Decrease In Current Amplitude Research Articles

A positively charged residue at the Kv1.1 T1 interface is critical for voltage-dependent activation and gating kinetics.

Within the tetramerization domain (T1) of most voltage-gated potassium channels (Kv) are highly conserved charged residues that line the T1-T1 interface. We investigated the Kv1.1 residue R86 located at the narrowest region of the T1 interface. A Kv1.1 R86Q mutation was reported in a child diagnosed with lower limb dyskinesia (Set KK, Ghosh D, Huq AHM, Luat AF. Mov Disord Clin Pract 4: 784-786, 2017). The child did not present with episodic ataxia 1 (EA1) symptoms typically associated with Kv1.1 loss-of-function mutations. We characterized the electrophysiological outcome of the R86Q substitution by expressing Kv1.1 in Xenopus laevis oocytes. Mutated α-subunits were able to form functional channels that pass delayed rectifier currents. Oocytes that expressed only mutated α-subunits produced a significant reduction in Kv1.1 current and showed a positive shift in voltage dependence of activation. In addition, there was substantially slower activation and faster deactivation implying a reduction in the time the channel is in its open state. Oocytes co-injected with both mutated and wild-type cRNA in equal amounts, to mimic the heterozygous condition of the disease, showed a decrease in current amplitude at -10 mV, a positive shift in activation voltage-dependence and faster deactivation kinetics when compared with the wild-type channel. These findings indicate that T1 plays a role in Kv1.1's voltage-dependent activation and in its kinetics of activation and deactivation.NEW & NOTEWORTHY This is the first Kv1.1 study to characterize the electrophysiological and structural phenotype of a tetramerization (T1) domain mutation. Surprisingly, the mutated α-subunits were able to tetramerize, albeit with different gating kinetics and voltage dependence. This novel finding points to a clear role of T1 in the channel's voltage dependence and gating. Mimicking the heterozygous condition resulted in milder alterations in channel function when compared with previously reported mutations. This is in agreement with the child's milder symptoms.

Negative self-regulation of transient receptor potential canonical 4 by the specific interaction with phospholipase C-δ1

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4β is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP2). In this study, we present the regulation mechanism of the TRPC4 channel with PIP2 hydrolysis which is mediated by a channel-bound PLCδ1 but not by the GqPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca2+ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP2 depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP2 level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca2+ ions into the cell and promoting the PIP2 hydrolyzing activity of PLCδ1.

Identification of a proton sensor that regulates conductance and open time of single hERG channels

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.

P1235Acetylcholine-activated inward-rectifier potassium currents in atrial cardiomyocytes derived from induced pluripotent stem cells and atrial engineered heart muscle preparations

Abstract Background Abnormal activity of muscarinic receptor (M-receptor) activated potassium current (IK,ACh) has been suggested as a potential atrial-specific drug target for antiarrhythmic therapy in patients with atrial fibrillation (AF). Recent work suggests that atrial myocytes derived from human induced pluripotent stem cells (iPSC-CM) hold great potential for in vitro disease modeling and patient-specific drug testing. Here we aim to investigate functional expression of IK,ACh channels in these cells. Methods Atrial differentiation was initiated by temporal WNT and retinoic acid signaling modulation. Action potentials (APs) were recorded using sharp electrode technique in engineered heart muscle preparations (EHM). Currents were measured with voltage-clamp technique in atrial and ventricular iPSC-CM. IK,ACh was activated with a saturating concentration of the non-selective M-receptor agonist carbachol (CCh, 2 μM). Results APs measured in atrial EHMs were shorter compared to their ventricular counterparts (AP duration at 90% repolarization [APD90]: 163.2±3.7 ms, n=6 [EHMs] vs. 210.2±3.1 ms, n=3, p<0.001). In addition, application of CCh resulted in APD90 reduction by ∼18% and hyperpolarisation of the resting membrane potential in atrial but not in ventricular EHMs, pointing to functional IK,ACh expression in atrial preparations. Basal inward-rectifier current, (at −100 mV) in the absence of muscarinic receptor agonists was larger in ventricular iPSC-CM compared to atrial differentiation (−26.1±3.8 pA/pF, n=82/5 [n = cells/line] vs. −15.6±1.6 pA/pF, n=71/5, p<0.02.). CCh-induced current increase, attributed to IK,ACh, was detectable in all myocytes from atrial preparations (−25.2±3.6 pA/pF, n=71/5). The time course of activation with a slow decrease in current amplitude during the continuous presence of CCh resembled measurements in freshly isolated human atrial myocytes. In ventricular iPSC-CM preparations only 13 out of 73 myocytes showed an increase in inward-rectifier current in response to CCh, suggesting that ventricular preparations may contain a minor fraction of atrial myocytes. Of note, in the presence of the selective IK,ACh blocker tertiapin (100 nM), CCh had no effect on basal inward-rectifier current, providing further evidence for the molecular identity of the CCh-induced current increase as IK,ACh. Interestingly, TTP reduced basal current amplitude in CM isolated from atrial EHMs by ∼54% suggesting that agonist-independent, constitutively active IK,ACh contributes to the basal inward-rectifier current amplitude in these myocytes. Conclusion Our data demonstrate that atrial iPSC-CMs express functional IK,ACh-channels, which can be activated by M-receptor agonists. These findings further outline the potential of atrial iPSC-CMs and EHMs as a promising tool for the investigation of channel-associated pathologies such as AF. Acknowledgement/Funding grants from the DFG (VO 1568/3-1, IRTG1816, and SFB1002 project A13), the Else-Kröner-Fresenius Foundation (EKFS 2016_A20) and the DZHK (DZHK GOE MD3)

Stable Interaction between Voltage-Activated Ca2+ Channel α1 and β Subunits Revealed by Translocatable β Systems

High-voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit and two auxiliary α2δ and β subunits. Although it is well established that CaV β promotes cell surface expression and regulates the gating properties of CaV channels, the stability of the α1-β interaction in vivo remains unclear. Here, we address this issue by engineering translocatable CaV β systems that allow the real-time measurement of the coupling in live imaging and patch-clamp. In cells without CaV α1B expression, all constructed β subunits except palmitoylated β2a were translocated to the intracellular target membranes by rapamycin application. However, in cells co-expressed with CaV α1B no translocation of the β subunits was measured up to 2 hrs. In addition, rapamycin-induced recruitment of CaV β subunits to the plasma membrane did not affected the gating properties of CaV channels. In contrast, double mutation of CaV β subunits was shown to be dissociated easily from CaV α1 by rapamycin application. In these mutant forms, dissociation of β subunit from CaV α1B lead to the decrease in current amplitude, PIP2 sensitivity and current recovery from Gβγ inhibition. Furthermore, it inhibited inactivation and shifted the voltage dependent IV curve to the right in the live cells. When cells were cotransfected with double mutated CaV β1b and β2a together, rapamycin lead to dissociation of β1b, but not β2a, from CaV α1B. The CaV α1B separated from β1b did not further interact with CaV β2a, suggesting that once dissociated, CaV α1 do not interact with other β. Taken together, our data demonstrate that the interaction of CaV α1 with β subunit is very stable in live cells.

A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.

First-principles simulations for excitation of currents in linear carbon chains under femtosecond laser pulse irradiation

We discuss the response of linear carbon chains to a femtosecond laser pulse. The influence of the chain structure on the excited currents was taken into account. The simulation results showed that excited currents were reversible when laser intensity was relatively weak. An unexpected decrease of current amplitude was found for a laser pulse with a wavelength of 200 nm, which may indicate the transient break of pi bonds in linear carbon chains. For chains with more than 10 atoms, the excited current oscillated with increasing amplitude during simulation time, which was due to the formation of plasma-like resonance.

Fabrication and Characterization of a Test Platform Integrating Nanoporous Structures With Biochemical Functionality

The application of solid-state nanopore technology for biosensing is a rapidly developing area of research with high commercial potential. Different synthetic materials, including silicon nitride, alumina, and polymers, are employed to fabricate single and multiple pores and offer a good platform for selective biomolecule detection. Two solid-state pore arrays, one with integrated silicon microfluidic system, were considered and an immobilization strategy suitable for detecting a single-stranded DNA (ssDNA) sequence was investigated. For the silicon nitride pores, a modification method based on the use of 3-aminopropyltriethoxysilane for silanization and 1,4-phenylene diisothiocyanate for amine crosslinking was applied to immobilize 100-nM ssDNA (amine C6) and a 100-nM limit of detection for complementary to probe ssDNA (Cy5) was estimated. The polycarbonate pores (the second type of the pore arrays) underwent surface modification based on an oxidation reduction reaction using sodium periodate and sodium borohydride and was used to immobilize 10-nM ssDNA and an estimated 100-nM limit of detection was also achieved. Linear sweep voltammetry was used to characterize the pores and a current potential profile was obtained after both immobilization of probe ssDNA and hybridization of complementary to probe ssDNA on the modified pore array surface. A decrease in current amplitude was measured after surface modification of both pore arrays, and this was attributed to the appearance of an additional layer on the pore surface reducing the pore opening and hindering the current flow. The hybridization event was also supported by contact angle measurements, where an increase in hydrophilicity was recorded at the different surface modification steps that were applied to produce the biofunctionalized nanopore. In addition, fluorescence was observed on the surfaces after hybridization, through incorporation of a CY5 fluorescent tag attached on the 5' end of the complementary to probe DNA. These results show the potential to use both silicon nitride and polycarbonate nanopores in DNA detection applications.

The ‘funny’ side of sepsis

The ‘funny’ current (If) of pacemaker cells is the main membrane mechanism generating the diastolic depolarization, the phase of the action potential responsible for cardiac spontaneous activity. If is also responsible for heart rate (HR) modulation by the autonomic nervous system (DiFrancesco, 2010). In the heart, both during development and in adulthood, the kinetic properties and expression levels of If match the need of the cells to perform pacemaker activity. While at immature stages of embryonic development all cardiomyocytes are autorhythmic and express a robust If current activated within the physiological range of voltages, shortly after birth the pacemaker activity and If expression become restricted to cells of the conduction system only, while in the working myocardium the current is downregulated and its activation is shifted to more negative, non-physiological voltages (DiFrancesco, 2010). The characteristics of native If are the results of a complex interplay of different factors. The kinetic properties and expression levels of the f-channels, encoded by the HCN genes (HCN1–4), are finely regulated by auxiliary proteins (caveolin-3, MiRP-1, KCR-1, SAP97, TRP8) and lipids (phosphatidylinositol 4,5-bisphosphate) and also by post-translational modifications (phosphorylation and glycosylation). Any alteration of these regulatory pathways may modify the contribution of If to the action potential and become arrhythmogenic (Baruscotti et al. 2010). It is known that lipopolysaccharides (LPS) released from Gram-negative bacteria during severe sepsis reduce both native If and HCN2-or HCN4-mediated currents and shift their activation to more negative potentials. However, the basic mechanism involved was still unexplored. In this issue of The Journal of Physiology, Klockner et al. have elucidated molecular aspects of the process underlying such modulation (Klockner et al. 2014). Using the cell-attached configuration of the patch-clamp technique, Klockner et al. have shown that the LPS effect is not mediated by any of the intracellular modulatory pathways affecting HCN channels, but is instead due to a direct interaction with the channel. Klockner et al. have also shown that alterations of HCN current require the integrity of the LPS molecule, since neither the pro-inflammatory lipid A alone nor the O-chain polysaccharidic region alone are effective (Klockner et al. 2014). The effect of LPS on HCN currents has some unusual features and may represent a novel channel modulatory mechanism. While the decrease of current amplitude may simply reflect a direct blocking action on the channel pore, the large (∼20 mV) negative shift of the activation curve implies an interaction with the voltage sensor or at least a distortion of the electrical field sensed by the voltage sensor. Another unusual feature of LPS action is that the HCN channel voltage dependence is modified by an extracellular agent. The extracellular action of LPS is confirmed by experiments showing that HCN channel glycosylation partly prevents LPS action, while de-glycosylation of HCN channels significantly amplifies it (Klockner et al. 2014). Further specific experiments will be required to more fully clarify the details of LPS action on the channel structure. HR and heart rate variability (HRV), the beat-to-beat change of the duration of the R–R interval, are important indicators of cardiovascular conditions, and it is well established that low HR and high HRV are associated with a lower risk of life-threatening cardiac events (Papaioannou et al. 2013). Two components affect HR and HRV: the balance between the sympathetic and parasympathetic outputs, and intrinsic cellular factors (Papaioannou et al. 2013). Because of its involvement in the generation of the heartbeat and HR modulation, the contribution of the funny current may be especially relevant to HRV. It is known for example that supplementation with fish oil (rich in ω-3 polyunsaturated fatty acids (ω-3 PUFAs)) decreases HR, an effect attributable to a reduced If current, and increases HRV (Billman, 2013). Also, the ivabradine-induced pharmacological blockade of If induces a HR reduction and a HRV increase (DiFrancesco & Camm, 2004; Papaioannou et al. 2013). During sepsis, If is specifically decreased by LPS but HRV is decreased too, rather than increased. This atypical behaviour can be explained since as well as inhibiting If, LPS also have a stimulating effect on sympathetic output. In the presence of LPS, the If current is essentially fully inhibited, since although the LPS-induced decrease of If conductance is quantitatively similar to that produced by ω-3 PUFAs or by ivabradine (about 30%), the concurrent hyperpolarizing shift in the voltage dependence effectively zeroes its contribution. An increased sympathetic drive and a concurrent abolishment of the If-mediated rate control narrow the range of physiological rate modulation and are likely to make the system more susceptible to failure under stressful conditions. In summary, the work of Klockner et al. indicates that LPS release during sepsis can modify the If properties by shifting them out of their physiological range, which may impair normal rate control and decrease the cardiac safety factor, making the system more prone to life-threatening perturbations.

Brugada Syndrome Disease Phenotype Explained in Apparently Benign Sodium Channel Mutations

Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac sodium channel. Typically, BrS mutations in SCN5A result in a reduction of sodium current with some mutations even exhibiting a dominant-negative effect on wild-type (WT) channels, thus leading to an even more prominent decrease in current amplitudes. However, there is also a category of apparently benign (atypical) BrS SCN5A mutations that in vitro demonstrates only minor biophysical defects. It is therefore not clear how these mutations produce a BrS phenotype. We hypothesized that similar to dominant-negative mutations, atypical mutations could lead to a reduction in sodium currents when coexpressed with WT to mimic the heterozygous patient genotype. WT and atypical BrS mutations were coexpressed in Human Embryonic Kidney-293 cells, showing a reduction in sodium current densities similar to typical BrS mutations. Importantly, this reduction in sodium current was also seen when the atypical mutations were expressed in rat or human cardiomyocytes. This decrease in current density was the result of reduced surface expression of both mutant and WT channels. Taken together, we have shown how apparently benign SCN5A BrS mutations can lead to the ECG abnormalities seen in patients with BrS through an induced defect that is only present when the mutations are coexpressed with WT channels. Our work has implications for risk management and stratification for some SCN5A-implicated BrS patients.

Hydrogen peroxide-induced reduction of delayed rectifier potassium current in hippocampal neurons involves oxidation of sulfhydryl groups

This study examined the effect of H2O2 on the delayed rectifier potassium current (IKDR) in isolated hippocampal neurons. Whole-cell voltage-clamp experiments were performed on freshly dissociated hippocampal CA1 neurons of SD rats before and after treatment with H2O2. To reveal the mechanism behind H2O2-induced changes in IKDR, cells were treated with different oxidizing and reducing agents. External application of membrane permeable H2O2 reduced the amplitude and voltage-dependence of IKDR in a concentration dependent manner. Desferoxamine (DFO), an iron-chelator that prevents hydroxyl radical (OH) generation, prevented H2O2-induced reduction in IKDR. Application of the sulfhydryl-oxidizing agent 5,5 dithio-bis-nitrobenzoic acid (DTNB) mimicked the effect of H2O2. Sulfhydryl-reducing agents dithiothreitol (DTT) and glutathione (GSH) alone did not affect IKDR; however, DTT and GSH reversed and prevented the H2O2-induced inhibition of IKDR, respectively. Membrane impermeable agents GSH and DTNB showed effects only when added intracellularly identifying intracellular sulfhydryl groups as potential targets for hydroxyl-mediated oxidation. However, the inhibitory effects of DTNB and H2O2 at the positive test potentials were completely and partially abolished by DTT, respectively, suggesting an additional mechanism of action for H2O2, that is not shared by DTNB. In summary, this study provides evidence for the redox modulation of IKDR, identifies hydroxyl radical as an intermediate oxidant responsible for the H2O2-induced decrease in current amplitude and identifies intracellular sulfhydryl groups as an oxidative target.

Human Ether-a-Go-Go-Related Gene Channel Blockers and its Structural Analysis for Drug Design

The human ether-a-go-go-related gene (hERG) is a K+ channel protein mainly expressed in the heart and the nervous systems and its blockade by non-cardiovascular acting drugs resulted in tachycardia and sudden death. In this present review, we have focused the physicochemical properties responsible for the hERG blocking activity of structurally different compounds. The reported research works showed that the hydrophobicity on the van der Waals (vdW) surface of the molecules (aroused from the aromatic ring) necessary for the hERG blocking activity along with topological and electronic properties. The quinolizidine alkaloids (natural products) such as oxymatrine, sophoridine, sophocarpine and matrine carry the common molecular structure of O=C=N-C-C-C-N that possessed positive ionotropic effect and hERG blocking activity. Acehytisine hydrochloride (previously named Guangfu base A) was isolated from the root of Aconitum coreanum (Levl.), is an anti-arrhythmic drug in phase IV clinical trial. The isoquinoline alkaloid, neferine (Nef) induces a concentration-dependent decrease in current amplitude (IC50 of 7.419 MM). Most of these natural product compounds contain non-flexible aromatic structures but have significant activity due to the presence of optimum hydrophobicity. Recent research works revealed that Eag and hERG channels are expressed by a variety of cancer cell lines and tissues. The Eag channel showed an oncogenic potential while hERG channels are associated with more aggressive tumors and have a role in mediating invasion. This review concluded that the consideration of physicochemical properties necessary for the hERG blocking activity will guide to develop novel drugs with less cardiotoxicity.

An evolutionarily conserved mode of modulation of Shaw ‐like K + channels

Voltage-gated K(+) channels of the Shaw family (also known as the KCNC or Kv3 family) play pivotal roles in mammalian brains, and genetic or pharmacological disruption of their activities in mice results in a spectrum of behavioral defects. We have used the model system of Caenorhabditis elegans to elucidate conserved molecular mechanisms that regulate these channels. We have now found that the C. elegans Shaw channel KHT-1, and its mammalian homologue, murine Kv3.1b, are both modulated by acid phosphatases. Thus, the C. elegans phosphatase ACP-2 is stably associated with KHT-1, while its mammalian homolog, prostatic acid phosphatase (PAP; also known as ACPP-201) stably associates with murine Kv3.1b K(+) channels in vitro and in vivo. In biochemical experiments both phosphatases were able to reverse phosphorylation of their associated channel. The effect of phosphorylation on both channels is to produce a decrease in current amplitude and electrophysiological analyses demonstrated that dephosphorylation reversed the effects of phosphorylation on the magnitude of the macroscopic currents. ACP-2 and KHT-1 were colocalized in the nervous system of C. elegans and, in the mouse nervous system, PAP and Kv3.1b were colocalized in subsets of neurons, including in the brain stem and the ventricular zone. Taken together, this body of evidence suggests that acid phosphatases are general regulatory partners of Shaw-like K(+) channels.

Human Ether-a-Go-Go-Related Gene Channel Blockers and its Structural Analysis for Drug Design

The human ether-a-go-go-related gene (hERG) is a K+ channel protein mainly expressed in the heart and the nervous systems and its blockade by non-cardiovascular acting drugs resulted in tachycardia and sudden death. In this present review, we have focused the physicochemical properties responsible for the hERG blocking activity of structurally different compounds. The reported research works showed that the hydrophobicity on the van der Waals (vdW) surface of the molecules (aroused from the aromatic ring) necessary for the hERG blocking activity along with topological and electronic properties. The quinolizidine alkaloids (natural products) such as oxymatrine, sophoridine, sophocarpine and matrine carry the common molecular structure of O=C=N–C–C–C–N that possessed positive ionotropic effect and hERG blocking activity. Acehytisine hydrochloride (previously named Guangfu base A) was isolated from the root of Aconitum coreanum (Levl.), is an anti-arrhythmic drug in phase IV clinical trial. The isoquinoline alkaloid, neferine (Nef) induces a concentration-dependent decrease in current amplitude (IC50 of 7.419 ΜM). Most of these natural product compounds contain non-flexible aromatic structures but have significant activity due to the presence of optimum hydrophobicity. Recent research works revealed that Eag and hERG channels are expressed by a variety of cancer cell lines and tissues. The Eag channel showed an oncogenic potential while hERG channels are associated with more aggressive tumors and have a role in mediating invasion. This review concluded that the consideration of physicochemical properties necessary for the hERG blocking activity will guide to develop novel drugs with less cardiotoxicity.

PIP 2 controls voltage-sensor movement and pore opening of Kv channels through the S4–S5 linker

Voltage-gated K(+) (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP(2) depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP(2). Gating current measurements revealed that PIP(2) constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP(2) controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.

Comparative Effects of Liensinine and Neferine on the Human Ether-a-go-go-related Gene Potassium Channel and Pharmacological Activity Analysis

Liensinine and neferine, a kind of isoquinoline alkaloid, can antagonize the ventricular arrhythmias. The human ether-a-go-go-related gene (hERG) is involved in repolarization of cardiac action potential. We investigated the effects of liensinine and neferine on the biophysical properties of hERG channel and the underlying structure–activity relationships. The effects of liensinine and neferine were examined on the hERG channels in the stable transfected HEK293 cells using a whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. The pharmacokinetics and tissue distribution determination of liensinine and neferine in rats were determined by a validated RP-HPLC method. Liensinine and neferine induced decrease of current amplitude in dose-dependent. Liensinine reduced hERG tail current from 70.3±6.3 pA/pF in control group to 56.7±2.8 pA/pF in the 1 µM group, 53.0±2.3 pA/pF (3 µM) and 17.8±0.7 pA/pF (30 µM); the corresponding current densities of neferine-treated cells were 41.9±3.1 pA/pF, 32.3±3.1 pA/pF and 16.2±0.6 pA/pF, respectively. Neferine had binding affinity for the open and inactivated state of hERG channel, liensinine only bound to the open state. The inhibitory effects of liensinine and neferine on hERG current were attenuated in the F656V or Y652A mutant channels. Neferine distributed more quickly than liensinine in rats, which was found to be in higher concentration than liensinine. Both liensinine and neferine had no effect on the generation and expression of hERG channels. In conclusion, neferine is a more potent blocker of hERG channels than liensinine at low concentration (<10 µM), which may be due to higher hydrophobic nature of neferine compared with liensinine. Neferine may be safety even for long-term treatment as an antiarrhythmic drug.

Calcium-dependent block of P2X7 receptor channel function is allosteric

Among purinergic P2X receptor (P2XR) channels, the P2X7R exhibits the most complex gating kinetics; the binding of orthosteric agonists at the ectodomain induces a conformational change in the receptor complex that favors a gating transition from closed to open and dilated states. Bath Ca2+ affects P2X7R gating through a still uncharacterized mechanism: it could act by reducing the adenosine triphosphate4− (ATP4−) concentration (a form proposed to be the P2X7R orthosteric agonist), as an allosteric modulator, and/or by directly altering the selectivity of pore to cations. In this study, we combined biophysical and mathematical approaches to clarify the role of calcium in P2X7R gating. In naive receptors, bath calcium affected the activation permeability dynamics indirectly by decreasing the potency of orthosteric agonists in a concentration-dependent manner and independently of the concentrations of the free acid form of agonists and status of pannexin-1 (Panx1) channels. Bath calcium also facilitated the rates of receptor deactivation in a concentration-dependent manner but did not affect a progressive delay in receptor deactivation caused by repetitive agonist application. The effects of calcium on the kinetics of receptor deactivation were rapid and reversible. A438079, a potent orthosteric competitive antagonist, protected the rebinding effect of 2’(3′)-O-4-benzoylbenzoyl)ATP on the kinetics of current decay during the washout period, but in the presence of A438079, calcium also increased the rate of receptor deactivation. The corresponding kinetic (Markov state) model indicated that the decrease in binding affinity leads to a decrease in current amplitudes and facilitation of receptor deactivation, both in an extracellular calcium concentration–dependent manner expressed as a Hill function. The results indicate that calcium in physiological concentrations acts as a negative allosteric modulator of P2X7R by decreasing the affinity of receptors for orthosteric ligand agonists, but not antagonists, and not by affecting the permeability dynamics directly or indirectly through Panx1 channels. We expect these results to generalize to other P2XRs.

Neuropathic Nav1.3-Mediated Sensitization to P2X Activation is Regulated by Protein Kinase C

BackgroundIncreased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na+ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.ResultsUsing the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na+ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Nav1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Nav1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.ConclusionNeuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Nav1.3 subunit, and the function of the Nav1.3 channel is regulated by PKC.

Blockade of HERG K+ channel by isoquinoline alkaloid neferine in the stable transfected HEK293 cells

We studied the effects of isoquinoline alkaloid neferine (Nef) extracted from the seed embryo of Nelumbo nucifera Gaertn on Human ether-à-go-go-related gene (HERG) channels stably expressed in human embryonic kidney (HEK293) cells using whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. Nef induced a concentration-dependent decrease in current amplitude according to the voltage steps and tail currents of HERG with an IC(50) of 7.419 microM (n(H) -0.5563). Nef shifted the activation curve in a significantly negative direction and accelerated recovery from inactivation and onset of inactivation, however, slowed deactivation. In addition, it had no significant influence on steady-state inactivation curve. Western blot and immunofluorescence results suggested Nef had no significant effect on the expression of HERG protein. In summary, Nef can block HERG K(+) channels that functions by changing the channel activation and inactivation kinetics. Nef has no effect on the generation and trafficking of HERG protein. A blocked-off HERG channel was one mechanism of the anti-arrhythmic effects by Nef.

Dopamine D4 Receptors Regulate GABAA Receptor Trafficking via an Actin/Cofilin/Myosin-dependent Mechanism

The GABA(A) receptor-mediated inhibitory transmission in prefrontal cortex (PFC) is implicated in cognitive processes such as working memory. Our previous study has found that GABA(A)R current is subject to the regulation of dopamine D(4) receptors, a PFC-enriched neuromodulator critically involved in various mental disorders associated with PFC dysfunction. In this study, we have investigated the cellular mechanism underlying D(4) modulation of GABA(A)Rs. We found that the density of surface clusters of GABA(A)R beta2/3 subunits was reduced by D(4), suggesting that the D(4) reduction of GABA(A)R current is associated with a decrease in functional GABA(A)Rs at the plasma membrane. Moreover, the D(4) reduction of GABA(A)R current was blocked by the actin stabilizer phalloidin and was occluded by the actin destabilizer latrunculin, suggesting that D(4) regulates GABA(A)R trafficking via an actin-dependent mechanism. Cofilin, a major actin depolymerizing factor whose activity is strongly increased by dephosphorylation at Ser(3), provides the possible link between D(4) signaling and the actin dynamics. Because myosin motor proteins are important for the transport of vesicles along actin filaments, we also tested the potential involvement of myosin in D(4) regulation of GABA(A)R trafficking. We found that dialysis with a myosin peptide, which competes with endogenous myosin proteins for actin-binding sites, prevented the D(4) reduction of GABA(A)R current. These results suggest that D(4) receptor activation increases cofilin activity presumably via its dephosphorylation, resulting in actin depolymerization, thus causing a decrease in the myosin-based transport of GABA(A)R clusters to the surface.