Outward Rectification Research Articles

The Outwardly Rectifying Current of Layer 5 Neocortical Neurons that was Originally Identified as "Non-Specific Cationic" Is Essentially a Potassium Current.

In whole-cell patch clamp recordings from layer 5 neocortical neurons, blockade of voltage gated sodium and calcium channels leaves a cesium current that is outward rectifying. This current was originally identified as a “non-specific cationic current”, and subsequently it was hypothesized that it is mediated by TRP channels. In order to test this hypothesis, we used fluorescence imaging of intracellular sodium and calcium indicators, and found no evidence to suggest that it is associated with influx of either of these ions to the cell body or dendrites. Moreover, the current is still prominent in neurons from TRPC1-/- and TRPC5-/- mice. The effects on the current of various blocking agents, and especially its sensitivity to intracellular tetraethylammonium, suggest that it is not a non-specific cationic current, but rather that it is generated by cesium-permeable delayed rectifier potassium channels.

Electrophysiological Properties of Magno‐and Parvo‐Cellular Neurons in Rat Hypothalamic Paraventricular Nucleus

The hypothalamic paraventricular nucleus (PVN) contains magnocellular neurosecretory neurons (PVN‐MC) projecting to posterior pituitary, and parvocellular neurons projecting to anterior pituitary (neurosecretory), medulla (PVN‐RVLM) and/or spinal cord (PVN‐IML) (presympathetic). These PVN neurons are different in their roles and projections, but the differences in excitability are not well known. In this study, we compared the basic electrophysiological properties of these neuron groups.The PVN‐MC, PVN‐IML and/or PVN‐RVLM neurons were identified by electrophysiological property (expressing a prominent transient outward rectification) and retrograde dye (FluoSphere‐Red). The spontaneous firing, EPSC and action potential were recorded in the cell‐attached or whole‐cell mode with Axopatch 200B. Data were sampled at 20 KHz using pClamp 8 data‐acquisition and analysis software.In the voltage clamp cell‐attached mode, the frequency of spontaneous firing in the PVN‐IML neurons was higher than those in the other two groups (PVN‐ RVLM, PVN‐IML, PVN‐MC: 1.4 vs. 2.8 vs. 1.0 Hz, n=30, 27, 29; p<0.05). The mean frequencies of IPSC (5.1Hz, n=11) was lowest and the 37~90% decay time of IPSC (5.6 ms, n=9) in PVN‐IML neurons was shortest, and the PVN‐MC neurons (191.0 mV n=12) had shown the largest amplitude of IPSC (PVN‐RVLM, PVN‐IML: 61 vs. 55 mV n=16, 11; p<0.05). The 37~90% decay time (5.8 ms, n=22) of EPSC in the PVN‐MC cells was also larger than those of the PVN‐RVLM and PVN‐IML neurons (3.2 vs. 2.88 ms, n=14, 16; p<0.05). The properties of action potential were determined in the whole cell current clamp. The amplitude in PVN‐MC was definitely the largest (PVN‐RVLM, PVN‐IML, PVN‐MC: 78 vs. 65 vs. 69 mV, n=20, 10, 17; p<0.05)These results indicate that electrophysiological properties of three neuron groups were also different. The PVN‐IML neurons were the highest of spontaneous firing, and PVN‐MC neurons have larger input resistance and action potential amplitude of action potential. Functional significance of these differences remains to be studied further.

L596–W733 bond between the start of the S4–S5 linker and the TRP box stabilizes the closed state of TRPV4 channel

Unlike other cation channels, each subunit of most transient receptor potential (TRP) channels has an additional TRP-domain helix with an invariant tryptophan immediately trailing the gate-bearing S6. Recent cryo-electron microscopy of TRP vanilloid subfamily, member 1 structures revealed that this domain is a five-turn amphipathic helix, and the invariant tryptophan forms a bond with the beginning of the four-turn S4-S5 linker helix. By homology modeling, we identified the corresponding L596-W733 bond in TRP vanilloid subfamily, member 4 (TRPV4). The L596P mutation blocks bone development in Kozlowski-type spondylometaphyseal dysplasia in human. Our previous screen also isolated W733R as a strong gain-of-function (GOF) mutation that suppresses growth when the W733R channel is expressed in yeast. We show that, when expressed in Xenopus oocytes, TRPV4 with the L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, these mutant channels have higher basal open probabilities and limited responses to the agonist GSK1016790A, explaining their biological GOF phenotypes. In addition, W733R current fails to inactivate during depolarization. Systematic replacement of W733 with amino acids of different properties produced similar electrophysiological and yeast phenotypes. The results can be interpreted consistently in the context of the homology model of TRPV4 molecule we have developed and refined using simulations in explicit medium. We propose that this bond maintains the orientation of the S4-S5 linker to keep the S6 gate closed. Further, the two partner helices, both amphipathic and located at the polar-nonpolar interface of the inner lipid monolayer, may receive and integrate various physiological stimuli.

Prolactin stimulates sodium and chloride ion channels in A6 renal epithelial cells.

Many hormonal pathways contribute to the regulation of renal epithelial sodium channel (ENaC) function, a key process for maintaining blood volume and controlling blood pressure. In the present study, we examined whether the peptide hormone prolactin (PRL) regulates ENaC function in renal epithelial cells (A6). Basolateral application of several different concentrations of PRL dramatically stimulated the transepithelial current in A6 cells, increasing both amiloride-sensitive (ENaC) and amiloride-insensitive currents. Using cell-attached patch clamp, we determined that PRL increased both the number (N) and open probability (Po) of ENaC present in the apical membrane. Inhibition of PKA with H-89 abolished the effect of PRL on amiloride-sensitive and insensitive transepithelial currents and eliminated the increase in ENaC NPo with PRL exposure. PRL also increased cAMP in A6 cells, consistent with signaling through the cAMP-dependent PKA pathway. We also identified that PRL induced activity of a 2-pS anion channel with outward rectification, electrophysiological properties consistent with ClC4 or ClC5. RT-PCR only detected ClC4, but not ClC5 transcripts. Here, we show for the first time that PRL activates sodium and chloride transport in renal epithelial cells via ENaC and ClC4.

A new look at the antibiotic amphotericin B effect on Candida albicans plasma membrane permeability and cell viability functions.

Amphotericin B (AmB) is an antifungal polyene for which the most accepted mode of action is formation of protein-like ion channels in the cell membrane. Patch-clamp research on Candida albicans protoplasts carried out in the outside-out configuration showed that application of 0.05 and 0.1μM AmB caused a decrease in seal resistance. Such a phenomenon can be correlated with a decrease in membrane tightness. AmB applied at a 0.05μM concentration also caused a decrease in the number of active TOK1 (two-pore outward rectifiers) potassium channels, but did not significantly change their open probability. The results indicate that in C. albicans protoplast AmB causes a decrease in cell membrane integrity by interaction with its lipid phase but not with ion channels. Fluorescence microscopy techniques showed that AmB treatment, in clinical concentrations, had no effect on the percentage of PI-positive protoplasts. AmB treatment in the concentrations tested did not cause a rapid reduction of the number of C. albicans protoplasts. However, there was a significant loss of replication competency and numerous morphological and physiological disorders, including cytoplasm shrinking, abnormal morphology of the nucleus and mitochondria, a sudden decrease in the MTT reduction level and oxidative stress. Our results show that the induction of yeast cell death by AmB, at therapeutic doses, is a multistage and long-term process involving multiple intracellular pathways.

The L596-W733 Bond between S4-S5 Linker and TRP Domain Maintains Basal Activity and Enables Inactivation of TRPV4

Unlike other cation channels, all TRP (Transient Receptor Potential) channel subunits have a TRP-domain helix immediately trailing S6 that bears the gate. The role(s) of TRP-domain helix is unclear. Recent cryo-EM TRPV1 structures revealed that this helix forms a bond with the beginning of the S4-S5 linker. By homology modeling, we identified the corresponding L596-W733 bond in TRPV4 (Vanilloid type 4). L596P, likely a gain-of-function (GOF) mutation, causes bone-developmental blockage of the spondylometaphyseal dysplasia Kozlowski type (SMDK) in human. Our previous screen also isolated W733R as a GOF that suppresses grows of yeast expressing TRPV4. Here we show, when expressed in Xenopus oocytes, TRPV4 with L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, as expected from their biological GOF phenotypes, these mutant channels indeed have higher basal open probabilities and limited responses to the strong agonist GSK1016790A. In addition, W733R current also fails to inactivate after activation during depolarization. Systematic substitutions of W733 with amino acids of different properties produce similar electrophysiological defects. The results can be consistently interpreted in the context of the homology model of TRPV4 that we have developed. Our results indicate that the TRP domain stabilizes various functional conformations by bonding to other structures, especially to the S4-S5 linker.

Identification of Significant Amino Acids in Multiple Transmembrane Domains of Human Transient Receptor Potential Ankyrin 1 (TRPA1) for Activation by Eudesmol, an Oxygenized Sesquiterpene in Hop Essential Oil

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca(2+) measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03-0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by β-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.

Activation of Ca2+-Dependent Cation Current by Fluid Shear Force in Atrial Myocytes

Atrial myocytes are subjected to fluid shear force (FSF) during each contraction and relaxation. Ionic currents regulated by shear force and their molecular integrity in cardiac myocytes have not been well-understood. We examined whether FSF activates specific current in atrial myocytes and underlying mechanisms for FSF-sensitive ionic current using whole-cell patch-clamp technique. A FSF of ∼16 dyne/cm2 was applied to entire single atrial myocyte using automated micro-puffing apparatus. A FSF-sensitive current (IFSF) was detected in lowly Ca2+-buffered (0.5 mM EGTA) atrial myocytes, but not in highly Ca2+-buffered (≥4 mM EGTA or 10 mM BAPTA) myocytes. The IFSF showed an outward rectification with a reversal potential of about -6 mV. The IFSF was inhibited by high concentrations (20-50 μM) of ryanodine and by replacement of external and internal cation with impermeant NMDG+, suggesting that IFSF is a Ca2+ release-dependent cation current. Application of either transient receptor potential melastatin subfamily 4 (TRPM4) inhibitor 9-phenanthrol or TRPM4-specific antibodies removed most of inward IFSF and ∼80% of outward IFSF. However, stretch-activated cation channel blocker GsMTx-4 did not affect IFSF. Interestingly, IFSF was strongly inhibited by inositol 1,4,5-trisphosphate receptor (IP3R) blockers, 2-APB (2 μM) or xestospongin C. In addition, in atrial myocytes isolated from type 2 IP3R (IP3R2) knock-out mouse, IFSF was not detected, although 9-phenanthrol-sensitive IFSF was recorded in wild-type myocytes. Co-immunostaining of TRPM4 and IP3R2 in rat atrial myocytes revealed peripheral localization of these proteins with some co-localizations. These results suggest that fluid shear stimuli may activate TRPM4 channels in atrial myocytes via Ca2+ releases triggered by the activation of nearby IP3R2.

Role of the Voltage Sensing Domain S1-S4 in TRPV1 Channels

Transient Receptor Potential Vallinoid type 1 (TRPV1) channel is a voltage-, heat-, and ligand-activated, nonselective cation channel. As a member of hexahelical cation channel superfamily, TRPV1 does not feature the positively charged S4, which is responsible for the voltage sensitivity of voltage-gated ion channels (VGICs). The origin for TRPV1's voltage sensitivity must lie elsewhere. The objective of this study was to investigate the role of “classical” S1-S4 voltage sensor, in particular S4, in the TRPV1, and obtain molecular information about the functional coupling between voltage, chemical and temperature sensors. To test whether the positive charge is involved in activation and to explore the function of the S4 segment in TRPV1 activation, we made a series of single point arginine replacements that scanned residues from E536 through T556 in the S4 and characterized their response to capsaicin, protons and heat using cut-open oocyte voltage-clamp. We also constructed TRPV1/Shaker chimeras in which the S1-S4, S3-S4, or S4 segments of the TRPV1 channel were replaced by the corresponding segments of Shaker channels. The introduction of positively charged residues in the S4 segment resulted in a steeper activation curve and a defective response to capsaicin and protons. Importantly, several of the mutant channels displayed strong inhibition of hyperpolarization-activated inward currents. Three chimeras gave rise to functional channels that exhibited stronger voltage dependence at positive voltages than TRPV1. The chimeras progressively lost inward current with increasing portions replaced by the corresponding Shaker region. They were activated by protons with higher sensitivity compared with TRPV1. These results suggest that re-established outward rectification was dominant over other gating stimuli; in other words, keeping the S1-S4 in the “deactivated” state prevented activation by protons, capsaicin or temperature. In wildtype TRPV1, the S4 seems to inhibit activation by protons.

Molecular Dynamics Simulations Describe the Mechanism of K Block in Bacterial Nav Channels

Although extensive electrophysiological characterization is available for eukaryotic voltage-gated Na+ channels (Nav), no high-resolution structures of these channels are available. Crystal structures of several bacterial Nav channels have been published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of the eukaryotic channels. Unlike eukaryotic Nav channels, however, the bacterial Nav channels are strongly outwardly rectifying, and the mechanism of this rectification has not previously been described. We used step-wise pulling protocols to implement Jarzynski's Equality in non-equilibrium molecular dynamics simulations of ion permeation through the bacterial NavAb channel to obtain a mechanistic description of this outward rectification. Results of the simulations indicate that two or three extracellular K+ ions bind tightly at the same z-coordinate along the selectivity filter of NavAb and can effectively block the channel in the presence of modest voltages or concentration driving forces. The configuration with two potassium ions located at the same z-coordinate is also found in the two-dimensional potential of mean forces generated from umbrella sampling and weighted histogram analysis. In contrast to K+, three Na+ ions move through the selectivity filter together as a unit in a “knock-on” mechanism of permeation. Differences in the amount of work required to move three Na+ ions through the selectivity filter of NavAb compared to three K+ ions predict the large negative reversal potentials observed for bacterial Nav channels in instantaneous current-voltage plots. The results of the simulations suggest that the block of bacterial voltage-gated Na+ channels by extracellular K+ does not occur in eukaryotic voltage-gated Na+ channels because of differences in the amino acids present in the selectivity filters of the different channels.

Regulation of the TRPM3 Channel in Planar Lipid Bilayers

Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective cation channel that can be activated with nifedipine and pregnenolone sulfate (PS). The role of TRPM3 in nociceptive neurons has been linked to the heat sensation and development of heat hyperalgesia during inflammation. However, the functional characterization of TRPM3 and its specific agonists remain poorly understood. To investigate biophysical and pharmacological properties of TRPM3 we aimed to incorporate the purified channel in planar lipid bilayers. TRPM3 was purified from HEK-293 cells using immunoprecipitation technique. The single channel activity was then examined under various conditions. We investigated TRPM3 activity with different agonists. Application of nifedipine (1-10 µM) resulted in dose-dependent increase of open probability of the channel. These results suggest that nifedipine can alone activate TRPM3. Unlike nifedipine, addition to the bilayers of PS, did not induce the channel openings alone and required co-application of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) or clotrimazole. TRPM3 demonstrated outward rectification upon activation with PS/PI(4,5)P2, but linear current-voltage relationship with PS/clotrimazole. In both conditions, TRPM3 tended to inactivate at higher voltages (100-120 mV) and with increased concentrations of PS. Interestingly, in the absence of any agonists TRPM3 exhibited basal channel activity that sustained for several minutes. Previously TRPM3 was proposed to serve as a temperature sensor. We tested temperature sensitivity of TRPM3 and found that in the framework of lipid bilayers the channel did not exhibit any temperature-induced activation in the absence of agonists. In the presence of nifedipine TRPM3 demonstrated weak temperature dependence with Q10of 1.5 at +100 mV and 1.7 at −100 mV. These results indicate that TRPM3 is unlikely to serve as a temperature sensor alone and may involve some alternative molecular mechanisms for temperature sensation.

Microglial K(+) channel expression in young adult and aged mice.

The K+ channel expression pattern of microglia strongly depends on the cells' microenvironment and has been recognized as a sensitive marker of the cells' functional state. While numerous studies have been performed on microglia in vitro, our knowledge about microglial K+ channels and their regulation in vivo is limited. Here, we have investigated K+ currents of microglia in striatum, neocortex and entorhinal cortex of young adult and aged mice. Although almost all microglial cells exhibited inward rectifier K+ currents upon membrane hyperpolarization, their mean current density was significantly enhanced in aged mice compared with that determined in young adult mice. Some microglial cells additionally exhibited outward rectifier K+ currents in response to depolarizing voltage pulses. In aged mice, microglial outward rectifier K+ current density was significantly larger than in young adult mice due to the increased number of aged microglial cells expressing these channels. Aged dystrophic microglia exhibited outward rectifier K+ currents more frequently than aged ramified microglia. The majority of microglial cells expressed functional BK-type, but not IK- or SK-type, Ca2+-activated K+ channels, while no differences were found in their expression levels between microglia of young adult and aged mice. Neither microglial K+ channel pattern nor K+ channel expression levels differed markedly between the three brain regions investigated. It is concluded that age-related changes in microglial phenotype are accompanied by changes in the expression of microglial voltage-activated, but not Ca2+-activated, K+ channels.

α-Synuclein forms non-selective cation channels and stimulates ATP-sensitive potassium channels in hippocampal neurons.

In Parkinson's disease, the protein α-synuclein (αS) is produced within neurons and also appears in the extracellular fluid. In this study in hippocampal neurons, αS formed non-selective cation channels with multiple levels of conductance and rectification depending on their insertion site. αS channels induced local spontaneous increases in intracellular Na(+) and Ca(2+), depolarized neurons, augmented bursting activity and stimulated the opening of ATP-sensitive K(+) channels. Non-selective channels were also observed in neurons transfected with either wild-type or mutant A53T αS, and after extracellular application of these proteins. The properties of αS channels in neuronal membranes suggest that extracellular αS is more toxic than αS produced within neurons. In Parkinson's disease and several other neurodegenerative diseases, the protein α-synuclein (αS) is produced within neurons and accumulates in the extracellular fluid. Several mechanisms of αS action are proposed, one of which is the formation of cation-permeable pores that may mediate toxicity. αS induces non-selective cation channels in lipid bilayers, but whether this occurs in living neurons and which properties the channels possess have not yet been examined. In this study the properties of αS channels in dissociated hippocampal neurons are documented. In cell-attached recordings the incorporation of αS into membranes was driven by applied negative potentials. These channels exhibited multiple levels of conductance (30, 70 and 120 pS at -100 mV) and inward rectification. The persistent activity of αS channels induced local changes in intracellular Na(+) and Ca(2+), depolarized neurons and augmented bursting activity. αS channels formed by adding αS to the intracellular membrane in inside-out patches exhibited outward rectification. αS channels were equally permeable to Na(+), K(+) and Ca(2+). These channels were also observed in neurons transfected with wild-type or mutant A53T αS, and after extracellular application of wild-type or mutant A53T αS proteins. Opening of αS channels stimulated opening of ATP-sensitive K(+) (KATP ) channels and did not interfere with the activity of delayed rectifier K(+) channels. The properties of αS channels in neuronal membranes suggest stronger toxicity of extracellularly applied αS than intracellular αS. Enhancement of neuronal excitability and distortions in ion homeostasis may underlie the toxic effects of αS that can be dampened by KATP channels.

Characteristics of GABAergic and cholinergic neurons in perinuclear zone of mouse supraoptic nucleus.

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons.

Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions

It is generally expected that 2-pore domain K+ (K2P) channels are open or outward rectifiers in asymmetric physiological K+ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K2P channels display 2 distinct types of I-V curves in asymmetric physiological K+ gradients. Two K2P isoforms in the TWIK subfamily conduct large inward K+ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K2P subfamilies conduct small inward K+ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K2P channels and suggest that TWIK K+ channels have a unique feature in regulating cellular function.

Carvacrol presynaptically enhances spontaneous excitatory transmission and produces outward current in adult rat spinal substantia gelatinosa neurons

Carvacrol, which is abundantly contained in oregano essential oils, has various pharmacological actions including antinociception. Although the oral administration of carvacrol results in antinociception, cellular mechanisms for this action have not been examined yet. We investigated the action of carvacrol on glutamatergic spontaneous excitatory transmission in substantia gelatinosa neurons which play a pivotal role in regulating nociceptive transmission from the periphery by using the patch-clamp technique in adult rat spinal cord slices. Carvacrol superfused for 2min produced either spontaneous excitatory postsynaptic current frequency increase or outward current at −70mV, or both of them in many of the neurons tested. The frequency increase and outward current had the EC50 values of 0.69mM and 0.55mM, respectively. The former action was inhibited by a selective TRPA1 antagonist HC-030031 but not a selective TRPV1 antagonist capsazepine, while the latter action was unaffected by their antagonists. The current–voltage relationship for the outward current indicated an involvement in the current of a change in the membrane permeability of K+ and its outward rectification. The outward current was inhibited in 10mM-K+ but not K+-channel blockers [tetraethylammonium and Ba2+]-containing and 11.0mM-Cl- Krebs solution. These results indicate that carvacrol increases the spontaneous release of l-glutamate from nerve terminals by activating TRPA1 but not TRPV1 channels and produces membrane hyperpolarization, which is possibly mediated by tetraethylammonium- and Ba2+-insensitive K+ channels, in substantia gelatinosa neurons. It is suggested that the hyperpolarizing effect of carvacrol could contribute to its antinociceptive action.

Functional analysis of acid-activated Cl− channels: Properties and mechanisms of regulation

Cl− channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acid-activated Cl− current (ICl(H)) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK-293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH5.15), permeability sequence (SCN−>I−>Br−>Cl−>gluconate), voltage dependence and sensitivity to blockers of ICl(H) were identical in all cells. These findings suggest a common molecular basis for ICl(H). We analysed the possible relationship of ICl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR, we found that ICl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca2+, ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of ICl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for ICl(H) regulation. Our results provide the first evidence about acid-activated Cl− channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.

Cisplatin activates volume-sensitive like chloride channels via purinergic receptor pathways in nasopharyngeal carcinoma cells.

Cisplatin-based concomitant chemoradiotherapy is considered as the standard treatment for locally advanced nasopharyngeal carcinoma patients. However, the curative efficacy of cisplatin-based chemotherapy is limited because of the occurrence of cisplatin resistance. Some researches indicate that activating the volume-sensitive Cl(-) channel might be a new strategy for the reduction of cisplatin resistance. However, little is known about the activation pathway of the Cl(-) channels activated by cisplatin. In this study, the cisplatin-activated chloride current was investigated using the whole cell patch-clamp technique in the poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells), and the activation pathway of the current was also discussed. The results showed that extracellular application of cisplatin activated a Cl(-) current, showing the properties of significant outward rectification, intracellular ATP dependency, and a selectivity sequence of I(-) > Br(-) > Cl(-) > gluconate, and being inhibited by the Cl(-) channel inhibitors tamoxifen and extracellular ATP. These characteristics are similar to those of the volume-sensitive Cl(-) current in CNE-2Z cells, indicating that cisplatin induces the Cl(-) current by activating the volume-sensitive like chloride channel. The cisplatin-activated current was blocked by suramin (a wide-spectrum purinergic antagonist) and RB2 (a relatively selective P2Y antagonist). In addition, the current was depressed by extracellular application of apyrase. The apoptotic volume decrease induced by cisplatin was also attenuated by RB2. P2Y receptors were expressed in CNE-2Z cells. These results suggest that cisplatin can induce a Cl(-) current by activating volume-sensitive like Cl(-) channels through the P2Y purinoceptor pathway.

What Makes You Can also Break You, Part III: Mitochondrial Permeability Transition Pore Formation by an Uncoupling Channel within the C-Subunit Ring of the F1FO ATP Synthase?

What Makes You Can also Break You, Part III: Mitochondrial Permeability Transition Pore Formation by an Uncoupling Channel within the C-Subunit Ring of the F1FO ATP Synthase?

The consequences of contracting early and often

Frequent premature ventricular contractions (PVCs) are associated with structural heart disease and an increase in sudden and overall mortality. PVCs themselves, if in sufficient numbers, may be associated with the development of a reversible form of cardiomyopathy (CM). A number of small series have suggested that PVC density and features, such as QRS duration, location of origin, and coupling, may increase the predilection to cause CM in structurally normal and diseased hearts. Moreover, elimination of PVCs by catheter ablation has improved ventricular function. Thus, frequent PVCs are a previously underappreciated target for therapeutic intervention in CM. However, the fundamental triggers for the development of myopathy and the details of structural and electrical remodeling have been impeded by the development of a suitable model system. Pacing has been exploited to generate reversible forms of tachycardia-induced CM. Huizar et al have used a premature pacing algorithm to create a model of PVC-induced CM in structurally normal hearts. The algorithm generates a high PVC burden, in this case in a bigeminal pattern pacing from the right ventricular apex. PVC pacing increased the heart rate, on average, from 80 bpm to approximately 130 bpm over 4 months of pacing. Previous characterization of this model demonstrated reversible cardiac dilation and reduced ejection fraction with a compromise in repolarizing reserve evidenced by an increase in ventricular refractory periods. This occurred in the absence of inflammation, fibrosis, increased programmed cell death, or compromised oxidative phosphorylation. In a study reported in this issue of HeartRhythm, Wang et al describe heterogeneously prolonged action potential durations (APD) in myocytes isolated from the left ventricular free wall, associated with reductions in the transient outward (Ito) and inward rectifier (IK1) potassium currents without significant changes in the delayed rectifier (IKr, IKs) currents. The decrease in repolarizing reserve is associated