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Introduction. In recent years, interactions of various polyamines with a number of ionotropic receptors have been reported. Such interactions can be either negative (inhibition) or positive (potentiation). It is proposed that hydrophilic polyamines act as open-channel blockers and bind sites deeply in the ion channel pore. Hydrophobic polyamines are believed to act in the shallower part of the pore. There has been cause to think that polyamines with two aromatic moieties block the nicotinic acetylcholine (nACh) receptor by adopting a U-shaped conformation, that is, a conformation in which the long positively charged polyamine chain enters the ion channel while aromatic moieties interact with extracellular parts of α-subunits. Objective. Our goal was to determine whether and how changes in the structure of methoctramine (a polyamine with two aromatic moieties) affect the way in which the nACh receptor is blocked. We synthesized derivatives of methoctramine which have a less flexible structure than methoctramine itself and may be less capable of adopting a U-shaped conformation within the ion channel. Materials and method. Whole-cell ACh-induced currents were recorded from mouse i28 satellite cells expanded in culture. Recordings were performed both in the presence and in the absence of polyamines. Results. All tested polyamines applied at a concentration of 5 mM blocked ACh-induced currents. Depending on the number of protonated nitrogen atoms, polyamines decreased the current amplitude and/or increased the decay rate of the current. Conclusions. We propose two possible mechanisms to explain the action of polyamines: desensitization, and displacement of agonist molecules from their binding sites. The impact of the number of protonated nitrogen atoms is discussed.

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.

Background: Previous 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. Results: The 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. Conclusions: The 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.

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.

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.

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

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 μM) 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 μM and 200 μM) 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.

The biophysical properties of the nicotinic acetylcholine receptor (AChR) are known to be modulated by some steroid hormones; their precise mechanism of action is, however, unclear. A possible direct effect of the glucocorticoid corticosterone (COR) on AChRs of mouse C2C12 myotubes was studied in the cell-attached patch-clamp configuration. When added to pipette solution containing acetylcholine, COR had no effect on the single channel conductance, but it reduced the longest time constant of both open time and burst duration histograms by 55% and 65%, respectively. COR also increased nearly by 10-fold the middle time constant of the closed time histogram. COR coupled to a hydrophilic molecule such as bovine serum albumin, however, affected only the closed time distribution. These results suggest that existence of specific recognition sites for COR on the surface of the cell membrane and/or the AChR protein.

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.

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)View Large Image | View Hi-Res Image | Download PowerPoint Slide

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.

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 -40 mV) potential of activation for the outward potassium current. We conclude that in human myoblasts biophysical properties of potassium currents change with donor age.

Recently, it has been reported that large-conductance Ca(2+)-activated potassium channels, also known as BK(Ca)-type potassium channels, are present in the inner mitochondrial membrane of the human glioma LN229 cell line. Hence, in the present study, we have investigated whether BK(Ca)-channel openers (BK(Ca)COs), such as the benzimidazolone derivatives NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one) and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), affect the functioning of LN229 glioma cell mitochondria in situ. We examined the effect of BK(Ca)COs on mitochondrial membrane potential, mitochondrial respiration and plasma membrane potassium current in human glioma cell line LN229. We found that BK(Ca)COs decrease the mitochondrial membrane potential with an EC(50) value of 3.6+/-0.4 microM for NS1619 and 5.4+/-0.8 microM for NS004. This mitochondrial depolarization was accompanied by an inhibition of the mitochondrial respiratory chain. Both BK(Ca)COs induced whole-cell potassium current blocked by charybdotoxin, as measured by the patch-clamp technique. The BK(Ca)COs had no effect on membrane bilayer conductance. Moreover, the inhibition of mitochondrial function by NS004 and NS1619 was without effect on cell survival, as measured by lactate dehydrogenase release from the cells.

There are 9 channels of the ClC family in mammals and few others in fishes, plants, yeast and bacteria. The ClC channels are present in different tissues and play a role in transmembrane potential stabilization, transepithelial transport, cell volume regulation, acidification of intracellular organelles. The genetic defects of ClC-1 chloride channel lead to myotonias, the defect in ClC-5 channel to the formation of stones in kidney, while the defect in ClC-Kb channel leads to the Bartter's syndrome.

Implantation of myoblasts is a strategy used to enhance the regeneration of skeletal muscle tissue in vivo. In mouse models, myogenic cell lines and primary cells have been employed with different yields of adult muscle tissue formed. The present work is a study of some developmental features of expanded primary mouse myoblasts (i28), which have been shown to form muscle tissue. i28 myoblasts were differentiated in vitro and the expression of acetylcholine receptor channels and maturation of the excitation–contraction coupling mechanism were investigated using patch clamp and videoimaging techniques. In all the developing cells the embryonic isoform of the acetylcholine receptors was present. Skeletal muscle-type excitation–contraction coupling (i.e., a mechanical link between voltage-dependent calcium channels and ryanodine receptor channels) was detected in about 75% of differentiating i28 myotubes. Only these cells showed spontaneous changes in cytosolic free calcium concentration associated with twitches. Our findings are the first description of the physiological properties of expanded primary myoblasts which are used for implantation and confirm that they are a heterogeneous cell population. In comparison to permanent cell lines, the Ca2+ signaling is more similar to that described in mature nonexpanded muscle fibers. This suggests that cultured primary cells are, so far, the most suitable cell type for muscle regeneration.

The kinetics of the nicotinic acetylcholine receptor (AChR) channel were analysed in the presence of hydrocortisone (HC, 100–400 μM), an electrically neutral steroid. The channel open time decreased, and in contrast to control conditions did not show any voltage dependency. However, HC induced a new (blocked) component in the closed time distribution, with a time constant that decreased with membrane hyperpolarization. HC decreased also, in a concentration-dependent way, the open time per burst. After coupling HC to bovine serum albumin, to restrict the place of steroid action at the external surface of the membrane, a voltage dependency of steroid action persisted. The effects of HC on the open and blocked time constants did not depend on agonist concentration, but was dependent on the type of agonist used (acetylcholine or nicotine). These results support the hypothesis that HC molecules bind near the agonist binding site.