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patch clamp - Top 30 Publications

The influence of acidic media on the effect of beta-amyloid peptide on the function of glycine receptor in hippocampal neurons.

We have previously shown that application of beta-amyloid peptide 1-42 (Aβ) at picomolar/nanomolar concentrations caused a decrease in the peak amplitude and acceleration of desensitization of the glycine-activated chloride current (IGly) in hippocampal pyramidal neurons (Bukanova et al., 2016). The aim of this work was to study the effect of Aβ on IGly in an acidified medium. The relevance of this work is determined by the fact that the pathogenic effects of Aβ in Alzheimer's disease are usually accompanied by inflammatory processes and acidosis. The IGly was induced by 600 ms application of 100 μM (nearly EC50) or 500 μM (nearly saturating) glycine on isolated rat hippocampal neurons. The solution of glycine was neutral (pH 7.4) or acidic over a pH range of 5.0-7.0. It was found that 600 ms application of protons rapidly, reversibly and in dose-dependent manner decreased the peak amplitude and accelerated the desensitization of IGly. The effect of H(+) on IGly desensitization did not depend on glycine concentration and may be considered noncompetitive, while the effect on IGly peak disappeared at saturating glycine concentration and can be regarded as a competitive. These characteristics of the proton effects on IGly coincide with the characteristics of the Aβ effects on IGly. Experiments with joint application of Aβ and H(+) showed interdependence of their effects. Addition of Aβ to perfusing solution reduced H(+) effects on IGly while long pretreatment of Aβ with acid solution prevented the effects of the peptide on IGly. Our results suggest the existence of common sites for Aβ and H(+) on the GlyR and indicate a mutual weakening of the inhibitory action of these molecules on IGly.

CRISPR correction of the PRKAG2 gene mutation in the patient's iPSC-derived cardiomyocytes eliminates the electrophysiological and structural abnormalities.

Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate-kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial-Wolff-Parkinson-White syndrome (WPW). Patients carrying the R302Q mutation in PRKAG2 present sinus bradycardia, escape rhythms, ventricular pre-excitation, supraventricular tachycardia and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW, HCM and arrhythmias remains unknown.

Electrophysiological heterogeneity of pacemaker cells in rabbit intercaval region, including SA node: insights from recording multiple ion currents in each cell.

Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology and electrophysiological characteristics. The exact extent to which these cells differ electrophysiologically is unclear, yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPC) sampled from the para-cristal, inter-caval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. Beating rate was measured at baseline and following inhibition of the Ca(2+) pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured density of If, IK (a surrogate of repolarization capacity) and ICa,L using whole-cell patch-clamp. The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero If Density of none of the currents was correlated with cell size, while ICa,L and If densities were related to baseline beating rates. If density was correlated with IK density, but not with that of ICa,L Inhibition of Ca(2+) cycling had a greater beating rate slowing effect in IPCs with lower If densities. Our numerical model simulation indicated that 1) IPC with small (or zero) If or small ICa,L can operate via a major contribution of Ca(2+)-clock, 2) If-Ca(2+)-clock interplay could be important for robust pacemaking function, 3) coupled If-IK function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of intercaval pacemaker cells. This heterogeneity is manifested in basal beating rate and response to interference of Ca(2+) cycling, which is linked to If.

Angiotensin II induces calcium-mediated autophagy in podocytes through enhancing reactive oxygen species levels.

As well known, abnormalities of Angiotensin II (Ang II) is closely related with glomerular damage. This study was to investigate whether Ang II could affect autophagy in podocytes via oxidative stress, and whether autophagy had a positive role in protecting podocytes impaired by Ang II. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that Ang II induced podocyte death. The measurements of malondialdehyde (MDA) and H2O2 levels, and flow cytometry assay revealed that Ang II considerably increased ROS generation in podocytes. Meaningfully, treatment with ROS scavenger N-(mercaptopropionyl)-glycine (N-MPG) could inhibit podocyte death and attenuate accumulation of ROS induced by Ang II. The patch-clamp experiments indicated that Ang II increased the current of transient receptor potential canonical 6 (TRPC6). Moreover, measurement of Fluo-3 image showed that Ang II increased intracellular Ca(2+) level, as N-MPG and La(3+) impeded Ang II induced Ca(2+) influx. Acridine orange staining indicated that Ang II induced accumulation of acidic vacuoles. Beclin-1 and LC3 are essential for autophagosome formation. Furthermore, as one of the selective substrates for autophagy, P62 plays a key role in the formation of cytoplasmic proteinaceous inclusion. Western blot assay presented that Ang II obviously elevated LC3-II/LC3-I ratio and expression of beclin-1, and reduced expression of P62. Meanwhile, N-MPG expectedly down-regulated autophagy in Ang II-treated podocytes. Rapamycin can enhance the level of autophagy by inhibiting mTOR, and 3-methyladenine (3-MA) can inhibit autophagosome formation through blocking class III phosphatidylinositol 3-kinase. MTT assay exhibited that rapamycin significantly enhanced the cell viability, while 3-methyladenine (3-MA) considerably reduced it in Ang II-treated podocytes. Consequently, this study demonstrated that Ang II could increase TRPC6 induced Ca(2+) influx and enhance autophagy through increasing ROS levels in podocytes, and autophagy could protect Ang II-treated podocytes. Improving TRPC6 channels and autophagy may become a new targeted therapy to relieve glomerular damage induced by Ang II.

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

Whole-Cell Recording of Neuronal Membrane Potential during Behavior.

Neuronal membrane potential is of fundamental importance for the mechanistic understanding of brain function. This review discusses progress in whole-cell patch-clamp recordings for low-noise measurement of neuronal membrane potential in awake behaving animals. Whole-cell recordings can be combined with two-photon microscopy to target fluorescently labeled neurons, revealing cell-type-specific membrane potential dynamics of retrogradely or genetically labeled neurons. Dual whole-cell recordings reveal behavioral modulation of membrane potential synchrony and properties of synaptic transmission in vivo. Optogenetic manipulations are also readily integrated with whole-cell recordings, providing detailed information about the effect of specific perturbations on the membrane potential of diverse types of neurons. Exciting developments for future behavioral experiments include dendritic whole-cell recordings and imaging, and use of the whole-cell recording pipette for single-cell delivery of drugs and DNA, as well as RNA expression profiling. Whole-cell recordings therefore offer unique opportunities for investigating the neuronal circuits and synaptic mechanisms driving membrane potential dynamics during behavior.

Heteromeric α/β glycine receptors regulate excitability in parvalbumin-expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.

The dorsal horn (DH) of the spinal cord is an important site for modality specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin-expressing inhibitory interneurons (PV + INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV + INs using targeted patch-clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV + INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV + INs. In addition, PV + INs expressed robust glycine-mediated tonic currents, however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV + IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/β glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV + INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV + INs. This article is protected by copyright. All rights reserved.

Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus).

The neuroepithelial cell (NEC) of the fish gill is an important model for O2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O2 chemoreception in vertebrates that are tolerant to anoxia has yet not been explored. Using whole-cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca(2+)-dependent K(+) current (IKCa) peaked at ~20 mV, was potentiated by increased intracellular Ca(2+), and was reduced by 100 μM Cd(2+) A voltage-dependent inward current in Ba(2+) solution, with peak at 0 mV, confirmed the presence of Ca(2+) channels. A voltage-dependent K(+) current (IKV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K(+) current (IKB) with open rectification. Mean resting membrane potential of -45.2 +/- 11.6 mV did not change upon administration of hypoxia (PO2 = 11 mmHg), nor were any of the K(+) currents sensitive to changes in PO2 during whole-cell recording. By contrast, when the membrane and cytosol were left undisturbed during Fura-2 or FM1-43 imaging experiments, hypoxia increased intracellular Ca(2+) concentration and initiated synaptic vesicle activity. 100 μM Cd(2+) and 50 μM nifedipine eliminated uptake of FM1-43. We conclude that Ca(2+) influx via L-type Ca(2+) channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of IKCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia.

Neuroprotective Effect of Amantadine on Corticosterone-induced Abnormal Glutamatergic Synaptic Transmission of CA3-CA1 pathway in rat's hippocampal slices.

Depression is a psychiatric disorder and chronic stress, leading to altered glucocorticoid secretion patterns, is one of the factors that induce depression. Our previous study showed that amantadine significantly attenuated the impairments of synaptic plasticity and cognitive function a rat model of CUS. However, little is known regarding the underlying mechanism. In the present study, the whole-cell patch-clamp technique was applied to examine the protection effect of amantadine on the hippocampus CA3-CA1 pathway. Evoked excitatory postsynaptic currents (eEPSCs), miniature excitatory postsynaptic currents (mEPSCs), paired-pulse ratio (PPR) and the action potentials of CA3 neurons were recorded. Our data showed that corticosterone increased the amplitude of eEPSCs and decreased the value of paired-pulse ratio (PPR), but both of them were significantly reversed by amantadine. In addition, the frequency of mEPSC was considerably increased by corticosterone, but it was reduced by amantadine. Moreover, we used the Fluo-3/AM image to detect the Ca(2+) influx in primary cultured hippocampal neurons. The results showed that the intracellular calcium levels were significantly decreased by amantadine in the corticosterone treated neurons. Additionally, the superoxide dismutase (SOD) and catalase (CAT) activities were reduced by corticosterone, while they were enhanced by either amantadine or low-calcium artificial cerebral spinal fluid (ACSF). These results suggest that amantadine significantly improves corticosterone-induced abnormal glutamatergic synaptic transmission of CA3-CA1 synapses presynaptically and alleviates the activities of antioxidant enzymes via regulating the calcium influx. This article is protected by copyright. All rights reserved.

4-Chloro-3-nitro-N-butylbenzenesulfonamide acts on KV3.1 channels by an open-channel blocker mechanism.

The effects of 4-chloro-3-nitro-N-butylbenzenesulfonamide (SMD2) on KV3.1 channels, heterologous expressed in L-929 cells, were studied with the whole cell patch-clamp technique. SMD2 blocks KV3.1 in a reversible and use-dependent manner, with IC50 around 10 µM, and a Hill coefficient around 2. Although the conductance vs. voltage relationship in control condition can be described by a single Boltzmann function, two terms are necessary to describe the data in the presence of SMD2. The activation and deactivation time constants are weakly voltage dependent both for control and in the presence of SMD2. SMD2 does not change the channel selectivity and tail currents show a typical crossover phenomenon. The time course of inactivation has a fast and a slow component, and SMD2 significantly decreased their values. Steady-state inactivation is best described by a Boltzmann equation with V 1/2 (the voltage where the probability to find the channels in the inactivated state is 50%) and K (slope factor) equals to -22.9 ± 1.5 mV and 5.3 ± 0.9 mV for control, and -30.3 ± 1.3 mV and 6 ± 0.8 mV for SMD2, respectively. The action of SMD2 is enhanced by high frequency stimulation, and by the time the channel stays open. Taken together, our results suggest that SMD2 blocks the open conformation of KV3.1. From a pharmacological and therapeutic point of view, N-alkylsulfonamides may constitute a new class of pharmacological modulators of KV3.1.

Serotonin gating of cortical and thalamic glutamate inputs onto principal neurons of the basolateral amygdala.

The basolateral amygdala (BLA) is a key site for crossmodal association of sensory stimuli and an important relay in the neural circuitry of emotion. Indeed, the BLA receives substantial glutamatergic inputs from multiple brain regions including the prefrontal cortex and thalamic nuclei. Modulation of glutamatergic transmission in the BLA regulates stress- and anxiety-related behaviors. Serotonin (5-HT) also plays an important role in regulating stress-related behavior through activation of both pre- and postsynaptic 5-HT receptors. Multiple 5-HT receptors are expressed in the BLA, where 5-HT has been reported to modulate glutamatergic transmission. However, the 5-HT receptor subtype mediating this effect is not yet clear. The aim of this study was to use patch-clamp recordings from BLA neurons in an ex vivo slice preparation to examine 1) the effect of 5-HT on extrinsic sensory inputs, and 2) to determine if any pathway specificity exists in 5-HT regulation of glutamatergic transmission. Two independent input pathways into the BLA were stimulated: the external capsule to mimic cortical input, and the internal capsule to mimic thalamic input. Bath application of 5-HT reversibly reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs) induced by stimulation of both pathways. The decrease was associated with an increase in the paired-pulse ratio and coefficient of variation of eEPSC amplitude, suggesting 5-HT acts presynaptically. Moreover, the effect of 5-HT in both pathways was mimicked by the selective 5-HT1B receptor agonist CP93129, but not by the 5-HT1A receptor agonist 8-OH DPAT. Similarly the effect of exogenous 5-HT was blocked by the 5-HT1B receptor antagonist GR55562, but not affected by the 5-HT1A receptor antagonist WAY 100635 or the 5-HT2 receptor antagonists pirenperone and MDL 100907. Together these data suggest 5-HT gates cortical and thalamic glutamatergic inputs into the BLA by activating presynaptic 5-HT1B receptors.

RHOA-ROCK signalling is necessary for lateralization and differentiation of the developing sinoatrial node.

RHOA-ROCK signalling regulates cell migration, proliferation, differentiation, and transcription. RHOA is expressed in the developing cardiac conduction system in chicken and mice. In early development, the entire sinus venosus myocardium, including both the transient left-sided and the definitive sinoatrial node (SAN), has pacemaker potential. Later, pacemaker potential is restricted to the right-sided SAN. Disruption of RHOA expression in adult mice causes arrhythmias including bradycardia and atrial fibrillation, the mechanism of which is unknown but presumed to affect the SAN. The aim of this study is to assess the role of RHOA-ROCK signalling in SAN development in the chicken heart.

Uncovering the arrhythmogenic potential of TRPM4 activation in atrial-derived HL-1 cells using novel recording and numerical approaches.

Transient receptor potential cation channel subfamily melastatin member 4 (TRPM4), a Ca2+-activated nonselective cation channel abundantly expressed in the heart, has been implicated in conduction block and other arrhythmic propensities associated with cardiac remodelling and injury. The present study aimed to quantitatively evaluate the arrhythmogenic potential of TRPM4.

Dynamic SERS nanosensor for neurotransmitter sensing near neurons.

Current electrophysiology and electrochemistry techniques have provided unprecedented understanding of neuronal activity. However, these techniques are suited to a small, albeit important, panel of neurotransmitters such as glutamate, GABA and dopamine, and these constitute only a subset of the broader range of neurotransmitters involved in brain chemistry. Surface-enhanced Raman scattering (SERS) provides a unique opportunity to detect a broader range of neurotransmitters in close proximity to neurons. Dynamic SERS (D-SERS) nanosensors based on patch-clamp-like nanopipettes decorated with gold nanoraspberries can be located accurately under a microscope using techniques analogous to those used in current electrophysiology or electrochemistry experiments. In this manuscript, we demonstrate that D-SERS can measure in a single experiment ATP, glutamate (glu), acetylcholine (ACh), GABA and dopamine (DA), among other neurotransmitters, with the potential for detecting a greater number of neurotransmitters. The SERS spectra of these neurotransmitters were identified with a barcoding data processing method and time series of the neurotransmitter levels were constructed. The D-SERS nanosensor was then located near cultured mouse dopaminergic neurons. The detection of neurotransmitters was performed in response to a series of K(+) depolarisations, and allowed the detection of elevated levels of both ATP and dopamine. Control experiments were also performed near glial cells, showing only very low basal detection neurotransmitter events. This paper demonstrates the potential of D-SERS to detect neurotransmitter secretion events near living neurons, but also constitutes a strong proof-of-concept for the broad application of SERS to the detection of secretion events by neurons or other cell types in order to study normal or pathological cell functions.

Molecular and functional characterization of the endothelial ATP-sensitive potassium channel.

ATP-sensitive potassium (KATP) channels are widely expressed in the cardiovascular system where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. KATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. KATP channels are also thought to be expressed in vascular endothelial cells (ECs), but their presence and function in this context is less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial KATP channels. We first generated mice with an endothelial specific deletion of the channel subunit Kir6.1 (eKO) using cre-loxP technology. Data from qRT-PCR, patch-clamp, ex-vivo coronary perfusion Langendorff heart experiments and endothelial cell Ca2+ imaging comparing eKO and wild type mice show that Kir6.1-containing KATP channels are indeed present in vascular endothelium. An increase in intracellular [Ca2+], which is central to changes in endothelial function such as mediator release, at least partly contributes to the endothelium-dependent vasorelaxation induced by the KATP channel opener pinacidil. The absence of Kir6.1 did not elevate basal coronary perfusion pressure in eKO mice. However, vasorelaxation was impaired during hypoxia in the coronary circulation and this resulted in greater cardiac injury during ischaemia-reperfusion. The response to adenosine receptor stimulation was impaired in eKO mice in single cells in patch-clamp recordings and in the intact coronary circulation. Our data support the existence of an endothelial KATP channel that contains Kir6.1, is involved in vascular reactivity in the coronary circulation and has a protective role in ischaemia reperfusion.

Modulation of hippocampal synapse maturation by activity-regulated E3 ligase via non-canonical pathway.

Development of functional synapses is crucial for the transmission and storage of information in the brain. Post establishment of the initial synaptic contact, synapses are stabilized through neuronal activity-induced signals. Emerging studies have implicated ubiquitination; a reversible posttranslational modification, as a key regulatory switch that modulates synapse development through proteasomal degradation. Ubiquitination of proteins is precisely regulated by E3 ligases, a set of enzymes that bind to specific substrates to facilitate the conjugation of monomeric or polymeric ubiquitin. However, the identity of specific E3 ubiquitin ligases that influence activity-dependent maturation of synapses and the mechanism by which ubiquitination of proteins regulate functional synapse development remain elusive. Here, we have identified a RING domain containing E3 ligase, Rnf2, asan activity-regulated factor that modulates glutamatergic synapse development in the hippocampus. Rnf2 is a synapse associated E3 ligase that is stabilized by neuronal activity through self-polyubiquitination. We have shown that neuronal activity shifts the balance toward stabilization of Rnf2 through self-polyubiquitination rather than triggering its degradation through polyubiquitination by Ube3A, an E3 ligase implicated in Angelman Syndrome. Our synapse density measurements and whole-cell patch-clamp recordings have revealed that the loss of Rnf2 function in cultured hippocampal neurons result in the development of 'silent' synapses that lack GluA1 containing functional AMPA receptors. These results provide a plausible mechanistic approach toward understanding how synapse maturation is regulated via the activity-dependent stabilization of Rnf2 through a non-canonical function of polyubiquitination.

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery.

Heterozygous loss of function mutations in the KCNK3 gene cause hereditary pulmonary arterial hypertension (PAH). KCNK3 encodes an acid-sensitive potassium channel, which contributes to the resting potential of human pulmonary artery smooth muscle cells. KCNK3 is widely expressed in the body, and dimerizes with other KCNK3 subunits, or the closely related, acid-sensitive KCNK9 channel.

Selective targeting of M-type potassium channel Kv7.4 demonstrates its key role in regulating of dopaminergic neuronal excitability and depression-like behaviour.

The mesolimbic dopamine (DA) system originating in the ventral tegmental area (VTA) is implicated in the development of depression, and firing patterns of VTA DA neurons are key determinants in this process. Here, we describe a crucial role of the M-type potassium channel Kv7.4 in modulation of VTA DA neuronal excitability and in the development of depressive behaviour in mice.

Running reorganizes the circuitry of one-week-old adult-born hippocampal neurons.

Adult hippocampal neurogenesis is an important form of structural and functional plasticity in the mature mammalian brain. The existing consensus is that GABA regulates the initial integration of adult-born neurons, similar to neuronal development during embryogenesis. Surprisingly, virus-based anatomical tracing revealed that very young, one-week-old, new granule cells in male C57Bl/6 mice receive input not only from GABAergic interneurons, but also from multiple glutamatergic cell types, including mature dentate granule cells, area CA1-3 pyramidal cells and mossy cells. Consistently, patch-clamp recordings from retrovirally labeled new granule cells at 7-8 days post retroviral injection (dpi) show that these cells respond to NMDA application with tonic currents, and that both electrical and optogenetic stimulation can evoke NMDA-mediated synaptic responses. Furthermore, new dentate granule cell number, morphology and excitatory synaptic inputs at 7 dpi are modified by voluntary wheel running. Overall, glutamatergic and GABAergic innervation of newly born neurons in the adult hippocampus develops concurrently, and excitatory input is reorganized by exercise.

Mechanosensitive ion channels (MSICs) in articular nociceptors drive mechanical allodynia in osteoarthritis (OA).

Osteoarthritis (OA) is a disabling and highly prevalent condition affecting millions worldwide. Pain is the major complaint of OA patients and is presently inadequately managed. It manifests as mechanical allodynia, a painful response to innocuous stimuli such as joint movement. Allodynia is due in part to the sensitization of articular nociceptors to mechanical stimuli. These nociceptors respond to noxious mechanical stimuli applied to their terminals via the expression of depolarizing high-threshold mechanosensitive ion channels (MSICs) that convert painful mechanical forces into electrical signals. In this study, we examined the contribution of MSICs to mechanical allodynia in a mouse model of OA.

Terbinafine is a novel and selective activator of the two-pore domain potassium channel TASK3.

Two-pore domain potassium channels (K2Ps) are characterized by their four transmembrane domain and two-pore topology. They carry background (or leak) potassium current in a variety of cell types. Despite a number of important roles there is currently a lack of pharmacological tools with which to further probe K2P function. We have developed a cell-based thallium flux assay, using baculovirus delivered TASK3 (TWIK-related acid-sensitive K(+) channel 3, KCNK9, K2P9.1) with the aim of identifying novel, selective TASK3 activators. After screening a library of 1000 compounds, including drug-like and FDA approved molecules, we identified Terbinafine as an activator of TASK3. In a thallium flux assay a pEC50 of 6.2 ( ±0.12) was observed. When Terbinafine was screened against TASK2, TREK2, THIK1, TWIK1 and TRESK no activation was observed in thallium flux assays. Several analogues of Terbinafine were also purchased and structure activity relationships examined. To confirm Terbinafine's activation of TASK3 whole cell patch clamp electrophysiology was carried out and clear potentiation observed in both the wild type channel and the pathophysiological, Birk-Barel syndrome associated, G236R TASK3 mutant. No activity at TASK1 was observed in electrophysiology studies. In conclusion, we have identified the first selective activator of the two-pore domain potassium channel TASK3.

Discovery and mode of action of a novel analgesic β-toxin from the African spider Ceratogyrus darlingi.

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Cav2.2 (IC50 2.6 μM) but not Cav1.3 or Cav3.1 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp electrophysiological assays Cd1a inhibited rat Cav2.2 with similar potency (IC50 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn't act on Cav2.2 as a classical gating modifier toxin. The Cd1a binding site on Cav2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Cav2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Nav1.1-1.2 and Nav1.7-1.8 (IC50 0.1-6.9 μM) but not Nav1.3-1.6 (IC50 > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Nav1.7 (IC50 16 nM) and produced a 29 mV depolarising shift in Nav1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Nav1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Cav2.2 inactivation gating, associated with the Cav2.2 α-subunit pore, while altering the activation gating of Nav1.7. Cd1a was inactive at some of the Nav and Cav channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

Optimization of an In silico Cardiac Cell Model for Proarrhythmia Risk Assessment.

Drug-induced Torsade-de-Pointes (TdP) has been responsible for the withdrawal of many drugs from the market and is therefore of major concern to global regulatory agencies and the pharmaceutical industry. The Comprehensive in vitro Proarrhythmia Assay (CiPA) was proposed to improve prediction of TdP risk, using in silico models and in vitro multi-channel pharmacology data as integral parts of this initiative. Previously, we reported that combining dynamic interactions between drugs and the rapid delayed rectifier potassium current (IKr) with multi-channel pharmacology is important for TdP risk classification, and we modified the original O'Hara Rudy ventricular cell mathematical model to include a Markov model of IKr to represent dynamic drug-IKr interactions (IKr-dynamic ORd model). We also developed a novel metric that could separate drugs with different TdP liabilities at high concentrations based on total electronic charge carried by the major inward ionic currents during the action potential. In this study, we further optimized the IKr-dynamic ORd model by refining model parameters using published human cardiomyocyte experimental data under control and drug block conditions. Using this optimized model and manual patch clamp data, we developed an updated version of the metric that quantifies the net electronic charge carried by major inward and outward ionic currents during the steady state action potential, which could classify the level of drug-induced TdP risk across a wide range of concentrations and pacing rates. We also established a framework to quantitatively evaluate a system's robustness against the induction of early afterdepolarizations (EADs), and demonstrated that the new metric is correlated with the cell's robustness to the pro-EAD perturbation of IKr conductance reduction. In summary, in this work we present an optimized model that is more consistent with experimental data, an improved metric that can classify drugs at concentrations both near and higher than clinical exposure, and a physiological framework to check the relationship between a metric and EAD. These findings provide a solid foundation for using in silico models for the regulatory assessment of TdP risk under the CiPA paradigm.

Mechanisms of Spontaneous Climbing Fiber Discharge-Evoked Pauses and Output Modulation of Cerebellar Purkinje Cell in Mice.

Climbing fiber (CF) afferents modulate the frequency and patterns of cerebellar Purkinje cell (PC) simple spike (SS) activity, but its mechanism is unclear. In the present study, we investigated the mechanisms of spontaneous CF discharge-evoked pauses and the output modulation of cerebellar PCs in urethane-anesthetized mice using in vivo whole-cell recording techniques and pharmacological methods. Under voltage-clamp recording conditions, spontaneous CF discharge evoked strong inward currents followed by small conductance calcium-activated potassium (SK) channels that mediated outward currents. The application of a GABAA receptor antagonist did not significantly alter the spontaneous SS firing rate, although an AMPA receptor blocker abolished complex spike (CS) activity and induced significantly increased SS firing rates and a decreased coefficient of variation (CV) SS value. Either removal of extracellular calcium or chelated intracellular calcium induced a decrease in amplitude of CS-evoked after-hyperpolarization (AHP) potential accompanied by an increase in SS firing rate. In addition, blocking SK channels activity with a selective antagonist, dequalinium decreased the amplitude of AHP and increased SS firing rate. Moreover, we found repeated CF stimulation at 1 Hz induced a significant decrease in the spontaneous firing rate of SS, and accompanied with an increase in CV of SS in cerebellar slices, which was also abolished by dequalinium. These results indicated that the spontaneous CF discharge contributed to decreasing SS firing rate via activation of SK channels in the cerebellar PCs in vivo in mice.

Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology.

The KCNJ13 gene encodes the inwardly rectifying potassium channel, Kir7.1. Mutations in this gene cause childhood blindness, in which the a- and b-wave responses of electroretinogram (ERG) are abolished. The ERG a-wave is the light-induced hyperpolarization of retinal photoreceptors, and the b-wave is the depolarization of ON-bipolar cells. The Kir7.1 channel is localized to the apical aspects of retinal pigment epithelium (RPE) cells and contributes to a delayed c-wave response. We sought to understand why a defect in an RPE ion-channel result in abnormal electrophysiology at the level of the retinal neurons. We have established the expression of Kir7.1 channels in the mouse RPE. ERGs recorded after mice Kir7.1 suppression by shRNA, or by blocking with VU590, showed reduced a-, b- and c-wave amplitudes. In contrast, the Kir7.1 blocker had no effect on the ex-vivo isolated mouse retina ERG where the RPE is not attached to the isolated retina preparation. Finally, we confirmed the specificity of VU590 action by inhibition of native mouse RPE Kir7.1 current in patch-clamp experiment. We propose that mutant RPE Kir7.1 channels contribute directly to the abnormal ERG associated with blindness via alterations in sub-retinal space K(+) homeostasis in the vicinity of the photoreceptor outer segment.

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation.

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.

Current-direction/amplitude-dependent single channel gating kinetics of mouse pannexin 1 channel: a new concept for gating kinetics.

The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl(-) influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl(-) efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl(-) with gluconate(-) diminishes the inward tail current (Cl(-) efflux) at a membrane potential of -100 mV due to the lowered outward current (gluconate(-) influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.

Induction of adipose and hepatic SWELL1 expression is required for maintaining systemic insulin-sensitivity in obesity.

Obesity is associated with a loss of insulin-sensitivity and systemic dysglycemia, resulting in Type 2 diabetes, however the molecular mechanisms underlying this association are unclear. Through adipocyte patch-clamp studies, we recently showed that SWELL1 is required for the Volume-Regulated Anion Current (VRAC) in adipocytes and that SWELL1-mediated VRAC is activated by both mechanical and pathophysiological adipocyte expansion. We also demonstrated that adipocyte SWELL1 is required for maintaining insulin signaling and glucose homeostasis, particularly in the setting of obesity. Here we show that SWELL1 protein expression is induced in subcutaneous fat, visceral fat and liver in the setting of obesity. Long- term AAV/rec2-shRNA mediated SWELL1 knock-down in both fat and liver are associated with increased weight gain, increased adiposity and exacerbated insulin resistance in mice raised on a high-fat diet. These data further support the notion that SWELL1 induction occurs in insulin- sensitive tissues (liver and adipose) in the setting of over-nutrition and contributes to improved systemic glycemia by supporting enhanced insulin-sensitivity.

Regulation of Hypothalamic CRH Neuron Excitability by Oxytocin.

Oxytocin (OT) is a neuropeptide that exerts multiple actions throughout the brain and periphery. Within the brain, OT regulates diverse neural populations, including neural networks controlling responses to stress. Local release of OT within the paraventricular nucleus (PVN) of the hypothalamus has been suggested to regulate stress responses by modulating the excitability of neighboring corticotropin-releasing hormone (CRH) neurons. However, the mechanisms by which OT regulates CRH neuron excitability are unclear. Here, we investigated the morphological relationship between OT and CRH neurons and determined the effects of OT on CRH neuron excitability. Morphological analysis revealed that the processes of OT and CRH neurons were highly intermingled within the PVN, possibly allowing for local cell-to-cell cross talk. Whole-cell patch-clamp recordings from CRH neurons was used to study the impact of OT on postsynaptic excitability and synaptic innervation. Bath-applied OT did not alter CRH neuron holding current, spiking output or any action potential parameters. Recordings of evoked excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) revealed no net effect of OT on current amplitude, however, subgroups of CRH neurons appeared to respond differentially to OT. Analysis of spontaneous EPSC events uncovered a significant reduction in spontaneous EPSC frequency but no change in spontaneous EPSC amplitude in response to OT. Together these data demonstrate that OT exerts a subtle modulation of synaptic transmission onto CRH neurons providing one potential mechanism by which OT could suppress CRH neuron excitability and stress axis activity. This article is protected by copyright. All rights reserved.

Effects of naftopidil on inhibitory transmission in substantia gelatinosa neurons of the rat spinal dorsal horn in vitro.

Naftopidil is used clinically for the treatment of voiding disorders in benign prostatic hyperplasia. Previous in vivo experiments in which naftopidil was applied intrathecally abolished rhythmic bladder contraction, suggesting that naftopidil might inhibit a voiding reflex through interaction with spinal dorsal horn neurons. Here we aimed to clarify the mechanism of action of naftopidil on dorsal horn neurons.