Patch Pipette Research Articles

Measurement of relative Ca²⁺ permeability during sustained activation of TRPV1 receptors.

Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.

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.

Estimating the magnitude of near-membrane PDE4 activity in living cells

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

Dynamics of T-Junction Solution Switching Aimed at Patch Clamp Experiments.

Solutions exchange systems are responsible for the timing of drug application on patch clamp experiments. There are two basic strategies for generating a solution exchange. When slow exchanges are bearable, it is easier to perform the exchange inside the tubing system upstream of the exit port. On the other hand, fast, reproducible, exchanges are usually performed downstream of the exit port. As both strategies are combinable, increasing the performance of upstream exchanges is desirable. We designed a simple method for manufacturing T-junctions (300 μm I.D.) and we measured the time profile of exchange of two saline solutions using a patch pipette with an open tip. Three factors were found to determine the timing of the solution switching: pressure, travelled distance and off-center distance. A linear relationship between the time delay and the travelled distance was found for each tested pressure, showing its dependence to the fluid velocity, which increased with pressure. The exchange time was found to increase quadratically with the delay, although a sizeable variability remains unexplained by this relationship. The delay and exchange times increased as the recording pipette moved away from the center of the stream. Those increases became dramatic as the pipette was moved close to the stream borders. Mass transport along the travelled distance between the slow fluid at the border and the fast fluid at the center seems to contribute to the time course of the solution exchange. This effect would be present in all tubing based devices. Present results might be of fundamental importance for the adequate design of serial compound exchangers which would be instrumental in the discovery of drugs that modulate the action of the physiological agonists of ion channels with the purpose of fine tuning their physiology.

Underestimated Sensitivity of Mammalian Cochlear Hair Cells Due to Splay between Stereociliary Columns

Current-displacement (I-X) and the force-displacement (F-X) relationships characterize hair-cell mechano-transduction in the inner ear. A common technique for measuring these relationships is to deliver mechanical stimulations to individual hair bundles with microprobes and measure whole cell transduction currents through patch pipette electrodes at the basolateral membrane. The sensitivity of hair-cell mechano-transduction is determined by two fundamental biophysical properties of the mechano-transduction channel, the stiffness of the putative gating spring and the gating swing, which are derived from the I-X and F-X relationships. Although the hair-cell stereocilia in vivo deflect <100 nm even at high sound pressure levels, often it takes >500 nm of stereocilia displacement to saturate hair-cell mechano-transduction in experiments with individual hair cells in vitro. Despite such discrepancy between in vivo and in vitro data, key biophysical properties of hair-cell mechano-transduction to define the transduction sensitivity have been estimated from in vitro experiments. Using three-dimensional finite-element methods, we modeled an inner hair-cell and an outer hair-cell stereocilia bundle and simulated the effect of probe stimulation. Unlike the natural situation where the tectorial membrane stimulates hair-cell stereocilia evenly, probes deflect stereocilia unevenly. Because of uneven stimulation, 1) the operating range (the 10–90% width of the I-X relationship) increases by a factor of 2–8 depending on probe shapes, 2) the I-X relationship changes from a symmetric to an asymmetric function, and 3) the bundle stiffness is underestimated. Our results indicate that the generally accepted assumption of parallel stimulation leads to an overestimation of the gating swing and underestimation of the gating spring stiffness by an order of magnitude.

Differential Gene Expression in Gonadotropin-Releasing Hormone Neurons of Male and Metestrous Female Mice

Background: Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the regulation of the hypothalamic-pituitary gonadal axis in a sex-specific manner. We hypothesized that the differences seen in reproductive functions of males and females are associated with a sexually dimorphic gene expression profile of GnRH neurons. Methods and Results: We compared the transcriptome of GnRH neurons obtained from intact metestrous female and male GnRH-green fluorescent protein transgenic mice. About 1,500 individual GnRH neurons from each sex were sampled with laser capture microdissection followed by whole-transcriptome amplification for gene expression profiling. Under stringent selection criteria (fold change >1.6, adjusted p value 0.01), Affymetrix Mouse Genome 430 PM array analysis identified 543 differentially expressed genes. Sexual dimorphism was most apparent in gene clusters associated with synaptic communication, signal transduction, cell adhesion, vesicular transport and cell metabolism. To validate microarray results, 57 genes were selected, and 91% of their differential expression was confirmed by real-time PCR. Similarly, 88% of microarray results were confirmed with PCR from independent samples obtained by patch pipette harvesting and pooling of 30 GnRH neurons from each sex. We found significant differences in the expression of genes involved in vesicle priming and docking (Syt1, Cplx1), GABAergic (Gabra3, Gabrb3, Gabrg2) and glutamatergic (Gria1, Grin1, Slc17a6) neurotransmission, peptide signaling (Sstr3, Npr2, Cxcr4) and the regulation of intracellular ion homeostasis (Cacna1, Cacnb1, Cacng5, Kcnq2, Kcnc1). Conclusion: The striking sexual dimorphism of the GnRH neuron transcriptome we report here contributes to a better understanding of the differences in cellular mechanisms of GnRH neurons in the two sexes.

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.

Plasma membrane mechanical stress activates TRPC5 channels.

Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP) channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca2+ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between −20 and −40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold.

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.

Functional roles of complexin in neurotransmitter release at ribbon synapses of mouse retinal bipolar neurons.

Ribbon synapses of photoreceptor cells and bipolar neurons in the retina signal graded changes in light intensity via sustained release of neurotransmitter. One molecular specialization of retinal ribbon synapses is the expression of complexin protein subtypes Cplx3 and Cplx4, whereas conventional synapses express Cplx1 and Cplx2. Because complexins bind to the molecular machinery for synaptic vesicle fusion (the SNARE complex) and modulate transmitter release at conventional synapses, we examined the roles of ribbon-specific complexin in regulating release at ribbon synapses of ON bipolar neurons from mouse retina. To interfere acutely with the interaction of native complexins with the SNARE complex, a peptide consisting of the highly conserved SNARE-binding domain of Cplx3 was introduced via a whole-cell patch pipette placed directly on the synaptic terminal, and vesicle fusion was monitored using capacitance measurements and FM-dye destaining. The inhibitory peptide, but not control peptides, increased spontaneous synaptic vesicle fusion, partially depleted reserve synaptic vesicles, and reduced fusion triggered by opening voltage-gated calcium channels under voltage clamp, without affecting the number of synaptic vesicles associated with ribbons, as revealed by electron microscopy of recorded terminals. The results are consistent with a dual role for ribbon-specific complexin, acting as a brake on the SNARE complex to prevent spontaneous fusion in the absence of calcium influx, while at the same time facilitating release evoked by depolarization.

Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons

Recent studies suggest that selective block of extrasynaptic N-methyl-d-aspartate (NMDA) receptors might protect against neurodegeneration. We recorded whole-cell currents with patch pipettes to characterize the ability of memantine, a low-affinity NMDA channel blocker, to block synaptic and extrasynaptic NMDA receptors in substantia nigra zona compacta (SNC) dopamine neurons in slices of rat brain. Pharmacologically isolated NMDA receptor-mediated EPSCs were evoked by electrical stimulation, whereas synaptic and extrasynaptic receptors were activated by superfusing the slice with NMDA (10µM). Memantine was 15-fold more potent for blocking currents evoked by bath-applied NMDA compared to synaptic NMDA receptors. Increased potency for blocking bath-applied NMDA currents was shared by the GluN2C/GluN2D noncompetitive antagonist DQP-1105 but not by the high-affinity channel blocker MK-801. Our data suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D subunits. Our results justify further investigations on the use of memantine as a neuroprotective agent in Parkinson׳s disease.

Physical Coupling between SERCA2 and PDE3A Regulates SERCA2 Activity in Cardiomyocytes

Rationale: SERCA2 controls cardiac contractility, and its activity is negatively regulated by the cAMP phosphodiesterase PDE3A through an unknown mechanism. Recent clinical trials have shown upregulation of SERCA2 gene therapy as beneficial in heart failure.Objective: To examine whether PDE3A is physically associated with SERCA, and to further evaluate whether this protein interaction represent a novel drug target to increase SERCA2 activity.Methods and results: PDE3 inhibition increased Ca2+ transients, SR Ca2+ load and SERCA2 activity without altering global cytosolic cAMP levels in field stimulated cardiomyocytes. SERCA2 activity was increased by PDE3 inhibition in cardiomyocytes dialyzed with 5 µmol/l cAMP by patch pipettes. Active PDE3A co-purified and precipitated with SERCA2 from left ventricular myocardium, and proximity ligation assay demonstrated co-localization of PDE3A and SERCA2 in intact cardiomyocytes. A combination of immunoprecipitation and peptide interaction experiments revealed interaction between specific cytosolic regions between amino acids 277 and 493 in PDE3A and amino acid 169 to 216 in SERCA2. By whole cell voltage clamp of intact cardiomyocytes, increased SERCA2 activity was induced by dialysis with disruptor peptides of the SERCA2-PDE3A interaction. TAT-labeled PDE3A-SERCA2 disruptor peptide fragments were further able to increase SERCA2 activity in field stimulated cardiomyocytes. PDE3A-SERCA2 disruptor peptides were able to increase SERCA2 activity in cardiomyocytes in presence of either PKI or Rp-cAMP and without concomitant phospholamban phosphorylation.Conclusion: PDE3A is physically associated to SERCA2, and this direct interaction regulates SERCA2 activity in cardiomyocytes possibly by direct regulation of SERCA2. Cell permeable disruptor peptides of the PDE3A-SERCA2 protein-protein interaction is able to increase SERCA2 activity and may potentially offer a new therapeutic approach in chronic heart disease.

Divergent Regulation of Cardiomyocyte Cav1.2 Currents by Calmodulin Mutants Associated with Human Sudden Death Syndromes

Recent reports identified six calmodulin (CaM) missense mutations associated with three distinct human arrhythmia syndromes: N54I and N98S are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT)-like clinical presentations, D96V, D130G and F142L with long QT syndrome (LQTS) and F90L with idiopathic ventricular fibrillation (IVF). We previously reported that all LQTS-CaMs exhibit reduced Ca affinity and normal or decreased (compared to WT CaM) ryanodine receptor (RyR2)-binding affinity, whereas CPVT-CaMs have normal or modestly lower Ca affinity but increase RyR2 single-channel open probability and show enhanced binding affinity to RyR2, causing significant increase in spontaneous Ca wave and spark activity in cardiac myocytes. Here, we investigate the effect of these mutant CaMs on another target potentially involved in arrhythmogenesis - Cav1.2 channels.Using whole-cell patch clamp, Cav1.2 current (ICa) was measured in freshly isolated murine ventricular myocytes pre-treated with ryanodine (50 µM) and thapsigargin (10 µM) to prevent SR Ca release. Cells were dialyzed with either wild-type or mutant CaM (6 µM) via patch pipette. All LQTS-CaMs and IVF-CaM caused significant impairment of ICa inactivation: τ=254±34 ms (D96V), 251±10 ms (F142L), 187±13 ms (D130G) and 190±8 ms (F90L) vs. 110±5 ms for WT (p<0.001, n=4-7 myocytes/group), whereas CPVT-CaMs had no effect on ICa inactivation(τ=108±3 ms (N54I), τ=132±14 ms (N98S) (NS, n=4-6 myocytes/group)). The effect of LQTS-CaMs and IVF-CaM was dominant, with a mixture of 25% mutant/75% WT CaM slowing ICa inactivation to a similar degree as 100% mutant CaM. Importantly, average peak current amplitude was unchanged in all groups. These results support the hypothesis that different targets are affected in arrhythmogenic disorders associated with LQTS-CaMs (impaired regulation of Cav1.2) vs. CPVT-CaMs (altered regulation of RyR2). The F90L CaM mutation shares characteristics with both CPVT and LQTS CaMs.

Viral Membrane Fusion at Single Pore Resolution

Fusion between membrane-enveloped viruses and their hosts is a critical step in infection, enabling the virus to release its genome and subsequently hijack the host cell's replication machinery. The genomes of enveloped viruses are packaged into multiple bulky ribonucleoprotein (RNP) particles whose release requires the dilation of the fusion pore - the initial, narrow, connection between fusing membranes. Electrophysiological studies of influenza A hemagglutinin (HA)-mediated fusion between HA-expressing fibroblasts and red blood cells and between HA-fibroblasts and voltage-clamped suspended bilayers comprise the most complete information to date about the dynamics of viral fusion pores. Small pores (∼2-5nm) were found to flicker repetitively and/or fluctuate in size prior to terminal dilation. A quantitative understanding of the biophysical driving forces of viral membrane fusion, including membrane contact area, membrane tension and protein fusogen cooperativity, however, remains elusive, and a number of questions - regarding the physical basis of pore flickering and the number of HA trimers required for successful pore dilation - remain open. Here we describe a novel electrophysiological approach, which has been successfully applied to study SNARE-mediated fusion pores, to probe the HA-mediated fusion pore. We voltage-clamped HA-expressing fibroblasts in the cell-attached configuration and included nanodiscs (NDs) in the patch pipette as fusion partners to probe fusion pore nucleation and evolution at single pore resolution. NDs are flat, bilayer discs whose composition and size can be varied. Current through voltage-clamped fusion pores reports pore size with sub-millisecond time resolution. Preliminary data shows that isolated fusion pores flicker, are long-lived (∼10s) and are of diameter ∼1.5nm.

Aging Alters cAMP Signaling and Membrane-Delimited Regulation of if in Sinoatrial Myocytes

We recently demonstrated that age-dependent declines in intrinsic and maximum heart rates are associated with slower action potential (AP) firing rate and a hyperpolarizing shift in the midpoint activation voltage (V1/2) of the funny current (If) in isolated sinoatrial myocytes (SAMs). Given that cAMP is an important intracellular modulator in SAMs, we hypothesized that reduced cAMP concentration may contribute to the slower APs and shifted If in aged SAMs. To test this hypothesis, we applied a saturating concentration of cAMP via the patch pipette in whole cell voltage-clamp and current clamp experiments in acutely isolated SAMs from young and old mice. Here, we show that this exogenous cAMP completely reversed the age-dependent changes in both AP firing rate and If in SAMs. In contrast, maximal stimulation of endogenous cAMP production, via co-application of the PDE inhibitor, IBMX and the adenylyl cyclase activator, forskolin, did not rescue either AP firing rate or If in aged SAMs. These data indicate that a deficit in cAMP production and/or responsiveness may contribute to the age-dependent changes in SAM excitability. To further determine how cAMP regulates If in aged versus young SAMs, we compared If activation in excised inside-out membrane patches from SAMs from young and old mice. Surprisingly, we found that the age-dependent hyperpolarized shift in the V1/2 of If persisted in the cell-free patches, indicating that soluble cAMP alone cannot account for the shifted If in aged SAMs. Rather, aging appears to alter an additional, membrane-associated factor that regulates If in SAMs. Future work will focus on resolving the underlying mechanism(s) responsible for the age-dependent shift in the V1/2 of If, and its relationship to AP generation in SAMs.

The Talk-1 C-Terminus is a Charge-Sensitive Module Regulating Channel Activity

Human beta-cell insulin secretion is dependent on Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs). K+ channels modulate the beta-cell membrane potential, which influences VDCC activity. The most abundant K+ channel of the human islet is the two-pore domain K+ (K2P) channel TALK-1; however, its function is unknown. We characterized TALK-1 currents in primary mouse beta-cells and find that K2P currents are significantly reduced after genetic ablation of TALK-1. To identify mechanisms that regulate TALK-1 channel activity, we used a Tl+-sensitive fluorescent screen to assess if TALK-1 is regulated by kinases. Cells expressing TALK-1 channels were monitored for changes in Tl+ flux in response to co-expression with a constitutively active kinase (examining a total of 192 kinases), revealing 9 kinases that modify Tl+ flux through TALK-1. Interestingly, three phosphatidylinositol phosphate kinases (PIKs) including PIK3CG, PI4K2B, and PIKFYVE, increased TALK-1 channel activity. To determine if activation of TALK-1 channels by PIKs is due to build-up of PIPs, we depleted membrane PIP2 with a rapamycin-recruitable phosphatase and found that TALK-1 channel activity was reduced. As charged residues of other K+ channels mediate PIP2 sensitivity, we assessed if PIP2 influences TALK-1 channel activity through a charged residue enriched domain in the C-terminus. We found that neutralization of these charged residues significantly reduced PIP2 activation of TALK-1 channels. Importantly, PIP2 introduced via patch pipette into primary mouse beta-cells also significantly enhanced TALK-1 currents. As glucose induces Ca2+-dependent oscillations in beta-cell PIP2, we then assessed how TALK-1 regulates islet Ca2+ dynamics. We find that Ca2+ oscillation frequency is significantly increased in islets lacking TALK-1, which results in enhanced glucose-stimulated insulin secretion (GSIS). These data provide compelling evidence that TALK-1 influences GSIS by limiting excitability, and suggests a novel mechanism for phospholipid-dependent regulation of beta-cell Ca2+ entry.

Mechanosensitivity of TRPC6 Ion Channels

The transient receptor potential (TRP) ion channel family is a diverse group of channels gated by various physical and chemical stimuli. One of the members, transient receptor potential canonical-6 (TRPC6), is a calcium permeable cation channel (PCa/PNa ∼ 6) located in the T-tubules of ventricular cardiomyocytes. Abnormal TRPC6 activity has been implicated in cardiac hypertrophy, a maladaptive process likely triggered by increased mechanical stress. As a result, it is important to elucidate the gating mechanism of TRPC6. In addition to activation by diacylglycerol (DAG) TRPC6 has been reported to be also activated by mechanical force, though this has not been supported by other studies. In this study, we investigated the mechanosensitivity of TRPC6 overexpressed in HEK293 cells using the patch clamp technique. Three TRPC6 constructs were utilised: N- terminal fusion GFP, C-terminal fusion GFP and non-GFP fusion TRPC6. We have successfully transfected these constructs into HEK293 cells with a 60% transfection efficiency rate using Lipofectamine® 3000 reagent. Confocal images of C-GFP TRPC6 demonstrated that the channel is localised in the plasma membrane. However, N-GFP and non-GFP TRPC6 both distributed uniformly throughout the cells suggestive of impaired trafficking. In our patch clamp experiments we recorded TRPC6 single channel activity in cell-attached and whole-cell configurations. By applying negative pressure (suction) of −50 mmHg to the patch pipette we were able to record single channel activity of TRPC6 channels. Moreover, the channels were spontaneously active without application of negative pressure to the patch pipette suggesting that the membrane tension resulting from the gigaohm seal formation was sufficient to activate TRPC6. These results demonstrate that TRPC6 expressed in the plasma membrane of HEK293 cells is mechanosensitive. Future studies will focus on purifying and incorporating the TRPC6 channel protein into liposomes to establish if TRPC6 is intrinsically mechanosensitive.

Liberation of Pser68-Plm Inhibition of NCX1 by an Optimized Anchoring Disruptor Peptide

The cardiac sodium-calcium exchanger (NCX1) is an important regulator of intracellular Ca2+ ([Ca2+]i) and a potential therapeutic target in heart failure. Among potential interacting partners regulating NCX1 activity is the transmembrane protein phospholemman (PLM); a substrate for protein kinase A and protein kinase C. Several reports have demonstrated that binding of phosphorylated PLM (pSer68-PLM) to NCX1 leads to NCX1 inhibition, while other studies have failed to demonstrate a functional interaction of these proteins. In present study, we aimed to analyze the biological function of the pSer68-PLM-NCX1 interaction by developing anchoring disruptor peptides. We observed that PLM co-fractionated and co-immunoprecipitated with NCX1 in rat left ventricle (LV) and in co-transfected HEK293 cells. Strong binding of pSer68-PLM to one of the previously reported NCX1-QKHPD regions was demonstrated in pull-down and overlay assays. Single amino acid substitutions of native NCX1 peptide sequence 1-KHPDKEIEQLIELANYQVLS-20 revealed that single substitutions at position 1-11 (particularly with tryptophan or tyrosine) increased binding affinity of the peptide to pSer68-PLM. Interestingly, we found that double substitution at position 4 and 6 with tyrosine (D4Y and E6Y) in the native peptide sequence enhanced the binding affinity to pSer68-PLM 8-fold, compared to native sequence. The optimized peptide 1-KHPYKYIEQLIELANYQVLS-20 was further tested in a series of electrophysiological experiments using whole-cell voltage clamp technique, with voltage-ramp protocol employed to elicit NCX current. Constitutively phosphorylated (S68D) PLM was observed to inhibit NCX1 current in both forward and reverse mode. Inclusion of the disruptor peptide in the patch pipette reversed S68D-PLM inhibition of NCX1, while NCX current was not altered by a scrambled peptide control. Taken together these data strongly suggest that PLM interacts directly with NCX1, and that when PLM is phosphorylated at serine 68, the interaction inhibits NCX1 activity.

Gβγ Subunits Modulation of Kv7.4 Channels Expressed in HEK293 Cells at the Single Channel Level

Recently it was shown that KCNQ4-encoded voltage-dependent potassium channels (Kv7.4) play an important role in the regulation of resting vascular tone and in response to endogenous vasodilators (Hypertension, 59:877–884, 2012). However there is lack of information about Kv7.4 basic biophysical properties on the single channel level and about their regulatory mechanisms. Therefore, using standard cell-attached and inside-out configurations of the patch-clamp technique we investigated the single channel properties of Kv7.4 in HEK293 cells stably expressing the channel. We found that NPo of Kv7.4 was 0.61±0.05 (n=9, mean±SEM) in the cell-attached configuration at −50 mV (voltage applied to the patch pipette), whereas in the inside-out configuration at 0 mV NPo of Kv7.4 was 0.04±0.01 (n=9). Based on the inside-out experiments, single channel conductance of Kv7.4 was calculated as 2.31 pS. Linopirdine (10 µM), the pan-Kv7 blocker, abolished the single channel activity in both cell-attached and inside-out configuration when added to the patch pipette, but did not produce significant changes when applied to the bath solution. Bath application of PI(4,5)P2 (1 µM) to the inside-out patches or application of active Gβγ subunits (50 ng/ml) resulted in significant increase of NPo of Kv7.4 correspondingly to 0.27±0.09 (n=3) or to 0.34±0.06 (n=6) without affecting channel conductance (p<0.001 in both cases, determined by 2-way ANOVA). Moreover, bath application of gallein (100 µM), an inhibitor of Gβγ activity, reduced significantly NPo of Kv7.4 channels to 0.03±0.01 (n=3, p<0.001) in the cell-attached experiments. Apart from revealing the basic biophysical properties of single Kv7.4 channels our results show that, similar to PI(4,5)P2, Gβγ subunits play a critical role in the basal activity of voltage-dependent Kv7.4 channels.Supported by the British Heart Foundation (PG/12/63/29824) and by the Medical Research Council (MR/K019074/1).

Calcium Influx through TRPV1 Inhibits Piezo Channels via Phosphoinositide Depletion

Capsaicin, the activator of the noxious heat sensor TPRV1 is clinically used to relieve chronic pain. Desensitization of TRPV1 involves downstream PLCδ activation, but the mechanism how capsaicin treatment can also alleviate mechanical allodynia is unknown. Piezo2 encodes rapidly adapting mechanically activated currents in sensory neurons. Expression of these novel mechanosensitive ion channels in a subset of TPRV1 positive neurons suggests their role in pain related sensory mechanotransduction.Our study shows that capsaicin application inhibited rapidly adapting mechanically activated currents in TRPV1 expressing sensory neurons. We also show that TRPV1 activation inhibited heterologously expressed Piezo1 and Piezo2 ion channels in whole-cell patch clamp experiments. Inclusion of either phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] or its precursor PI(4)P in the patch pipette alleviated this inhibitory effect. Activation of PLCβ by stimulating muscarinic receptors only marginally inhibited mechanically activated Piezo1 currents. Experiments using phosphoinositide sensors revealed that activation of PLCδ by a robust calcium influx through TRPV1 severely depleted PI(4,5)P2 and PI(4)P. On the other hand, muscarinic stimulation of PLCβ significantly decreased PI(4,5)P2 levels, but only induced a small decrease of PI(4)P. This differential activation of PLC isoforms may explain the difference between the inhibitory effects of these PLC pathways. Targeted depletion of PI(4,5)P2 and PI(4)P using a chemically inducible lipid phosphatase replicated the inhibition of Piezo1 currents. Additionally, PI(4,5)P2 and PI(4)P applied to excised inside-out patches inhibited the rundown of Piezo1 activity further emphasizing the significance of these phosphoinositides in Piezo channel regulation.Here we demonstrate that the activity of Piezo channels require the presence of either PI(4,5)P2 or PI(4)P and severe depletion of both phosphoinositides by TRPV1 activation limits channel activity. In conclusion our data may explain how capsaicin alleviates mechanically induced pain.