Whole-cell Current Recordings Research Articles

Molecular determinants of hERG potassium channel inhibition by disopyramide

The Class Ia antiarrhythmic drug disopyramide (DISO) causes QT interval prolongation that is potentially dangerous in acquired Long QT Syndrome but beneficial in short QT syndrome, through inhibition of the hERG-encoded channels responsible for rapid delayed rectifier K+ current (IKr). In this study, alanine mutants of hERG S6 and pore helix residues and MthK-based homology modelling and ligand docking were used to investigate molecular determinants of DISO binding to hERG. Whole-cell hERG current (IhERG) recordings were made at 37°C from HEK-293 cells expressing WT or mutant hERG channels. WT outward IhERG tails were inhibited with an IC50 of 7.3μM, whilst inward IhERG tails in a high [K+]e of 94mM were blocked with an IC50 of 25.7μM. The IC50 for the Y652A mutation was ~55-fold that of WT IhERG; this mutation also abolished a leftward shift in voltage-dependent IhERG activation present for WT hERG. The IC50 for F656A IhERG was ~51 fold its corresponding WT control. In contrast to previously studied methanesulphonanilide hERG inhibitors, neither the G648A S6 nor the T623A and S624A pore helical mutations modified DISO IC50. Computational docking with the hERG model showed that DISO did not exhibit a single unique binding pose; instead several low energy binding poses at the lower end of the pore cavity favoured interactions with Y652 and F656. In the WT hERG model DISO did not interact directly with residues at the base of the pore helix, consistent with the minimal effect of mutation of these residues on drug block.

Dopamine oxidation facilitates rotenone-dependent potentiation of N-methyl-d-aspartate currents in rat substantia nigra dopamine neurons

Rotenone is a mitochondrial poison that causes dopamine cell death and is used as a model of Parkinson's disease in rodents. Recently, we showed that rotenone augments currents evoked by N-methyl-D-aspartate (NMDA) by relieving voltage-dependent Mg(2+) block in rat substantia nigra compacta (SNC) dopamine neurons. Because rotenone is well known to generate reactive oxygen species (ROS), we conducted the present experiments to evaluate the role of ROS in mediating the effect of rotenone on NMDA current augmentation. Using patch pipettes to record whole-cell currents from SNC neurons in slices of rat brain, we found that the ability of rotenone (100 nM) to increase NMDA (3-30 μM) current was antagonized by the antioxidant agent n-acetylcysteine (1 mM). In contrast, mercaptosuccinate (1 mM), which blocks glutathione peroxidase and raises tissue levels of H(2)O(2), mimicked rotenone by augmenting inward currents evoked by NMDA. Because oxidation of dopamine can also generate ROS, we explored the role of dopamine on this action of rotenone. We prepared dopamine-depleted midbrain slices from rats that had been pretreated with reserpine (5 mg/kg ip) and alpha-methyl-para-tyrosine (AMPT, 250 mg/kg ip). Dopamine depletion blocked the ability of rotenone (100 nM) to increase inward current evoked by NMDA (30 μM). Rotenone-dependent augmentation of NMDA current was also blocked by the monoamine oxidase inhibitor pargyline (100 μM) in slices prepared from normal rats. In contrast, the dopamine precursor levodopa potentiated the action of rotenone on NMDA current. These results suggest that ROS and/or dopamine oxidation products mediate the ability of rotenone to potentiate NMDA currents. Because excessive NMDA receptor stimulation can produce excitotoxicity, our results suggest that oxidative metabolism of dopamine might facilitate the neurotoxicity of rotenone.

Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons

Mild uncoupling of oxidative phosphorylation has been reported to reduce generation of reactive oxygen species (ROS) and therefore may be neuroprotective. We reported previously that the mitochondrial poison rotenone enhanced currents evoked by N-methyl- d-aspartate (NMDA) by a ROS-dependent mechanism in rat midbrain dopamine neurons. Thus, rotenone, which produces a model of Parkinson's disease in rodents, may increase the risk of dopamine neuron excitotoxicity. The purpose of this study was to test the hypothesis that oxidative phosphorylation uncoupling agents would antagonize the effect of rotenone on NMDA current. We used patch pipettes to record whole-cell currents under voltage-clamp (− 60 mV) in substantia nigra dopamine neurons in slices of rat brain. Rotenone, NMDA and uncoupling agents were added to the brain slice superfusate. Inward currents evoked by NMDA (30 μM) more than doubled in amplitude after slices were superfused for 30 min with 100 nM rotenone. Continuous superfusion with the uncoupling agent carbonyl cyanide- p-trifluoromethoxy-phenylhydrazone (1–3 nM) or 2,4-dinitrophenol (100 nM) significantly antagonized and delayed the ability of rotenone to potentiate NMDA currents. Coenzyme Q 10 (1–10 nM), which has been reported to facilitate uncoupling protein activity, also antagonized this action of rotenone. These results suggest that mild uncoupling of oxidative phosphorylation may protect dopamine neurons against injury from mitochondrial poisons such as rotenone.

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.

Quantal Nature of Excitatory Synaptic Transmission in Cultured Hippocampal Cells of Rat and AMPAR Channel Kinetics

Fast excitatory postsynaptic currents (EPSCS) mediated by glutamate arise from AMAPR channels in the membrane of cultured hippocampal cells in culture, incubated for 2-3 weeks after the cultural procedure of dissected cells from embryonic rat brain (E 17), were identified under Nomarski optics and investigated electrophysiologically. Whole-cell current recording using the patch-clamp technique revealed the synaptic currents ranging from less than 10 to more than 200 pA at a holding potential of −80 mV. These currents were blocked by 4μM CNQX, indicating that they results from the activation of postsynaptic AMPA receptor channels. Addition of tetrodotoxin (TTX, 1μM) resulted in the loss of most currents of more than 50 pA in amplitude. The currents, which disappeared after these treatments, were seemed to be the results of spontaneous presynaptic action potentials. Moreover, 2μM-bicuculline was added in order to eliminate the inhibitory postsynaptic currents (IPSC) that were mediated by GABAA receptor channels. The peak of spontaneous miniature EPSC amplitude histograms was asymmetrical and a tail of larger amplitude miniature EPSCs was observed. The decay time courses of miniature EPSCs were fitted with single exponential component. The histogram of the decay time constants also conformed to asymmetrical distribution with a tail of longer time constant.We also recorded the currents responses activated by (IR and/or UV) laser photolysis which have been already shown previously. It was shown that the miniature EPSCs and laser-evoked current responses have the similar physiological properties, which assumed that these currents are mediated by a AMPA receptor channel kinetics model that has been already presented.

Caveolin-3 regulates the Volume-Regulated Anion Channel in Mouse Ventricular Cells

Caveolae are small invaginated microdomains located with a variety of signal transduction molecules on the plasma membrane. Recent reports showed that the knockout mice of a muscle-specific protein caveolin-3 (Cav-3 KO), a principal component of the caveolae in heart, displayed an enlargement of ventricular cells. Volume-regulated outwardly rectifying anion channel (VRAC) is activated by membrane stretch, and play a significant role in cell volume regulation in cardiac cells. However, it is unknown the properties of VRAC in the enlarged cardiac cells from Cav-3 KO mice. In this study, we examined that VRAC current and the cell volume regulation in freshly isolated single ventricular cells from Cav-3 KO mice (Hagiwara et al. 2000). Whole-cell current recording showed that the density of VRAC current induced by extracellular hypotonic solution (HYPO) is markedly reduced in the cells from Cav-3 KO mice, compared to that from wild-type (WT) mice. Video-image analysis revealed that the degree of HYPO-induced cell swelling in Cav-3 KO mice is significantly bigger than that in WT mice, and the regulatory volume decrease, which was seen in WT cells after osmotic swelling, is almost lost in cells from Cav-3 KO mice. This result is in parallel with the VRAC inhibition. In contrast, acidic extracellular pH-activated chloride current and extracellular UTP-activated CFTR current were affected less by the deficiency of caveoline-3. The attenuated VRAC current was restored by intracellular application of a VRAC modulator, phosphatidylinositol 3,4,5-trisphosphate (PIP3). These findings suggested that the attenuation of cardiac VRAC current is due to the PIP3 depletion in Cav-3 KO mice.

Berberine Reduces cAMP-Induced Chloride Secretion in T84 Human Colonic Carcinoma Cells through Inhibition of Basolateral KCNQ1 Channels

Berberine is a plant alkaloid with multiple pharmacological actions, including antidiarrhoeal activity and has been shown to inhibit Cl− secretion in distal colon. The aims of this study were to determine the molecular signaling mechanisms of action of berberine on Cl− secretion and the ion transporter targets. Monolayers of T84 human colonic carcinoma cells grown in permeable supports were placed in Ussing chambers and short-circuit current measured in response to secretagogues and berberine. Whole-cell current recordings were performed in T84 cells using the patch-clamp technique. Berberine decreased forskolin-induced short-circuit current in a concentration-dependent manner (IC50 80 ± 8 μM). In apically permeabilized monolayers and whole-cell current recordings, berberine inhibited a cAMP-dependent and chromanol 293B-sensitive basolateral membrane K+ current by 88%, suggesting inhibition of KCNQ1 K+ channels. Berberine did not affect either apical Cl− conductance or basolateral Na+–K+-ATPase activity. Berberine stimulated p38 MAPK, PKCα and PKA, but had no effect on p42/p44 MAPK and PKCδ. However, berberine pre-treatment prevented stimulation of p42/p44 MAPK by epidermal growth factor. The inhibitory effect of berberine on Cl− secretion was partially blocked by HBDDE (∼65%), an inhibitor of PKCα and to a smaller extent by inhibition of p38 MAPK with SB202190 (∼15%). Berberine treatment induced an increase in association between PKCα and PKA with KCNQ1 and produced phosphorylation of the channel. We conclude that berberine exerts its inhibitory effect on colonic Cl− secretion through inhibition of basolateral KCNQ1 channels responsible for K+ recycling via a PKCα-dependent pathway.

Amoxapine Inhibits Delayed Outward Rectifier K+ Currents in Cerebellar Granule Cells via Dopamine Receptor and Protein Kinase A Activation

Background: Although tricyclic antidepressants amoxapine is proposed to target 5-HT and D2 receptors, very few studies have addressed the effect of amoxapine on molecular and cellular mechanisms via receptor pathways. In this study, we test the effect of amoxapine on rat cerebellar granule neurons (CGNs) to address this possibility. Methods: CGNs cell culture, whole-cell current recording using a patch-clamp technique, western blot and non-radioactive detection analysis of phosphorylated protein kinase A (PKA) were used. Results: Amoxapine inhibits delayed rectifier potassium (I_K) current in a dose-dependent manner and modulates inactivation properties in CGNs. Those effects were not eliminated by preincubation with 5-HT or 5-HT receptor antagonists, but abolished by dopamine and D1/D5 receptor antagonists. Application of GTPγ-S and inhibitor of the Gs signalling cascade abolished the amoxapine-induced effect on I_K. The application of forskolin or dibutyryl-cAMP mimicked the inhibitory effect of amoxapine on I_K. Western blotting for phosphorylated PKA revealed that amoxapine significantly increased the intracellular levels of phosphorylated PKA, a marker of PKA activation. Conclusion: Amoxapine inhibits I_K currents in rat CGNs via cAMP/PKA-dependent pathways, as in mouse cortical neurons we reported earlier, but that involves D1-like receptors instead of 5-HT receptors.

Homer regulation of native plasma membrane calcium channels in A431 cells

Homers are adapter proteins that play a significant role in the organization of calcium signaling protein complexes. Previous functional studies linked Homer proteins to calcium influx in nonexcitable cells. These studies utilized calcium imaging or whole-cell current recordings. Because of limited resolution of these methods, an identity of Homer-modulated ion channels remained unclear. There are several types of plasma membrane calcium influx channels in A431 cells. In the present study, we demonstrated that Homer dissociation resulted in specific activation of I min channels but not of I max channels in inside-out patches taken from A431 cells. In contrast, inositol 1,4,5-trisphosphate activated both I min and I max channels in inside-out patches. Short (1a) and long (1c) forms of Homer had different effects on I min channel activity. Homer 1a but not Homer 1c activated I min in the patches. This study indicates that I min channels are specifically regulated by Homer proteins in A431 cells.

A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

We report on a simple and high-yield manufacturing process for silicon planar patch-clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high-quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high-impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high-fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole-cell current recordings obtained from a voltage-clamp stimulation protocol, and in current-clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch-clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high-information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity.

Mechanism of Potentiation of a Dysfunctional Epilepsy-Linked Mutated GABAA Receptor by a Neurosteroid (3α, 21-Dihydroxy-5α-pregnan-20-one): Transient Kinetic Investigations

The malfunction of a mutated GABA(A) receptor (alpha1beta2gamma2L(K289M)) in an inheritable form of epilepsy (GEFS+, generalized epilepsy with febrile seizures plus) in humans [Baulac, S., Huberfeld, G., Gourfinkel-An, I., Mitropoulou, G., Beranger, A., Prud'homme, J. F., Baulac, M., Brice, A., Bruzzone, R., and LeGuern, E. (2001) Nat. Genet. 28, 46-48] has been accounted for by a 5-fold decrease in the channel-opening equilibrium of the mutated receptor compared to the wild type [Ramakrishnan, L., and Hess, G. P. (2004) Biochemistry 43, 7534-7540]. Here we describe the mechanism by which the neurosteroid 3alpha, 21-dihydroxy-5alpha-pregnan-20-one (5alpha-THDOC) alleviates this malfunction of the mutated receptor transiently expressed in HEK293 cells. Two rapid reaction techniques, the cell-flow and the laser-pulse photolysis methods, were used in combination with whole-cell current recordings. 150-muM 5alpha-THDOC does not affect the rate constant for channel opening (k(op)) of approximately 250 s(-1) but does decrease the rate constant for channel closing (k(cl)) from 121 +/- 11 s(-1) to 56 +/- 21 s(-1). This results in an increase in the channel-opening equilibrium constant ((Phi(-1) = k(op)/k(cl)) by a factor of about 2, leading to about 50% alleviation of the malfunction of the inheritable mutated (alpha1beta2gamma2L(K289M)) GABA(A) receptor linked to GEFS+.

The role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca2+ channels in non-excitable cells has been studied. Receptor- and store-operated Ca2+ influxes have been measured using the fluorescent method of detection of cytosolic Ca2+ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca2+ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels Imax and non-selective channels Ins in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance Imin channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant alpha7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha3, alpha5, alpha7, beta2 and beta4 subunits was present during the course of differentiation, while mRNAs coding for alpha2, alpha4 and beta3 subunits were not expressed in PC12 cells. alpha7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha7 agonist choline. Increased alpha7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Less is more, or enough is enough? Ca2+‐dependent inactivation revisited

Less is more, or enough is enough? Ca²⁺‐dependent inactivation revisited

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR alpha7 subunit without compromising formation of functional receptors. A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplasmic loop between the third and fourth transmembrane domains of the mouse nAChR alpha7 subunit (alpha7Y). SH-EP1 cells were transfected with mouse nAChR wild type alpha7 subunits (alpha7) or with alpha7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with (125)I-labeled alpha-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Whole-cell currents revealed that alpha7Y nAChRs and alpha7 nAChRs were functional with comparable EC(50) values for the alpha7 nAChR-selective agonist, choline, and IC(50) values for the alpha7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that alpha7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little alpha7Y localized to the plasma membrane, typical of alpha7 nAChRs. nAChRs composed as homooligomers of alpha7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of alpha7 nAChRs. alpha7Y nAChRs may be used to elucidate properties of alpha7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

Rat neuronal nicotinic acetylcholine receptors containing α7 subunit: pharmacological properties of ligand binding and function

To compare pharmacological properties of heterologously expressed homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) with those of native nAChRs containing alpha7 subunit (alpha7* nAChRs) in rat hippocampus and cerebral cortex. We established a stably transfected HEK-293 cell line that expresses homomeric rat alpha7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat alpha7* nAChRs in rat hippocampus and cerebral cortex. We used [(125)I]-alpha-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the alpha7 nAChRs expressed in this cell line were studied using whole-cell current recording. The newly established cell line, KXalpha7R1, expresses homomeric alpha7 nAChRs that bind [(125)I]-alpha-bungarotoxin with a K(d) value of 0.38+/-0.06 nmol/L, similar to K(d) values of native rat alpha7* nAChRs from hippocampus (K(d)=0.28+/-0.03 nmol/L) and cerebral cortex (K(d)=0.33+/-0.05 nmol/L). Using whole-cell current recording, the homomeric alpha7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC(50) values of 280+/-19 micromol/L and 180+/-40 micromol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of alpha7 nAChRs, alpha-bungarotoxin (IC(50)=19+/-2 nmol/L) and methyllycaconitine (IC(50)=100+/-10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric alpha7 nAChRs and native alpha7* receptors in rat brain, but it also revealed several notable differences. This newly established stable cell line should be very useful for studying the properties of homomeric alpha7 nAChRs and comparing these properties to native alpha7* nAChRs.

Comparative effects of ultra-short-acting β1-blockers on voltage-gated tetrodotoxin-resistant Na+ channels in rat sensory neurons

To examine a possible mechanism for their antinociceptive actions, we compared the effects of two clinically used ultra-short-acting beta1-blockers, landiolol and esmolol, on tetrodotoxin-resistant sodium (TTX-r Na) channels in rat dorsal root ganglion neurons, which are important for nociception. In small (<30 microm) dorsal root ganglion neurons from Sprague-Dawley rats, recordings of whole-cell membrane currents were made using the patch-clamp technique. To examine the effects of landiolol and esmolol on TTX-r Na currents, whole-cell membrane Na currents were evoked every 10 s by stepping for 50 ms from a holding potential of -70 to -10 mV. Each drug was applied at stepwise-increased concentrations every 2 min. The voltage dependence of the steady-state inactivation of the TTX-r Na current was investigated by using a conventional double-pulse protocol. To test for use-dependent blockade of TTX-r Na channels by beta-blockers, trains of depolarizing pulses (to -10 from a holding potential of -70 mV) were applied at one of three frequencies (0.2, 5 or 20 Hz) in the absence or presence of drug (landiolol 8 mmol l, esmolol 140 micromol l). Esmolol blocked TTX-r Na currents in a dose-dependent and use-dependent manner, but a very high concentration of landiolol was required to block TTX-r Na channel activities. The half-maximal inhibitory concentrations (IC50) for the TTX-r Na current were (holding potential, -70 mV) landiolol 7.66 +/- 0.62 mmol l (n = 6) and esmolol 145 +/- 7.5 micromol l (n = 6), and the Hill coefficients were landiolol 1.06 +/- 0.09 (n = 6) and esmolol 0.96 +/- 0.05 (n = 6). Esmolol, but not landiolol, may have useful effects against pain related to TTX-r Na channel activity.

G019 Cholesterol depletion enhances Kv1.5-encoded K+ current by increasing Rab11-mediated recycling

Membrane lipid composition is a major determinant of protein organisation in the cell membrane. In a previous study, we reported that depletion of membrane cholesterol by methyl- f Ò-cyclodextrin (MCD) causes a marked increase in Kv1.5-current (Ikur) in neonatal cardiac myocytes. Here, we examined the mechanisms of the cholesterol effects on potassium current in adult rat cardiomyocytes (ARC). GFP-tagged Kv1.5 channels were transduced in ARC using adenoviral vectors and patch clamp experiments were performed to record whole-cell currents and single channel activity. Fluorescence recovery after photobleaching (FRAP) technique was used to investigate GFP-Kv1.5 channels mobility; 3D-epifluorescence microscopy was conducted to follow Kv1.5 channels trafficking. In both freshly isolated and cultured ARC over-expressing GFP-Kv1.5 channels, MCD induced a rapid (< 7 min) increase in Ikur but not Ito. On the contrary, incubation with the cholesterol donor LDL reduced Ikur. Single channel experiments revealed that MCD application caused a progressive and drastic increase of the number of active channels. Moreover, FRAP experiments showed that MCD reduced both mobility and recovery of GFP-Kv1.5. Several steps of the trafficking process of ion channels were studied. Blocking SNARE-mediated exocytosis with N-ethylmaleimide prevented the MCD-effect on Ikur. While disruption of Golgi complex/secretion pathway with brefeldine-A had no effect, manipulation of GTP-ases activity with GTP- f ×-S suppressed the MCD effect. Transfection with a dominant negative (DN) form of Rab11 effect but not Rab4 DN prevented the MCD. Moreover, Kv1.5 channels co-immunoprecipitated with Rab11 which is stringly expressed in myocardium and ARC (qPCR and western blot). Finally, 3D-microscopy evidenced that Kv1.5 channels association with Rab11-positive recycling endosomes observed in control condition disappeared following cholesterol depletion. Lowering cholesterol rapidly induces the insertion of Kv1.5 channels by a process that involves vesicle fusion and trafficking processes, particularly the Rab11-associated slow recycling pathway. Given the role of Kv1.5 channel in normal and pathological atrial electrical properties, this study opens news perspectives for therapeutic modulation of cardiac myocytes excitability.

Investigations of Ligand-gated Ion Channels Using Caged Neurotransmitters Reveal Mechanism-based Approaches to Modulating Receptor Function

Recognizing the problem of rapidly equilibrating membrane-bound proteins on cell surfaces with activating ligands due to the water layer that tightly adheres to the surface of spherical objects (1), we began to develop caged (2) neurotransmitters (3,4). A caged neurotransmitter can be equilibrated with receptors on a cell surface before photolysis releases the neurotransmitter in the microsecond time region (3,4). Using caged neurotransmitters in whole-cell current recordings enables the rate constants for the opening (kop) and closing (kcl) of the receptor channel, and thus the channel-opening equilibrium constant (Phe−1), to be measured without interference from diffusional delays or receptor desensitization.The rate constants and the channel-opening equilibrium constant of the excitatory nicotinic acetylcholine receptor (5) are altered in the presence of noncompetitive inhibitors, such as cocaine, as are those of a mutated inhibitory gamma-aminobutyric acid (GABA) receptor (6) linked to epilepsy (7). Based on the mechanisms of the two receptors, we developed compounds that bind with higher affinity to the open-channel than to the closed-channel conformation of each receptor, alleviating (8,9) the inhibition of the acetylcholine receptor and potentiating (10) the function of a mutated GABAA receptor linked to epilepsy.1. Landau & Lifshitz (1959) Fluid Mechanics (Pergamon, Oxford)2. Kaplan et al. (1978) Biochemistry 17, 19293. Milburn et al. (1989) Biochemistry 28, 514. Shembekar et al. (2007) Biochemistry 46, 54795. Hess et al. (2000) Proc. Natl. Acad. Sci. USA 97, 138956. Ramakrishnan. & Hess (2004) Biochemistry 43, 75347. Baulac et al. (2001) Nature Genetics 28, 468. Hess et al. (2003) Biochemistry 42, 61069. Chen et al. (2004) Biochemistry 43, 1014910. Krivoshein & Hess (2006) Biochemistry 45, 11632

Regulation of Nav1.7 and Nav1.8 peripheral nervous system sodium channels by auxiliary &amp;beta; subunits

Event Abstract Back to Event Regulation of Nav1.7 and Nav1.8 peripheral nervous system sodium channels by auxiliary β subunits Juan Zhao1, Cojen Ho2, Michael E. O'Leary2 and Mohamed Chahine1* 1 Le Centre de Recherche Universite Laval Robert-Giffard, Canada 2 Jefferson Medical College, United States The Nav1. 7 and Nav1. 8 Na channel isoforms are preferentially expressed in small-diameter sensory neurons of the dorsal root ganglia (DRG) where they are known to play a central role in nociception. The goal of these studies was to investigate the β subunit regulation of the Nav1.7 and Nav1.8 channels. The Na channel α and β subunits were heterologously expressed in HEK 293 cells and the functional properties assessed from whole-cell Na current recordings. Co-expressing the β3 subunit produced a hyperpolarizing shift in Nav1.7 activation (-5 mV) and slowed channel gating. The β1 subunit produced a depolarizing shift (+ 7 mV) in the midpoint of steady-state inactivation but no change in current kinetics. The β2 and β3 subunit did not alter Nav1.7 gating and none of the β subunits produced significant changes in Na current density. Co-expressing the β1 subunit increased the Nav1.8 current density 2-fold and produced hyperpolarizing shifts in both activation (-4 mV) and inactivation (-5 mV). This contrasted with the β3 subunit, which produced a 30% reduction in Nav1.8 current amplitude. The β2 and β3 subunits did not alter the gating or current density of the Nav1.8 channel. Co-expressing the β4 subunit produced hyperpolarizing shifts in the activation (-18 mV) and inactivation (-6 mV) of the Nav1.8 channel. To further investigate the contribution of the auxiliary subunits in vivo the transcripts encoding for β subunits were quantitatively measured in small-diameter sensory neurons isolated from the rat DRG. The β3 subunit is widely expressed in DRG nociceptors where it produces a hyperpolarizing shift in Nav1.7 activation and a reduction in Nav1.8 peak current amplitude. Although the β1 and β4 subunits differentially regulate the Nav1.7 and Nav1.8 channels these subunits are expressed at comparatively low levels in sensory neurons. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Neurobiology of pain and depression Citation: Zhao J, Ho C, O'Leary ME and Chahine M (2009). Regulation of Nav1.7 and Nav1.8 peripheral nervous system sodium channels by auxiliary β subunits. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.115 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Nov 2009; Published Online: 23 Nov 2009. * Correspondence: Mohamed Chahine, Le Centre de Recherche Universite Laval Robert-Giffard, Beauport, Qc G1J 2G3, Canada, Mohamed.Chahine@phc.ulaval.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine Google Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine Google Scholar Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine PubMed Juan Zhao Cojen Ho Michael E O'Leary Mohamed Chahine Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.