of valproic acid is, among others, inhibition of TTX-resistant sodium currents. (C) 2017 Elsevier Inc. All rights reserved.

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na+ current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20-day old), late adolescent (60-day old), and adult (6- to 7-month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX-resistant, low-threshold, noninactivating, and voltage-dependent Na+ current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1371-1384, 2017.

TREK-2-like channels were identified on the basis of electrophysiological and pharmacological tests performed on freshly isolated and enzymatically/mechanically dispersed pyramidal neurons of the rat medial prefrontal cortex (mPFC). Single-channel currents were recorded in cell-attached configuration and the impact of adrenergic receptors (α1, α2, β) stimulation on spontaneously appearing TREK-2-like channel activity was tested. The obtained results indicate that noradrenaline decreases the mean open probability of TREK-2-like channel currents by activation of β1 but not of α1- and α2-adrenergic receptors. Mean open time and channel conductance were not affected. The system of intracellular signaling pathways depends on the activation of protein kinase A. We also show that adrenergic control of TREK-2-like channel currents by adrenergic receptors was similar in pyramidal neurons isolated from young, adolescent, and adult rats. Immunofluorescent confocal scans of mPFC slices confirmed the presence of the TREK-2 protein, which was abundant in layer V pyramidal neurons. The role of TREK-2-like channel control by adrenergic receptors is discussed.

We transfected HEK-293 cells with cDNA for homomeric human α1 glycine receptors. 2-3 days after transfection, we recorded currents and used a submillisecond perfusion system to apply glycine to outside-out patches. The extracellular solution contained 150 mM chloride but the intracellular solution contained either 150 mM chloride (symmetric) or 9 mM chloride and 127 mM acetate ("physiological"). We clamped at a range of potentials near the current reversal potential for each condition. With symmetric chloride, currents were nearly constant during the 70 ms application of 3 mM glycine. With "physiological" chloride, currents reversed direction from inward to outward on the 5 ms time scale during agonist application. Similar biphasic currents were seen with gluconate as the substitute intracellular anion. Biphasic currents were observed at external pH values from 6.4-8.0, with HEPES, TRIS or PIPES as external buffer and with external choline replacing sodium. Activation of GlyR channels is dependent on Clin (J Neurosci 28:11454, 2008) but this cannot explain the observed reversal of currents. We are considering that local changes in Clin can cause this effect or that time-dependent changes in pore selectivity are possible. Support from NIH NS045095 (JPD)

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K+ (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K+ channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.

We present a new approach to nicotinic receptor kinetics and a new model explaining random variabilities in the duration of open events. The model gives new interpretation on brief and long receptor openings and predicts (for two identical binding sites) the presence of three components in the open time distribution: two brief and a long. We also present the physical model of the receptor block. This picture naturally and universally explains receptor desensitization, the phenomenon of central importance in cellular signaling. The model is based on single-channel experiments concerning the effects of hydrocortisone (HC) on the kinetics of control wild-type (WT) and mutated alphaD200Q mouse nicotinic acetylcholine receptors (nAChRs), expressed in HEK 293 cells. The appendix contains an original result from probability renewal theory: a derivation of the probability distribution function for the duration of a process performed by two independent servers.

Ageing in humans is accompanied by a reduction in the capacity of satellite cells to proliferate and the forming myoblasts to fuse. The processes of myoblast differentiation and fusion are associated with specific changes in the cells electrical properties. We wanted to elucidate the possible effects of ageing on these parameters and performed whole-cell patch-clamp recordings on human myoblasts obtained from biopsies of skeletal muscles from 2-, 48- and 76-year-old donors. First, we found that resting membrane potential on the 4th day of differentiation in vitro is less negative in the older than in the younger cells. Moreover, the oldest cells showed a smaller density of outward and inward potassium currents. More cells from the old and middle-age donors have a low (less than −40mV) potential of activation for the outward potassium current. We conclude that in human myoblasts biophysical properties of potassium currents change with donor age.