Pipette Solution Research Articles

Inhibition of RYR2 Activity by Intracellular Flecainide Effectively Suppresses Arrhythmogenic Ca Waves in Intact Ventricular Myocytes from Casq2 -/- Mice

We reported previously that flecainide suppresses Ca waves in MEMBRANE-PERMEABILIZED ventricular myocytes from Casq2-/- mice and that flecainide is more potent (i.e., lower IC50) in Casq2-/-compared to WT myocytes. Other groups reported that in INTACT myocytes flecainide acts only by blocking Na+ channels and has no effect on RyR2. Here we test the effect of flecainide on INTACT ventricular myocytes from Casq2-/- and WT littermates using voltage-clamp. To eliminate any contribution of Na channels, all solutions contained 30 µM TTX. Complete block INa was confirmed for each cell tested. Spontaneous Ca waves were elicited by a pacing train followed by a holding potential of −70mV for 45 s. 6 µM flecainide or vehicle (H2O) was added to both external and pipette solutions. Under such conditions, flecainide had no significant effect on the average number of spontaneous Ca waves in WT myocytes (Vehicle: 3.1±0.6, n=24, Flecainide: 3.0±0.8, n=20, p=NS). In Casq2-/- myocytes, however, flecainide significantly reduced Ca waves (Vehicle: 6.6±1.2, n=23; Flecainide: 2.1±0.4, n=19, p 0.5 vs. Casq2-/- vehicle), suggesting that intracellular dialysis by the patch pipette removed flecainide from the cytosol. We conclude that intracellular concentrations of 6 µM flecainide effectively inhibit arrhythmogenic Ca waves in Casq2-/- but not in WT myocytes, indicating that flecainide action is dependent on the state of RyR2. Our results further demonstrate that flecainide suppression of Ca waves is independent from its Na channel block.

Using Action Potential Clamp Data to Determine the Calcium Fluxes and Contributions in Excitation-Contraction Coupling in Vivo in Cardiomyocytes

Aim: The significance of the different calcium influx pathways like L-type Ca2+-channels (LTCC), reverse Na+/Ca2+-exchange (NCXrev) or Ca2+-induced Ca2+-release (CICR) varies with species and the state of a cell. For instance, in case of heart failure, CICR decreases and influx via NCXrev increases. The aim of this study is to develop a method to quantify calcium fluxes in cardiac excitation-contraction coupling under physiological conditions.Methods: We used action potential clamp to measure ILTCC and INCX currents in rainbow trout ventricular myocytes. Fluorescence changes induced by Ca2+-transients were recorded using 1μM Fluo-4 AM. To measure ILTCC and/or INCX, we used 10μM nifedipine and 1 or 5mM NiCl2 in extracellular solution. Ryanodine receptors and SR calcium ATPase were inhibited by incubating cells in the presence of 10μM ryanodine and 2μM thapsigargin. We used 240μg/ml amphotericin B in pipette solution for the perforated-patch clamp configuration. Also, we composed a simplified mathematical model of Ca2+-dynamics. By fitting model solution to the recorded experimental data, we determined contribution of LTCC, NCX and CICR currents in trout.Results: From experiments where CICR was inhibited we determined the calcium buffering capacity in the cells that is 55μM (+/- 8 μM). With this method we quantify the contribution and kinetics of LTCC (10-20%), NCX (20-40%) and CICR (50-70%).Conclusions: We have developed a method to quantify calcium fluxes under physiological conditions in cardiomyocytes. Furthermore, we found that SR plays a significant role in trout EC coupling. This is in sharp contrast with previous estimates. Also, the method is not specific to trout and is easily applicable to different species.

Intracellular Zinc and Ascorbate Potentiate KCNQ Currents via Distinct Mechanisms

KCNQ (Kv7) potassium voltage-gated channels are expressed in the dorsal root ganglion (DRG) sensory neurons where they play an important role in controlling DRG neuron excitability. Therefore, any substance modulating the activity of these channels could significantly affect the thresholds and firing rates of DRG neurons, including nociceptors. KCNQ channels are potently augmented by reactive oxygen species, therefore, here we investigated potential effects on KCNQ currents of two redox-related compounds, ascorbate and zinc. Potassium currents were measured in CHO cells overexpressing KCNQ4 subunit, using perforated patch clamp technique. Extracellular perfusion of ascorbate increases KCNQ4 currents by 1.96±0.27. This effect was completely reverted by DTT (0.90±0.29), but not by the Zn chelator TPEN. The removal of the redox-sensitive cysteine cluster in the S2-S3 linker (KCNQ4CCC156-158AAA) completely abolished the ascorbate effect. Application of extracellular ZnCl2 (1-100 μM) was without an effect on KCNQ4 currents while inclusion of 10 μM ZnCl2 in the pipette solution increased the KCNQ4 current amplitude from 536±135 pA to 927±177 pA. Extracellular perfusion of zinc ionophore zinc pyrithione (ZnPy) increased the intracellular concentration of Zinc, as shown with FluoZin3 imaging in HEK cells. Additionally, ZnPy increased the KCNQ4 currents by 2.40±0.40 fold, similarly ZnPy also significantly augmented KCNQ2/3 currents. This increase was completely reverted by TPEN (0.89±0.18) but not by DTT. The increase of KCNQ4 currents by ZnPy was identical between the wild-type and the KCNQ4CCC156-158AAA mutant channel. Taken together, these results suggest that both extracellular ascorbate and intracellular Zn can increase KCNQ currents via distinct mechanisms.

Interferon-gamma potentiates NMDA receptor signaling in spinal dorsal horn neurons via microglia-neuron interaction.

BackgroundGlia–neuron interactions play an important role in the development of neuropathic pain. Expression of the pro-inflammatory cytokne →cytokine Interferon-gamma (IFNγ) is upregulated in the dorsal horn after peripheral nerve injury, and intrathecal IFNγ administration induces mechanical allodynia in rats. A growing body of evidence suggests that IFNγ might be involved in the mechanisms of neuropathic pain, but its effects on the spinal dorsal horn are unclear. We performed blind whole-cell patch-clamp recording to investigate the effect of IFNγ on postsynaptic glutamate-induced currents in the substantia gelatinosa neurons of spinal cord slices from adult male rats.ResultsIFNγ perfusion significantly enhanced the amplitude of NMDA-induced inward currents in substantia gelatinosa neurons, but did not affect AMPA-induced currents. The facilitation of NMDA-induced current by IFNγ was inhibited by bath application of an IFNγ receptor-selective antagonist. Adding the Janus activated kinase inhibitor tofacitinib to the pipette solution did not affect the IFNγ-induced facilitation of NMDA-induced currents. However, the facilitatory effect of IFNγ on NMDA-induced currents was inhibited by perfusion of the microglial inhibitor minocycline. These results suggest that IFNγ binds the microglial IFNγ receptor and enhances NMDA receptor activity in substantia gelatinosa neurons. Next, to identify the effector of signal transmission from microglia to dorsal horn neurons, we added an inhibitor of G proteins, GDP-β-S, to the pipette solution. In a GDP-β-S–containing pipette solution, IFNγ-induced potentiation of the NMDA current was significantly suppressed after 30 min. In addition, IFNγ-induced potentiation of NMDA currents was blocked by application of a selective antagonist of CCR2, and its ligand CCL2 increased NMDA-induced currents.ConclusionOur findings suggest that IFNγ enhance the amplitude of NMDA-induced inward currents in substantia gelatinosa neurons via microglial IFNγ receptors and CCL2/CCR2 signaling. This mechanism might be partially responsible for the development of persistent neuropathic pain.

Baicalin Activates Glycine and γ-Aminobutyric Acid Receptors on Substantia Gelatinosa Neurons of the Trigeminal Subsnucleus Caudalis in Juvenile Mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) receives nociceptive afferent inputs from thin-myelinated A[Formula: see text] fibers and unmyelinated C fibers and has been shown to be involved in the processing of orofacial nociceptive information. Scutellaria baicalensis Georgi (Huang-Qin, SbG), one of the 50 fundamental herbs of Chinese herbology, has been used historically as anti-inflammatory and antineoplastic medicine. Baicalin, one of the major compounds of SbG, has been reported to have neuroprotective, anti-inflammatory and analgesic effects. However, the receptor type activated by baicalin and its precise action mechanism on the SG neurons of Vc have not yet been studied. The whole-cell patch clamp technique was performed to examine the ion channels activated by baicalin on the SG neurons of Vc. In high Cl[Formula: see text] pipette solution, the baicalin (300[Formula: see text][Formula: see text]M) induced repeatable inward currents ([Formula: see text][Formula: see text]pA, [Formula: see text]) without desensitization on all the SG neurons tested. Further, the inward currents showed a concentration (0.1-3[Formula: see text]mM) dependent pattern. The inward current was sustained in the presence of tetrodotoxin (0.5[Formula: see text][Formula: see text]M), a voltage sensitive Na[Formula: see text] channel blocker. In addition, baicalin-induced inward currents were reduced in the presence of picrotoxin (50[Formula: see text][Formula: see text]M), a GABAA receptor antagonist, flumazenil (100[Formula: see text][Formula: see text]M), a benzodiazepine-sensitive GABAA receptor antagonist, and strychnine (2[Formula: see text][Formula: see text]M), a glycine receptor antagonist, respectively. These results indicate that baicalin has inhibitory effects on the SG neurons of the Vc, which are due to the activation of GABAA and/or the glycine receptor. Our results suggest that baicalin may be a potential target for orofacial pain modulation.

Evidence for glucagon-like peptide-1 receptor signaling to activate ATP-sensitive potassium channels in pancreatic beta cells

Glucagon-like peptide-1 (GLP-1) is a gut peptide that promotes insulin release from pancreatic beta cells. GLP-1 has been shown to confer glucose-insensitive beta cells with glucose sensitivity by modulation of the activity of the ATP-sensitive potassium (KATP) channel. The channel closing effect of GLP-1, interacting with corresponding G-protein-coupled receptors, has been well established; however, to our knowledge, no study has shown whether GLP-1 directly induces activation of beta-cell KATP channels. Here, we aimed to evaluate whether the activation of beta-cell KATP channels by GLP-1 exists and affects intracellular Ca2+ levels ([Ca2+]i). KATP channel activity was measured in isolated rat pancreatic beta cells by whole-cell perforated patch-clamp recordings with a diazoxide-containing pipette solution. Changes in [Ca2+]i and the subcellular localization of KATP channels were observed using the calcium-sensitive dye fura-4/AM and anti-Kir6.2 antibodies in INS-1 beta cells, respectively. To eliminate the well-known inhibitory effects of GLP-1 on KATP channel activity, channels were fully inhibited by pretreatment with methyl pyruvate and epigallocatechin-3-gallate. In the pretreated beta cells, GLP-1 and exendin-4 promptly activated the channels, reducing [Ca2+]i. The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 blocked the effects of GLP-1 on channel activity. Moreover, phosphatidylinositol-3,4,5-trisphosphate mimicked the effects of GLP-1. These results suggested that beta-cell GLP-1 receptor signaling involved activation of KATP channels via a PI3K-dependent pathway. This alternative mechanism of GLP-1 function may act as a negative feedback pathway, modulating the glucose-dependent GLP-1 inhibition on KATP channel activity.

Abstract 14587: Proarrhythmic Effects of Ibrutinib, a Clinically Approved Inhibitor of Bruton’S Tyrosine Kinase (BTK) Used in Cancer Therapy

Introduction: Ibrutinib is a novel and potent BTK inhibitor which has been approved for several types of B-cell malignancies. Our meta-analysis of the published clinical trials finds that atrial fibrillation occurs in ~7% of patients after chronic ibrutinib exposure. Ventricular tachycardia has also been reported. Recently, multi-targeted tyrosine kinase inhibitors (TKIs) have been associated with arrhythmias; one implicated mechanism is increased late sodium current (INa-L) after chronic but not acute drug exposure. This effect appears to be mediated by inhibition of PI3Kinase since it is rescued by intra-pipette PIP3, a PI3K effector. Hypothesis: In this study, we tested the hypothesis that chronic exposure to ibrutinib exerts arrhythmogenic effects by this mechanism. Methods: Isolated adult mouse cardiac atrial/ventricular and rabbit ventricular myocytes as well as KCNH2 (HERG/Kv11.1)- or Kv4.3-transfected CHO cells were used for the electrophysiological studies pre- and post-ibrutinib (0.3 μ or 1 μM) acutely (5-10 min) and chronically (5 hrs in myocytes or 48 hrs in CHO cells). In some experiments, 1 μM PIP3 was added into the pipette solution. Results: Chronic but not acute ibrutinib prolonged cardiac action potentials (APs), triggered abnormal APs (EADs and DADs; Figure), and markedly increased cardiac INa-L (1.68±0.27% vs control 0.21±0.04% of peak current, P<0.01) in mouse atrial cells. A similar INa-L increase was also seen in mouse and rabbit ventricular cells. The abnormal APs and increased INa-L were antagonized by adding 1 μM PIP3 into the pipette and by the INa-L blocker ranolazine (10 μM). Ibrutinib has minor and no effects on cardiac HERG and Kv4.3 currents. Conclusion: Ibrutinib, like other TKIs, increased cardiac INa-L, an effect reversed by intracellular PIP3. These data further reinforce the importance of PIP3 signaling as a proximate mechanism leading to arrhythmogenesis by emerging targeted oncology therapies.

Gap junction coupling confers isopotentiality on astrocyte syncytium.

Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K(+) free pipette solutions that alter the K(+) equilibrium potential to non-physiological voltages. We show that an astrocyte's associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte's VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K(+) and thereby maintains a sustained driving force for highly efficient K(+) uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

Ca2+ Diffusion through Endoplasmic Reticulum Supports Elevated Intraterminal Ca2+ Levels Needed to Sustain Synaptic Release from Rods in Darkness.

Vertebrate rod and cone photoreceptors both release vesicles at synaptic ribbons, but rods also exhibit substantial slow release at non-ribbon sites triggered by Ca(2+)-induced Ca(2+) release (CICR). Blocking CICR inhibits >50% of release from rods in darkness. How do rods maintain sufficiently high [Ca(2+)] in terminal endoplasmic reticulum (ER) to support sustained CICR-driven synaptic transmission? We show that maintained depolarization creates a [Ca(2+)] gradient within the rod ER lumen that promotes soma-to-terminal diffusion of Ca(2+) to replenish intraterminal ER stores. This mechanism allows CICR-triggered synaptic release to be sustained indefinitely while rods remain depolarized in darkness. Free diffusion of Ca(2+) within the ER may also communicate synaptic Ca(2+) changes back to the soma to influence other critical cell processes.

Effects of Dangkwisoo‑san, a traditional herbal medicine for treating pain and blood stagnation, on the pacemaker activities of cultured interstitial cells of Cajal.

The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoo‑san (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The whole‑cell patch‑clamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+i) was analyzed in cultured ICCs using fura‑2‑acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4‑diphenylacetoxy‑N‑methyl‑piperidine methiodide muscarinic M3 receptor antagonist inhibited DS‑induced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'‑[β‑thio] diphosphate (GDP‑β‑S; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DS‑induced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DS‑induced pacemaker depolarizations. Pretreatment with Ca2+‑free solution and thapsigargin, a Ca2+‑ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DS‑induced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G protein‑dependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na(+), H(+) (OH(-)), bicarbonate, borate, and NH4 (+). Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH4 (+), Na(+), and H(+) contributions to electrogenic ion transport through SLC4A11 stably expressed in Na(+)/H(+) exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH4Cl were determined by the [NH3]o, not [NH4 (+)]o, and current amplitudes varied with the [H(+)] gradient. These currents were relatively unaffected by removal of Na(+), K(+), or Cl(-) from the bath but could be reduced by inclusion of NH4Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH4Cl. Reversal potential shifts in response to changing [NH3]o and pHo suggested an NH3/H(+)-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH3 but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH3/H(+)). NH3-dependent currents were insensitive to 10 μM ethyl-isopropyl amiloride or 100 μM 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid. We propose that SLC4A11 is an NH3/2H(+) co-transporter exhibiting unique characteristics.

CAMP‐dependent protein kinase inhibits α7 nicotinic receptor activity in layer 1 cortical interneurons through activation of D1/D5 dopamine receptors

Protein kinases can modify the function of many proteins including ion channels. However, the role of protein kinaseA in modifying nicotinic receptors in the CNS has never been investigated. We showed through whole-cell recordings of layer1 prefrontal cortical interneurons that α7 nicotinic responses are negatively modulated by protein kinaseA. Furthermore, we show that stimulation of dopamine receptors can similarly attenuate α7 nicotinic responses through the activation of protein kinaseA. These results suggest how the interaction of the cholinergic and dopaminergic systems may influence neuronal excitability in the brain. Phosphorylation of ion channels, including nicotinic acetylcholine receptors (nAChRs), by protein kinases plays a key role in the modification of synaptic transmission and neuronal excitability. α7 nAChRs are the second most prevalent nAChR subtype in the CNS following α4β2. Serine 365 in the M3-M4 cytoplasmic loop of the α7 nAChR is a phosphorylation site for protein kinase A (PKA). D1/D5 dopamine receptors signal through the adenylate cyclase-PKA pathway and play a key role in working memory and attention in the prefrontal cortex. Thus, we examined whether the dopaminergic system, mediated through PKA, functionally interacts with the α7-dependent cholinergic neurotransmission. In layer1 interneurons of mouse prefrontal cortex, α7 nicotinic currents were decreased upon stimulation with 8-Br-cAMP, a PKA activator. In HEK 293T cells, dominant negative PKA abolished 8-Br-cAMP's effect of diminishing α7 nicotinic currents, while a constitutively active PKA catalytic subunit decreased α7 currents. In brain slices, the PKA inhibitor KT-5720 nullified 8-Br-cAMP's effect of attenuating α7 nicotinic responses, while applying a PKA catalytic subunit in the pipette solution decreased α7 currents. 8-Br-cAMP stimulation reduced surface expression of α7 nAChRs, but there was no change in single-channel conductance. The D1/D5 dopamine receptor agonist SKF83822 similarly attenuated α7 nicotinic currents from layer1 interneurons and this attenuation of nicotinic current was prevented by KT-5720. These results demonstrate that dopamine receptor-mediated activation of PKA negatively modulates nicotinic neurotransmission in prefrontal cortical interneurons, which may be a contributing mechanism of dopamine modulation of cognitive behaviours such as attention or working memory.

Inhibitory effects of endomorphin-2 on excitatory synaptic transmission and the neuronal excitability of sacral parasympathetic preganglionic neurons in young rats.

The function of the urinary bladder is partly controlled by parasympathetic preganglionic neurons (PPNs) of the sacral parasympathetic nucleus (SPN). Our recent work demonstrated that endomorphin-2 (EM-2)-immunoreactive (IR) terminals form synapses with μ-opioid receptor (MOR)-expressing PPNs in the rat SPN. Here, we examined the effects of EM-2 on excitatory synaptic transmission and the neuronal excitability of the PPNs in young rats (24–30 days old) using a whole-cell patch-clamp approach. PPNs were identified by retrograde labeling with the fluorescent tracer tetramethylrhodamine-dextran (TMR). EM-2 (3 μM) markedly decreased both the amplitude and the frequency of the spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) of PPNs. EM-2 not only decreased the resting membrane potentials (RMPs) in 61.1% of the examined PPNs with half-maximal response at the concentration of 0.282 μM, but also increased the rheobase current and reduced the repetitive action potential firing of PPNs. Analysis of the current–voltage relationship revealed that the EM-2-induced current was reversed at −95 ± 2.5 mV and was suppressed by perfusion of the potassium channel blockers 4-aminopyridine (4-AP) or BaCl2 or by the addition of guanosine 5′-[β-thio]diphosphate trilithium salt (GDP-β-S) to the pipette solution, suggesting the involvement of the G-protein-coupled inwardly rectifying potassium (GIRK) channel. The above EM-2-invoked inhibitory effects were abolished by the MOR selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), indicating that the effects of EM-2 on PPNs were mediated by MOR via pre- and/or post-synaptic mechanisms. EM-2 activated pre- and post-synaptic MORs, inhibiting excitatory neurotransmitter release from the presynaptic terminals and decreasing the excitability of PPNs due to hyperpolarization of their membrane potentials, respectively. These inhibitory effects of EM-2 on PPNs at the spinal cord level may explain the mechanism of action of morphine treatment and morphine-induced bladder dysfunction in the clinic.

CsCl-Based Internal Pipette Solution

CsCl-Based Internal Pipette Solution

K-Aspartate-Based Internal Pipette Solution

K-Aspartate-Based Internal Pipette Solution

Potentiation of tonic GABAergic inhibition by activation of postsynaptic kainate receptors

Presynaptic kainate-type glutamate ionotropic receptors (KARs) that mediate either the depression or the facilitation of GABA release have been intensively studied. Little attention has been given to the modulation of GABAA receptors (GABAARs) by postsynaptic KARs. Recent studies suggest that two GABAAR populations, synaptic (sGABAAR) and extrasynaptic (eGABAAR) GABAARs, mediate phasic and tonic forms of inhibition, respectively. Tonic inhibition plays an important role in the excitability of neuronal circuits and the occurrence of epileptic seizures. For this study, we are the first to report that the activation of postsynaptic KARs by the KAR agonist, Kainic acid (KA, 5μM), enhanced tonic inhibition by potentiating eGABAARs. KA enhanced THIP-induced eGABAAR currents and prolonged the rise and decay time of muscimol-induced sGABAAR/eGABAAR currents, but also depressed the amplitude of evoked inhibitory postsynaptic currents (IPSCs), unitary IPSCs (uIPSCs), and muscimol-induced sGABAAR/eGABAAR currents. The PKC inhibitor, staurosporine (1μM), in the patch pipette solution fully blocked the KA-induced potentiation of tonic inhibition, suggesting the involvement of an intracellular PKC pathway. Our study suggests that the activation of postsynaptic KARs potentiates eGABAARs but depresses sGABAARs. By activating postsynaptic KARs, synaptically released glutamate depresses phasic inhibition to facilitate neuronal plasticity, but potentiates tonic inhibition to protect neurons from over-excitation.

Synergistic modulation of KCNQ1/KCNE1 K+ channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP

Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion.Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.

K-Aspartate-Based Pipette Solution

K-Aspartate-Based Pipette Solution

ANO1 and TRPC6 Channels are Functionally Coupled in Cerebral Artery Smooth Muscle Cells

Activation of anoctamin 1 (ANO1 or TMEM16A), a calcium (Ca2+)‐activated chloride channel, in myocytes contributes to pressure‐induced vasoconstriction (the myogenic response) in small arteries. ANO1 is activated due to Ca2+ influx through a stretch‐activated non‐selective cation channel that has not been identified. We used biochemical, fluorescence and patch‐clamp techniques to identify the cation channel associated with ANO1 in cerebral artery myocytes. ImmunoFRET experiments indicated that transient receptor potential channel 6 (TRPC6) and ANO1 were located in close spatial proximity in arterial myocytes. ANO1 antibodies co‐immunoprecipitated both ANO1 and TPRC6 protein from arterial lysate. A TRPC6 antibody that is a selective TRPC6 channel inhibitor blocked swelling‐activated ANO1 currents. HYP9, a TPRC6‐specific channel activator, activated Cl‐ currents that were blocked by ANO1‐inh, a ANO1‐specific inhibitor. Substitution of EGTA with BAPTA, a fast Ca2+ chelator in the patch pipette solution inhibited HYP9‐induced ANO1 current activation. ANO1 knockdown using siRNA, reduced both ANO1 protein and the amplitude of HYP9‐induced ANO1 currents. These data indicate that TRPC6 and ANO1 channels are spatially localized and that Ca2+ influx via TRPC6 channels activates ANO1 currents in arterial myocytes.