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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.

Simple Summary The pyramidal neurons of rat prefrontal cortex express potassium channels identified as a non-canonical splice variant of the TREK-2 channel. The main function of TREK channels is to regulate the resting membrane potential. We showed that cytoplasmic Ca2+ upregulates the activity of TREK-2-like channels. Previous studies have indicated that the activation of TREK-2 channels is mediated by PI(4,5)P2, a polyanionic lipid in the inner leaflet of the plasma membrane. While TREK channels are believed to not be regulated by calcium, our work shows otherwise. We propose a model in which calcium ions enable the formation of PI(4,5)P2 nanoclusters, which stabilize active conformation of the channel. Abstract 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.

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.

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.

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

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.

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.