Pipette Solution Research Articles

OR-014 Chronic exercise potentiates anorectic effects of leptin in hypothalamic Pomc neurons

OR-014 Chronic exercise potentiates anorectic effects of leptin in hypothalamic Pomc neurons

Analyzing synaptic plasticity at the single cell level with STDP

Abstract Using patch clamp recordings in acutely isolated brain slices allows to investigate molecular processes that are involved in long-term potentiation (LTP) and long-term depression (LTD) at the level of a single postsynaptic neuron. Via the pipette solution in the recording pipette, it is possible to apply inhibitors of signaling cascades selectively into the postsynaptic neuron to unravel the molecular mechanisms of synaptic plasticity. In recent years, LTP research has been increasingly focused on induction protocols for LTP and LTD that rely on a minimal number of repeated synaptic stimulations at low frequency to induce synaptic plasticity. This is where spike timing-dependent plasticity (STDP) comes into play. STDP can be induced by repetitive pairings of action potential firing in presynaptic (first neuron) – and postsynaptic (second synaptically connected) neurons, that occurs with a delay of roughly 5–20 ms in forward or backward manner. While forward pairing with short positive time delays (“pre before post”) leads to LTP, backward pairing (“post before pre”) leads to LTD. In addition to the exact sequence and timing of pre- and postsynaptic spiking, the presence of neuromodulatory transmitters in the extracellular space (e. g., dopamine, acetylcholine, noradrenaline) and the synaptic release of intercellular mediators of synaptic plasticity (e. g., BDNF, endocannabinoids) critically regulate the outcome of STDP protocols. In this review we focus on the role of these mediators and modulators of synaptic plasticity in STDP.

TMEM16F/ANO6, a Ca2+-activated anion channel, is negatively regulated by the actin cytoskeleton and intracellular MgATP

Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca2+]i. In addition to the low sensitivity to Ca2+, the activation of ANO6 Cl− conductance is very slow (>3–5 min to reach peak level at 10 μM [Ca2+]i), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (IANO6,w-c), disruption of the actin cytoskeleton with cytochalasin-D (cytoD) significantly accelerated the activation kinetics, while actin filament-stabilizing agents (phalloidin and jasplakinolide) commonly inhibited IANO6,w-c. Inside-out patch clamp recording of ANO6 (IANO6,i-o) showed immediate activation by raising [Ca2+]i. We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of IANO6,w-c, and also prevented the inactivation of IANO6,w-c. However, the addition of cytoD still accelerated both activation and inactivation of IANO6,w-c. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca2+-dependent activation and inactivation of IANO6,w-c, respectively.

Long-Term IL-2 Incubation-Induced L-type Calcium Channels Activation in Rat Ventricle Cardiomyocytes.

The following study examined the impact of IL-2 on Ca2+ channel activity in the event of several hours' incubation in IL-2. The right ventricle free wall for action potential measurements was isolated and perfused with Tyrode solution. The whole-cell voltage clamp experiments were performed on enzymatically isolated single cardiomyocytes. The whole-cell voltage clamp recording of Ca2+ currents was performed using the Cs+-based pipette and bath solutions. The protocol with depolarizing prepulse (- 40mV) was used to inactivate both Na+ current and Ca2+T-type current. The L-type Ca2+ current was elicited by a series of 250ms depolarizing square pulses with 10mV increments. At the 15th minute of continuous recording, the peak density at 0mV was - 3.036 ± 0.3015 pA/pF under IL-2 and - 3.008 ± 0.3452 pA/pF in control conditions. The IL-2 in moderate concentration (1ng/mL) has no acute effects on ICa.L in rat ventricular cells. In contrast, to the lack of acute effects, the long-term incubation with IL-2 (2h or more) produced a prominent enhancement of Ca2+L-type current. In rat, ventricular myocardium IL-2 (1ng/mL) produced a very gradual prolongation of subendocardial APs which reached a maximal extent after 3-4h of treatment. The patch clamp study shows an IL-2-induced ICa.L current activation, while the action potential studies on multicellular ventricular preparations suggest an IL-2-induced L-type Ca2+ channel participation in the development of AP.

Noradrenaline up-regulates volume-regulated chloride current by PKA-independent cAMP/exchange protein activated by cAMP pathway in human atrial myocytes.

Adrenergic regulation of cell volume-regulated chloride current (ICl.vol ) is species-dependent. The present study investigates the mechanism underlying adrenergic regulation of ICl.vol in human atrial myocytes. Conventional whole-cell patch voltage-clamp techniques were used to record membrane current in human atrial myocytes. ICl.vol was evoked by hyposmotic bath solution (0.6 times isosmotic, 0.6T). ICl.vol was augmented by noradrenaline (1μM) during cell swelling in 0.6T but not under isosmotic (1T) conditions. Up-regulation of ICl.vol in 0.6T was blocked by the β-adrenoceptor antagonist propranolol (2μM), but not by the α1 -adrenoceptor antagonist prazosin (2μM). This β-adrenergic response involved cAMP but was independent of PKA; the protein kinase inhibitor H-89 (2μM) or PKI (10μM in pipette solution) failed to prevent ICl.vol up-regulation by noradrenaline. Moreover, the PI3K/PKB inhibitor LY294002 (50μM) and the PKG inhibitor KT5823 (10μM) did not affect noradrenaline-induced increases in ICl.vol . Interestingly, the exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2'-O-Me-cAMP (50μM) also up-regulated ICl.vol , and the noradrenaline-induced increase of ICl.vol in 0.6T was reversed or prevented by the Epac inhibitor ESI-09 (10μM). These data show that ICl.vol evoked by cell swelling of human atrial myocytes is up-regulated by noradrenaline via a PKA-independent cAMP/Epac pathway in human atrial myocytes. cAMP/Epac-induced ICl.vol is expected to shorten action potential duration during human atrial myocytes swelling and may be involved in abnormal cardiac electrical activity during cardiac pathologies that evoke β-adrenoceptor signalling.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Transient receptor potential melastatin 4 channel inhibitor 9-phenanthrol inhibits K+ but not Ca2+ currents in canine ventricular myocytes.

The role of transient receptor potential melastatin 4 (TRPM4) channels has been frequently tested using their inhibitor 9-phenanthrol in various cardiac preparations; however, the selectivity of the compound is uncertain. Therefore, in the present study, the concentration-dependent effects of 9-phenanthrol on major ionic currents were studied in canine isolated ventricular cells using whole-cell configuration of the patch-clamp technique and 10 mM BAPTA-containing pipette solution to prevent the Ca2+-dependent activation of TRPM4 channels. Transient outward (Ito1), rapid delayed rectifier (IKr), and inward rectifier (IK1) K+ currents were suppressed by 10 and 30 μM 9-phenanthrol with the blocking potency for IK1 < IKr < Ito1 and partial reversibility. L-type Ca2+ current was not affected up to the concentration of 30 μM. In addition, a steady outward current was detected at voltages positive to -40 mV in 9-phenanthrol, which was larger at more positive voltages and larger 9-phenanthrol concentrations. Action potentials were recorded using microelectrodes. Maximal rate of depolarization, phase-1 repolarization, and terminal repolarization were decreased and the plateau potential was depressed by 9-phenanthrol (3-30 μM), congruently with the observed alterations of ionic currents. Significant action potential prolongation was observed by 9-phenanthrol in the majority of the studied cells, but only at 30 μM concentration. In conclusion, 9-phenanthrol is not selective to TRPM4 channels in canine ventricular myocardium; therefore, its application as a TRPM4 blocker can be appropriate only in expression systems but not in native cardiac cells.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel.

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

Dialysis of an Unknown Cytosolic Cofactor Results in Partial, Delayed Activation of TRPA1

In many organ systems, a subset of sensory nerve fibers termed nociceptors are stimulated by noxious stimuli and evoke defensive sensations, behaviors and reflexes. Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in sensory neurons and evokes action potentials in nociceptive fibers when activated. Agonists of TRPA1 are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1 subunits, which result in activation. Electrophiles can modify cysteines through reversible or irreversible reactions based on the structure of the compound. Whole‐cell patch clamp and live‐cell Ca2+ imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of electrophiles. Ca2+ imaging experiments show that irreversible electrophiles like N‐ethylmaleimide (NEM) rapidly increase Ca2+ influx, which suggests TRPA1 activation. However, whole‐cell patch clamp studies show a delayed, partial activation of TRPA1 with NEM treatment, but display rapid activation by reversible electrophiles. Furthermore, inside‐out single channel patch clamp experiments exhibit a reduction in open probability with NEM treatment that is comparable to the whole‐cell patch clamp experiments. To determine the mechanism of TRPA1 activation, the cause for the disparity of TRPA1 activation between whole‐cell patch clamp and Ca2+ imaging assays must be identified. We hypothesized that dialysis of cellular components in whole‐cell patch clamp that promote rapid, robust TRPA1 activation with NEM. To test our hypothesis, I performed whole‐cell patch clamp and gramicidin‐perforated patch clamp, a non‐invasive patch clamp method, to observe and compare the rate‐of‐change of NEM‐induced TRPA1 responses. Both methods contain 5mM Na‐polyphosphates in the pipette solution to prevent rundown of the TRPA1 channel. Studies using gramicidin‐perforated patch clamp exhibit a significant increase in the rate‐of‐change of NEM‐induced TRPA1 responses compared to activation in whole‐cell experiments. We also hypothesize that glutathione is the unidentified cofactor that is dialyzed out of the cell in whole‐cell experiments. Future whole‐cell experiments with glutathione (5mM) in the pipette solution will be performed to determine if rapid TRPA1 activation can be rescued. Identification of the unknown cytosolic cofactor will promote a complete understanding of the electrophilic cysteine modification events that underlie TRPA1 activation.Support or Funding InformationR01 from the NHLBIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Subunit composition critically determines pH sensitivity of epithelial sodium channels in Xenopus laevis

The Epithelial Sodium Channel (ENaC) mediates the selective flux of sodium ions across the apical membrane of epithelial cells. ENaC consists of three subunits (α,β,γ) and its activity at the cell surface is subject to a multitude of regulatory mechanisms, including proteolytic cleavage, extracellular pH and Na+ ions. Channel regulation may also depend on ENaC subunit composition, as an additional δ‐subunit can replace α‐ENaC in the heterotrimeric configuration. As the physiological implications of ENaC containing the δ‐subunit are poorly understood, we characterized the regulation of Xenopus δβγ‐ENaC by extracellular pH and Na+.Regulation of Xenopus δβγ‐ENaC by extracellular Na+ and pH was investigated by two‐electrode voltage‐clamp and cell‐attached Patch‐Clamp electrophysiology using the Xenopus oocyte expression system. Successive reduction of extracellular pH from pH 8 – 6 increased amiloride‐sensitive transmembrane currents of oocytes expressing δβγ‐ENaC by &gt; 7 fold. In cell‐attached Patch‐Clamp recordings the pH of the pipette solution (pHpip = extracellular) affected δβγ‐ENaC open probability and kinetics. Compared to pHpip at 7.4, alkaline conditions (pHpip 8) decreased open probability as well as the mean channel open time, whereas under acidic conditions (pHpip 6) an increased open probability was accompanied by a reduction of average channel closed times. Transmembrane currents mediated by Xenopus δβγ‐ENaC are sensitive to sodium self‐inhibition, a phenomenon describing the fast inhibition of ENaC in the presence of high extracellular Na+ concentrations. Channel activation under acidic conditions (pH 7 &amp; 6) was accompanied by a decrease in magnitude and apparent Na+‐affinity of self‐inhibition, whereas these effects were reversed under alkaline conditions (pH 8). Hypothesizing mutual regulation of δβγ‐ENaC activity by extracellular Na+ and pH, we examined whether neutralization of pH‐sensitive aspartates in the δ‐subunit affected channel self‐inhibition and regulation by pH. Specific aspartates in a subunit domain corresponding to a putative Na+‐sensing site of mouse α‐ENaC (Kashlan et al., 2015, J Biol Chem 290:568–76) were replaced by asparagines via site‐directed mutagenesis (δD293N, δD296N, δD293,6N). All mutations decreased pH‐mediated channel activation. Notably, ENaC containing the δD296N mutation also displayed a decreased sodium self‐inhibition, which was not observed in channels bearing a single δD293N mutation. Furthermore, human δβγ‐ENaCs which have a reduced sodium self‐inhibition were not activated by acidic pH as observed with the Xenopus orthologue displaying a strong sodium self‐inhibition.In conclusion, these results demonstrate that activity of ENaC containing the δ‐subunit in Xenopus laevis is sensitive to changes in the extracellular pH, whereas ENaC containing the α‐subunit is not. Proton‐mediated activation of ENaC involves changes in single channel kinetics and a reduction of sodium self‐inhibition. Thus, sensitivity of this amphibian ENaC to extracellular pH is critically determined by its subunit composition.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased Hydrogen Peroxide After Traumatic Brain Injury Disrupts Phosphatidylinositol 4,5‐Bisphosphate Metabolism Causing Impaired Inward Rectifier Potassium Channel Function

Severe trauma, including traumatic brain injury (TBI), can result in several deleterious outcomes including coagulopathy, multi‐organ failure and death. We previously showed that TBI in rodents causes uncoupling of mesenteric artery (MA) nitric oxide production, resulting in excessive reactive oxygen species (ROS) generation and impaired endothelial‐dependent vasodilation. The inward‐rectifier potassium channel (KIR) is an essential component of agonist‐ and flow‐induced vasodilation and signal transduction in mesenteric arteries (MAs); however, its function following trauma has not been assessed. Phosphatidylinositol 4,5‐bisphosphate (PIP2) is a membrane phospholipid which is required for normal KIR channel function in MAs. We hypothesized that oxidative stress, specifically hydrogen peroxide (H2O2), was driving down PIP2 levels, causing KIR channelopathies. We studied male rats 24 hours after fluid percussion TBI, compared to controls. Plasma oxidative‐reduction potential (ORP), an indicator of oxidative stress, was significantly elevated in TBI rats compared to controls. In TBI but not control plasma, catalase application significantly reduced the ORP signal to control values, suggesting that H2O2 contributes to global oxidative stress following TBI. We then measured KIR currents in isolated endothelial cells (ECs) from MAs, using the whole‐cell patch‐clamp technique from control and TBI rats. KIR channel currents were significantly diminished in ECs from TBI rats compared to controls. KIR currents in ECs from TBI MAs were rescued with the addition of PIP2 to the pipette solution when compared to untreated TBI cells, and showed no difference from their control, treated counterparts, implying depletion of PIP2 following TBI is a contributing factor to KIR channel dysfunction. Next, diameter responses of arteries using pressure myography were measured. In control arteries, increasing concentrations of extracellular potassium from 6 to 10 mM K+ caused dilations; concentrations between 14 to 60 mM K+ caused constrictions. Dilations to 10 mM K+ were significantly reduced following TBI, consistent with an impaired KIR channel response. Next, basal KIR function was assessed using the KIR and KIR2.1 antagonists BaCl2 (100 μM) and ML 133 (20 μM). Following TBI, arteries constricted significantly less to both BaCl2 and ML 133. Furthermore, dilations to 10 mM K+ and constrictions to ML 133 were restored in TBI arteries in the presence of catalase. Lastly, To further quantify cellular pathology following TBI, the metabolomic phenotype was measured by liquid chromatography‐mass spectrometry (LC‐MS) in plasma obtained from control and TBI rats. Rats exhibited a profound metabolopathy following TBI; within the lipidome: arachidonate metabolism, glycerophospholipid synthesis, and fatty acid mobilization were significantly altered. These data suggest H2O2 production serves an antagonistic role on KIR channel function through changes in second messenger and lipid signaling pathways in the mesenteric circulation following TBI.Support or Funding InformationThis work was supported by the Totman Medical Research Trust and the National Institute of Health (K08‐GM‐098795 and UM1‐HL‐120877).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of the α-Subunit of Phosphoinositide 3-Kinase in Heart Increases Late Sodium Current and Is Arrhythmogenic.

Although inhibition of phosphoinositide 3-kinase (PI3K) is an emerging strategy in cancer therapy, we and others have reported that this action can also contribute to drug-induced QT prolongation and arrhythmias by increasing cardiac late sodium current (INaL). Previous studies in mice implicate the PI3K-α isoform in arrhythmia susceptibility. Here, we have determined the effects of new anticancer drugs targeting specific PI3K isoforms on INaL and action potentials (APs) in mouse cardiomyocytes and Chinese hamster ovary cells (CHO). Chronic exposure (10-100 nM; 5-48 hours) to PI3K-α-specific subunit inhibitors BYL710 (alpelisib) and A66 and a pan-PI3K inhibitor (BKM120) increased INaL in SCN5A-transfected CHO cells and mouse cardiomyocytes. The specific inhibitors (10-100 nM for 5 hours) markedly prolonged APs and generated triggered activity in mouse cardiomyocytes (9/12) but not in controls (0/6), and BKM120 caused similar effects (3/6). The inclusion of water-soluble PIP3, a downstream effector of the PI3K signaling pathway, in the pipette solution reversed these arrhythmogenic effects. By contrast, inhibition of PI3K-β, -γ, and -δ isoforms did not alter INaL or APs. We conclude that inhibition of cardiac PI3K-α is arrhythmogenic by increasing INaL and this effect is not seen with inhibition of other PI3K isoforms. These results highlight a mechanism underlying potential cardiotoxicity of PI3K-α inhibitors.

Activation of Endogenous Piezo1 Channels by Shear Stress in Excised Membrane Patches

The sensing of shear stress arising because of blood flow is critical in vascular development and maintenance of a healthy vasculature in the adult. The identity of molecules which sense and transduce this force into appropriate vascular anatomy and function is therefore keenly sought. A central question is whether there is a force sensor protein (“receptor”) which directly detects the force, acting either alone or in complex with other proteins. Piezo1 channels are Ca2+-permeable non-selective cationic channels which are activated by membrane stretch. These channels are important for shear stress-sensing and vascular function in embryonic and adult mice (Li et al 2014 Nature 515, 279-; Rode et al 2017 Nature Communications 8, 350-). We have used patch-clamp recording to study the channels in freshly isolated endothelium of second-order mesenteric arteries of adult mice. The pipette solution contained (mM): 145 KCl, 1 MgCl2, 0.5 EGTA, 10 HEPES. Whole-cell perforated patch recordings from endothelial fragments revealed a resting membrane potential of −43 mV which was depolarized by fluid flow. When Piezo1 was conditionally deleted in endothelium the resting potential was hyperpolarized and fluid flow-evoked depolarization was absent (Rode et al 2017). Outside-out patches were then excised from endothelium and found to contain constitutive single channel activity with the expected 25-pS unitary conductance of Piezo1 channels; the channel activity could be further enhanced by fluid flow and inhibited by the non-selective Piezo1 channel inhibitor, Gd3+, and was absent in Piezo1 knockout mice (Rode et al 2017). The data suggest Piezo1 channels of the endothelium can be activated by shear stress in a sparse excised membrane environment, consistent with direct sensing of shear stress or a closely-associated force. The research was supported by grants from the British Heart Foundation, MRC, Wellcome Trust.

CrossTalk proposal: CNNM proteins are Na+ /Mg2+ exchangers playing a central role in transepithelial Mg2+ (re)absorption.

CrossTalk proposal: CNNM proteins are Na+ /Mg2+ exchangers playing a central role in transepithelial Mg2+ (re)absorption.

Modeling sources of interlaboratory variability in electrophysiological properties of mammalian neurons.

Patch-clamp electrophysiology is widely used to characterize neuronal electrical phenotypes. However, there are no standard experimental conditions for in vitro whole cell patch-clamp electrophysiology, complicating direct comparisons between data sets. In this study, we sought to understand how basic experimental conditions differ among laboratories and how these differences might impact measurements of electrophysiological parameters. We curated the compositions of external bath solutions (artificial cerebrospinal fluid), internal pipette solutions, and other methodological details such as animal strain and age from 509 published neurophysiology articles studying rodent neurons. We found that very few articles used the exact same experimental solutions as any other, and some solution differences stem from recipe inheritance from advisor to advisee as well as changing trends over the years. Next, we used statistical models to understand how the use of different experimental conditions impacts downstream electrophysiological measurements such as resting potential and action potential width. Although these experimental condition features could explain up to 43% of the study-to-study variance in electrophysiological parameters, the majority of the variability was left unexplained. Our results suggest that there are likely additional experimental factors that contribute to cross-laboratory electrophysiological variability, and identifying and addressing these will be important to future efforts to assemble consensus descriptions of neurophysiological phenotypes for mammalian cell types. NEW & NOTEWORTHY This article describes how using different experimental methods during patch-clamp electrophysiology impacts downstream physiological measurements. We characterized how methodologies and experimental solutions differ across articles. We found that differences in methods can explain some, but not all, of the study-to-study variance in electrophysiological measurements. Explicitly accounting for methodological differences using statistical models can help correct downstream electrophysiological measurements for cross-laboratory methodology differences.

KATP Channels Mediate Differential Metabolic Responses to Glucose Shortage of the Dorsomedial and Ventrolateral Oscillators in the Central Clock

The suprachiasmatic nucleus (SCN) central clock comprises two coupled oscillators, with light entraining the retinorecipient vasoactive intestinal peptide (VIP)-positive ventrolateral oscillator, which then entrains the arginine vasopressin (AVP)-positive dorsomedial oscillator. While glucose availability is known to alter photic entrainment, it is unclear how the SCN neurones respond to metabolic regulation and whether the two oscillators respond differently. Here we show that the ATP-sensitive K+ (KATP) channel mediates differential responses to glucose shortage of the two oscillators. RT-PCR and electrophysiological results suggested the presence of Kir6.2/SUR1 KATP channels in the SCN neurones. Immunostaining revealed preferential distribution of Kir6.2 in the dorsomedial subregion and selective colocalization with AVP. Whole cell recordings with ATP-free pipette solution indicated larger tolbutamide-induced depolarisation and tolbutamide-sensitive conductance in dorsal SCN (dSCN) than ventral SCN (vSCN) neurones. Tolbutamide-sensitive conductance was low under perforated patch conditions but markedly enhanced by cyanide inhibition of mitochondrial respiration. Glucoprivation produced a larger steady-state inhibition in dSCN than vSCN neurones, and importantly hypoglycemia via opening KATP channels selectively inhibited the KATP-expressing neurones. Our results suggest that the AVP-SCN oscillator may act as a glucose sensor to respond to glucose shortage while sparing the VIP-SCN oscillator to remain in synch with external light-dark cycle.

Quantitative analysis of the Ca2+ -dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes.

[Ca2+ ]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr ) amplitude and voltage dependence were unaffected by high [Ca2+ ]i . When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+ ]i was buffered to 500nm. The slowly activating delayed rectifier K+ current (IKs ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+ ]i ) and β-adrenergic receptor (β-AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+ ]i dependence of IKs in steady-state conditions and with dynamically changing membrane potential and [Ca2+ ]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+ ]i , we found that raising [Ca2+ ]i from 100 to 600nm produced similar increases in IKs as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca2+ ]i increased maximally activated tail IKs , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+ ]i clamped at 500-600nm. Thus, our study provides novel quantitative data as to how physiological [Ca2+ ]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+ ]i , in the submembrane or junctional cleft space, is not required to maximize [Ca2+ ]i -dependent IKs activation during normal Ca2+ transients.

Dilation of Fusion Pores by SNARE Protein Crowding

Hormones and neurotransmitters are released through exocytotic fusion pores that can fluctuate in size and flicker open and shut multiple times. The kinetics and the amount of cargo released, and the mode of vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Pore nucleation requires zippering between vesicle-associated v- and target membrane t-SNAREs, but the mechanisms governing the subsequent pore dilation are not known. Past approaches either monitored single exocytotic pores in live cells with unknown biochemistry, or used reconstitutions that lacked single pore sensitivity. Here, we probed dilation of single fusion pores using v-SNARE-reconstituted ∼23 nm diameter discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells ectopically expressing cognate, “flipped” t-SNAREs. A flipped t-SNARE cell is patch-clamped in the cell-attached configuration with the vNLPs included in the pipette solution. Fusion of a vNLP with the cell surface produces a pore connecting the cytosol with the pipette solution, through which direct-currents are measured under voltage clamp. The magnitude of the current reports pore size with sub-millisecond time resolution (Wu, Z. et al. Sci. Rep. 2016). We found that pore nucleation required a minimum of 2, and reached a maximum above ∼4 copies of v-SNAREs per NLP face. In contrast, the mean conductance of single pores increased as copy number was increased and was far from saturating at 15 copies, the NLP capacity. Thus, very different numbers of SNARE complexes cooperate at the distinct stages of fusion pore nucleation and pore dilation. We calculated pore size distributions and free energy profiles versus pore size. Combined with a mathematical model, these results suggest crowding of SNARE complexes at the pore waist drive pore expansion.

Dynamics of volume-averaged intracellular Ca2+ in a rat CNS nerve terminal during single and repetitive voltage-clamp depolarizations.

The intracellular concentration of free calcium ions ([Ca2+ ]i ) in a nerve terminal controls both transmitter release and synaptic plasticity. The rapid triggering of transmitter release depends on the local micro- or nanodomain of highly elevated [Ca2+ ]i in the vicinity of open voltage-gated Ca2+ channels, whereas short-term synaptic plasticity is often controlled by global changes in residual [Ca2+ ]i , averaged over the whole nerve terminal volume. Here we describe dynamic changes of such global [Ca2+ ]i in the calyx of Held - a giant mammalian glutamatergic nerve terminal, which is particularly suited for biophysical studies. We provide quantitative data on Ca2+ inflow, Ca2+ buffering and Ca2+ clearance. These data allow us to predict changes in [Ca2+ ]i in the nerve terminal in response to a wide range of stimulus protocols at high temporal resolution and provide a basis for the modelling of short-term plasticity of glutamatergic synapses. Many aspects of short-term synaptic plasticity (STP) are controlled by relatively slow changes in the presynaptic intracellular concentration of free calcium ions ([Ca2+ ]i ) that occur in the time range of a few milliseconds to several seconds. In nerve terminals, [Ca2+ ]i equilibrates diffusionally during such slow changes, such that the globally measured, residual [Ca2+ ]i that persists after the collapse of local domains is often the appropriate parameter governing STP. Here, we study activity-dependent dynamic changes in global [Ca2+ ]i at the rat calyx of Held nerve terminal in acute brainstem slices using patch-clamp and microfluorimetry. We use low concentrations of a low-affinity Ca2+ indicator dye (100μm Fura-6F) in order not to overwhelm endogenous Ca2+ buffers. We first study voltage-clamped terminals, dialysed with pipette solutions containing minimal amounts of Ca2+ buffers, to determine Ca2+ binding properties of endogenous fixed buffers as well as the mechanisms of Ca2+ clearance. Subsequently, we use pipette solutions including 500μm EGTA to determine the Ca2+ binding kinetics of this chelator. We provide a formalism and parameters that allow us to predict [Ca2+ ]i changes in calyx nerve terminals in response to a wide range of stimulus protocols. Unexpectedly, the Ca2+ affinity of EGTA under the conditions of our measurements was substantially lower (KD =543±51nm) than measured in vitro, mainly as a consequence of a higher than previously assumed dissociation rate constant (2.38±0.20 s-1 ), which we need to postulate in order to model the measured presynaptic [Ca2+ ]i transients.

The Effects of Extracellular Protons on the hERG Potassium Channel

The α-subunit of channels mediating the cardiac rapid delayed rectifier current (IKr) is encoded by the human Ether-à-go-go-Related Gene (hERG). Macroscopic hERG current (IhERG) amplitude is reduced and deactivation kinetics are accelerated with extracellular acidosis. We have investigated the single channel basis for the effects of acidic external pH (pHe) on the isoforms of IhERG expressed in myocytes (hERG1a and 1b). Patch clamp recordings were made at room temperature with the extracellular superfusate (whole-cell) or pipette solution (cell attached) acidified to pH 6.3 compared with control (pH 7.4). A decrease in pHe to 6.3 caused acceleration in deactivation and a reduction in maximal whole-cell conductance of ∼34% for IhERG1a (n=8 cells) and of ∼36% for IhERG1b (n=5 cells). Single channel recordings were made with isotonic potassium (140 mM) bathing the cells and in the electrode. Channel amplitude and open state kinetics were measured at a series of repolarisation voltages following a depolarising command to +40mV. Slope conductance values derived from amplitude current-voltage relationships between −120 and −40mV were 12.3±0.2pS for pH 7.4 (n=10 cells) and 9.3±0.1pS for pH 6.3 (n=9 cells) (P<0.01, two-tailed t-test) for hERG1a. The corresponding values for hERG1b were 11.4±0.2pS for pH 7.4 (n=6 cells) and 7.6±0.4pS for pH 6.3 (n=5 cells) (P<0.0001; two-tailed t-test). Open-time kinetics at −120mV for hERG1a were reduced from 8.49±1.0ms in control (n=8 cells) to 4.2±0.4ms in pHe 6.3 (n=7 cells) (P<0.05; two-tailed t-test). The hERG1b open state kinetics were 5.7±0.6ms in pHe 7.4 (n=6 cells) and reduced to 3.1±0.7ms in pHe 6.3 (n=5 cells) (P<0.05; two-tailed t-test). Thus, it can be concluded that a reduction in the single channel conductance and acceleration of open-times contribute to the attenuation of macroscopic IhERG when exposed to acidic pHe.