Cell-attached Patch Recordings Research Articles

Protein kinase A (PKA) activation reorganizes microtubules and microtubule-plus-end-binding-protein 1 (EB1) and enriches Nav1.5 at the intercalated discs (ICDs) of adult ventricular myocytes.

In cardiomyocytes β-adrenergic stimulation increases Na current (INa) through PKA activation, and EB1 targets Nav1.5 to ICDs where INa mediates impulse propagation through gap-junctions. To understand how persistent PKA activation affects INa and Nav1.5 protein expression/distribution in cardiomyocytes, and the role of EB1 and microtubules. Sprague-Dawley ventricular myocytes cultured without or with 8cpt-cAMP (100 uM) plus serine/threonine phosphatase inhibitor (okadaic acid, 100 nM) for 10-20 hr were subject to immunostaining/imaging, subcellular fractionation/immunoblot, and patch clamp recordings. These were supplemented by HEK293 experiments. Cell-attached patch recordings from ICD areas (≤5 sarcomeres from cell ends) showed 10-fold increase in INa in PKA-activated vs control myocytes, without change in total Nav1.5 protein level. To understand Nav1.5 distribution and relationship to EB1 and microtubules at ICDs, we imaged the 3 with n-cadherin (ICD marker) simultaneously using Airyscan at xyz pixel dimension 50 nm. Image reconstruction allowed us to quantify protein signals in en-face views of ICDs. PKA activation (a) increased %ICD area occupied by Nav1.5, β-tubulin and EB1, and (b) increased correlation between Nav1.5/EB1, Nav1.5/β-tubulin, and EB1/β-tubulin. These were supported by HEK293 data: Nav1.5 co-immunoprecipitated more EB1 and β-tubulin in PKA activated (4-6 hr) than control cells. Although PKA activation increased EB1 capture of microtubules at ICD, it decreased microtubule density in the cytoplasmic area, consistent with biochemical data: PKA activation did not change total α-tubulin level but reduced detyrosinated α-tubulin. Myocytes have >10 fold free β-tubulin in cytosol than in microtubules, and >150 fold free EB1 in cytosol than bound to microtubules/membranes. Spatial/temporal dynamics of microtubules and EB1 modulation and PKA-promoted Nav1.5/tubulin/EB1 interactions enrich Nav1.5 at ICDs, facilitating impulse propagation in β-adrenergic stimulated heart.

Intrinsic plasticity of cerebellar stellate cells is mediated by NMDA receptor regulation of voltage-gated Na+ channels.

We show that NMDA receptors (NMDARs) elicit a long-term increase in the firing rates of inhibitory stellate cells of the cerebellum NMDARs induce intrinsic plasticity through a Ca2+ - and CaMKII-dependent pathway that drives shifts in the activation and inactivation properties of voltage-gated Na+ (Nav ) channels An identical Ca2+ - and CaMKII-dependent signalling pathway is triggered during whole-cell recording which lowers the action potential threshold by causing a hyperpolarizing shift in the gating properties of Nav channels. Our findings open the more general possibility that NMDAR-mediated intrinsic plasticity found in other cerebellar neurons may involve similar shifts in Nav channel gating. Memory storage in the mammalian brain is mediated not only by long-lasting changes in the efficacy of neurotransmitter receptors but also by long-term modifications to the activity of voltage-gated ion channels. Activity-dependent plasticity of voltage-gated ion channels, or intrinsic plasticity, is found throughout the brain in virtually all neuronal types, including principal cells and interneurons. Although intrinsic plasticity has been identified in neurons of the cerebellum, it has yet to be studied in inhibitory cerebellar stellate cells of the molecular layer which regulate activity outflow from the cerebellar cortex by feedforward inhibition onto Purkinje cells. The study of intrinsic plasticity in stellate cells has been particularly challenging as membrane patch breakthrough in electrophysiology experiments unintentionally triggers changes in spontaneous firing rates. Using cell-attached patch recordings to avoid disruption, we show that activation of extrasynaptic N-methyl-d-aspartate receptors (NMDARs) elicits a long-term increase in the firing properties of stellate cells by stimulating a rise in cytosolic Ca2+ and activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). An identical signalling pathway is triggered during whole-cell recording which lowers the action potential threshold by causing a hyperpolarizing shift in the gating properties of voltage-gated sodium (Nav ) channels. Together, our findings identify an unappreciated role of Nav channel-dependent intrinsic plasticity in cerebellar stellate cells which, in concert with non-canonical NMDAR signalling, provides the cerebellum with an unconventional mechanism to fine-tune motor behaviour.

Cation-chloride cotransporters and the polarity of GABA signalling in mouse hippocampal parvalbumin interneurons.

Cation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABAA receptor (GABAA R)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked. We compared the polarity of GABA signalling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons. Under resting conditions, GABAA R activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABAA R-mediated currents often above action potential threshold. However, during repetitive GABAA R activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride build-up is not responsible for this form of plasticity. Our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons. Transmembrane chloride gradients govern the efficacy and polarity of GABA signalling in neurons and are usually maintained by the activity of cation-chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAA receptor (GABAA R) activation in adult mouse hippocampal parvalbumin interneurons (PV-INs) and pyramidal cells (PCs). Loose cell-attached, tight-seal and gramicidin-perforated patch recordings all show GABAA R-mediated transmission is slightly depolarizing and yet inhibitory in both PV-INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV-INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAA R-mediated currents in PV-INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAA Rs. Finally, we show that repetitive 10Hz activation of GABAA Rs in both PV-INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride build-up may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV-INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signalling in both cell types.

Mechanism of Modulation of AMPA Receptors by TARPγ8

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate activated AMPA receptors (AMPARs). The core GluA subunits of AMPARs in neurons co-assemble with auxiliary subunits, including transmembrane AMPAR regulatory proteins (TARPs), several of which modifiy AMPA receptor-channel gating kinetics. In the case of AMPARs containing four copies of TARP γ8, achieved by expression of the tandem GluA2Q∗TARPγ8 subunit developed in the lab, AMPARs exhibit resensitization, or partial recovery from desensitization in continued presence of glutamate. We have recorded single channel currents in cell-attached patch recordings and show that under resensitizing conditions, these AMPARs show transitions primarily from closed to higher conductance levels, similar to activation of homomeric GluA receptors in the presence of cyclothiazide. To study the conformation associated with these states we used single molecule FRET and show that this high conductance state produced by GluA2Q∗TARPγ8 channels exhibits tighter coupling between subunits in the extracellular parts of the receptor.

Cholestane-3β, 5α, 6β-triol suppresses neuronal hyperexcitability via binding to voltage-gated sodium channels

Neuronal hyperexcitability is identified as a critical pathological basis of epileptic seizures. Cholestane-3β, 5α, 6β-triol (Triol) is a major metabolic oxysterol of cholesterol. Although its neuroprotective effect on ischemia-induced neuronal injury and negative modulation of voltage-gated sodium (Nav) channels were well established, the physical binding site of triol to sodium channels and its effects on neuronal hyperexcitability have not yet been explored. In this study, we utilized molecular docking and molecular dynamics simulation to investigate the interaction between triol and Nav Channels. Our results demonstrated that triol binds to the indole ring of Trp122 of the Nav Channel in silico with a high biological affinity. We further found that triol negatively modulates the action potentials bursts of hippocampal neurons by cell-attached patch recording. Moreover, triol significantly inhibits low Mg2+-induced hyperexcitability in vitro. In addition, triol attenuates pentylenetetrazole (PTZ)-induced convulsive-form behavioral deficits in vivo. Together, our results suggest that triol suppresses neuronal hyperexcitability via binding to Nav channel, indicating that triol might be an attractive lead compound for the treatment of neuronal hyperexcitability-related neurological disorders, especially epileptic seizures.

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses.

It has been known that Ca2+ plays an essential role in mediating different modes of neurotransmitter release via different sensing mechanisms. Synaptotagmin 1, 2, and 9 were found to act as the Ca2+ sensors for synchronous release and synaptotagmin 7 and Doc-2 were proposed as the Ca2+ sensors for asynchronous release. Comparatively, the Ca2+ sensor for spontaneous release remains a mystery. At the Calyx of Held synapse, the Ca2+ sensor for spontaneous release was found not identical to the sensor for synchronous release, synaptotagmin 2. As Ca2+ sensors have different sensitivity to Sr2+ and Ca2+ and induce significantly different rate of vesicle release, Sr2+ is traditionally used as a tool to examine the intrinsic properties of different Ca2+ sensors. Here, we employed cell-attached patch recording and presynaptic/postsynaptic whole-cell recording at the Calyx of Held synapses of synaptotagmin 2 knock-out mice to assay the Sr2+ and Ca2+ influx into the nerve terminal at resting potential and observed the effects of Ca2+ and Sr2+ on spontaneous neurotransmitter release. We found that the dwell time of single voltage gated Ca2+ channel opening increased around threefold for Sr2+ than Ca2+ with the channel conductance unchanged; the divalent cation sensing machinery in regulating spontaneous release has much lower sensitivity to Sr2+ than Ca2+ . Thus, our study reveals some of the intrinsic properties of Ca2+ sensor(s) of spontaneous transmitter release and provided an insight into the underlying mechanisms.

Localization of Anomalous Mechano-Sensitive Ion Channels in Cochlear Hair Cells

Two types of mechanosensitive (MS) currents have been described in cochlear hair cells: conventional MS currents evoked by displacements of the hair bundle towards its tallest edge, and anomalous MS currents, elicited by bundle displacements in the opposite direction, referred to as reversed-polarity currents. Conventional MS transducer channels are located at the bottom end of the tip links, but reverse-polarity MS currents persist after severing tip links with BAPTA, suggesting a different site for this channel. We investigated localization of reverse-polarity MS channels using cell-attached patch recordings on cochlear outer hair cells (OHC) of neonatal mice, and also by detecting calcium influx with Fluo5F indicator using swept-field confocal imaging. Single mechano-sensitive channels were recorded in cell-attached mode on the apical surface around the base of the hair bundle and were activated by suction through the patch pipette. In OHCs of wild-type mice, MS channels appeared a few minutes after BAPTA exposure with a mean conductance of 61 ± 6 pS in Na-saline with 1.5 mM CaCl2 and 94 ± 13 pS in Na-saline with reduced, 0.07 mM, CaCl2. Their reversal potential was 0 mV as expected for a non-selective cationic channel. Similar MS channels were also seen in Tmc1:Tmc2 double mutants, with mean conductance of 58 ± 4 pS in 1.5 mM CaCl2. In both cases, the apical surface of the OHC was confirmed as the main site of calcium entry during reverse polarity MS currents by imaging calcium influx. The properties of the reverse-polarity MS channels closely resemble conventional MS channels but they are concentrated on the apical plasma membrane of the hair cell. We suggest they reflect insertion of new MS channels before their transport up the bundle to the transduction complex.

Biophysical Factors that Promote Mechanically-Induced Action Potentials in Neocortical and Hippocampal Pyramidal Neurons

Pressure activation of single mechanosensitive (MS) cation channel currents can trigger action potentials in neocortical and hippocampal pyramidal neurons (Nikolaev et al., 2015). Here we describe several factors that underlie this powerful influence on neuronal activity. First, cell-attached patch recordings indicate the MS cation channels are located on the soma and therefore inject a depolarizing current close to the axonal initial segment/spike initiation zone (SIZ) where action potential threshold is lowest. Second, the rapid rise of the single MS channel current (< 50 µs) generates a rapid depolarizing (dV/dt) event that lowers spike threshold by favoring fast Na+ channel activation over slower inactivation. Finally, with pressure activation the gating of the MS channel switches from single brief spontaneous openings (< 1 ms) to longer bursts of openings (∼ 10 ms) during which a spike may be triggered by each stochastic opening within the burst. Thus, the combination of SIZ proximity and rapid, burst gating underlies the powerful influence of single MS channels on central neuronal activity. For comparison, whereas a unitary excitatory post-synaptic current activated on a distal dendrite typically fails to trigger an action potential even when generating as much as 1000 fC charge transfer across the synaptic membrane, a single MS channel current that generates less than 200 fC can trigger repetitive spiking.Nikolaev, Y.A. et al., Single mechanically-gated cation channel currents can trigger action potentials in neocortical and hippocampal pyramidal neurons (2015) Brain Research. 1608: 1-13.

High dendritic expression of Ih in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons

The hyperpolarization-activated cation current Ih is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown. We used cell-attached patch recordings to determine the localization profile of Ih along the somatodendritic axis of nigral dopamine neurons in slices from young rats. Ih density is higher in axon-bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon-lacking dendrites. Dual current-clamp recordings revealed a similar contribution of Ih to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain. The Ih blocker ZD7288 increased the temporal summation in all dendrites with a comparable effect in axon- and non-axon dendrites. The strategic position of Ih in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine. Dendrites of most neurons express voltage-gated ion channels in their membrane. In combination with passive properties, active currents confer to dendrites a high computational potential. The hyperpolarization-activated cation current Ih present in the dendrites of some pyramidal neurons affects their membrane and integration properties, synaptic plasticity and higher functions such as memory. A gradient of increasing h-channel density towards distal dendrites has been found to be responsible for the location independence of excitatory postsynaptic potential (EPSP) waveform and temporal summation in cortical and hippocampal pyramidal cells. However, reports on other cell types revealed that smoother gradients or even linear distributions of Ih can achieve homogeneous temporal summation. Although the existence of a robust, slowly activating Ih current has been repeatedly demonstrated in nigral dopamine neurons, its subcellular distribution and precise role in synaptic integration are unknown. Using cell-attached patch-clamp recordings, we find a higher Ih current density in the axon-bearing dendrite than in the soma or in dendrites without axon in nigral dopamine neurons. Ih is mainly concentrated in the dendritic membrane area surrounding the axon origin and decreases with increasing distances from this site. Single EPSPs and temporal summation are similarly affected by blockade of Ih in axon- and non-axon-bearing dendrites. The presence of Ih close to the axon is pivotal to control the integrative functions and the output signal of dopamine neurons and may consequently influence the downstream coding of movement.

9-Phenanthrol inhibits recombinant and arterial myocyte TMEM16A channels.

In arterial smooth muscle cells (myocytes), intravascular pressure stimulates membrane depolarization and vasoconstriction (the myogenic response). Ion channels proposed to mediate pressure-induced depolarization include several transient receptor potential (TRP) channels, including TRPM4, and transmembrane protein 16A (TMEM16A), a Ca(2+) -activated Cl(-) channel (CaCC). 9-Phenanthrol, a putative selective TRPM4 channel inhibitor, abolishes myogenic tone in cerebral arteries, suggesting that either TRPM4 is essential for pressure-induced depolarization, upstream of activation of other ion channels or that 9-phenanthrol is non-selective. Here, we tested the hypothesis that 9-phenanthrol is also a TMEM16A channel blocker, an ion channel for which few inhibitors have been identified. Patch clamp electrophysiology was used to measure rat cerebral artery myocyte and human recombinant TMEM16A (rTMEM16A) currents or currents generated by recombinant bestrophin-1, another Ca(2+) -activated Cl(-) channel, expressed in HEK293 cells. 9-Phenanthrol blocked myocyte TMEM16A currents activated by either intracellular Ca(2+) or Eact , a TMEM16A channel activator. In contrast, 9-phenanthrol did not alter recombinant bestrophin-1 currents. 9-Phenanthrol reduced arterial myocyte TMEM16A currents with an IC50 of ∼12 μM. Cell-attached patch recordings indicated that 9-phenanthrol reduced single rTMEM16A channel open probability and mean open time, and increased mean closed time without affecting the amplitude. These data identify 9-phenanthrol as a novel TMEM16A channel blocker and provide an explanation for the previous observation that 9-phenanthrol abolishes myogenic tone when both TRPM4 and TMEM16A channels contribute to this response. 9-Phenanthrol may be a promising candidate from which to develop TMEM16A channel-specific inhibitors.

Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel‐containing membrane microdomains

Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that β-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP‐sensitive potassium channels by nitric oxide in ventricular cardiomyocytes

The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in intact cardiomyocytes, but the H2O2-induced KATP channel stimulation was obliterated when ERK1/2 or CaMKII activity was suppressed, implying that H2O2 is positioned upstream of ERK1/2 and CaMKII for K(ATP) channel modulation. Furthermore, genetic ablation (i.e., knockout) of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished ventricular sarcK(ATP) channel stimulation elicited by activation of PKG, unveiling CaMKIIδ as a crucial player. Additionally, evidence from kinase activity and Western blot analyses revealed that activation of NO-PKG signalling augmented CaMKII activity in rabbit ventricular myocytes and, importantly, CaMKII activation by PKG occurred in an ERK1/2-dependent manner, placing ERK1/2 upstream of CaMKII. Taken together, these findings suggest that NO modulates ventricular sarcK(ATP) channels via a novel sGC-cGMP-PKG-ROS(H2O2)-ERK1/2-calmodulin-CaMKII (δ isoform in particular) signalling cascade, which heightens K(ATP) channel activity by destabilizing the long closed states while facilitating closed-to-open state transitions. This pathway may contribute to regulation of cardiac excitability and cytoprotection against ischaemia-reperfusion injury, in part, by opening myocardial sarcK(ATP) channels.

A Family of HCN Channel Homologs in Bacteria

HCN channels belong to the superfamily of six transmembrane domain voltage-gated ion channels. Functionally, these channels activate upon membrane hyperpolarization and carry an inward current that is weakly selective between potassium and sodium and can be modulated by cyclic nucleotides. At the structural level, insight into these channels is limited to a crystal structure of the intracellular C-linker and cyclic nucleotide binding domain, which assembles into a tetramer. To advance our structural understanding of HCN channels, we investigated bacterial homologs of HCN channels as candidates for large-scale expression and future structural studies. Using a BLAST search of the bacterial genome database we identified different homologs, which have a sequence identity of 24-32% with the human HCN2 channel. Using a C-terminal fusion with green fluorescent protein (GFP) we investigated whether the bacterial HCN homologs could be expressed in E. coli. We identified homologs that could be expressed at a whole-cell fluorescence level of ∼40% compared to KcsA-GFP as a positive control. Next, we conducted detergent screening using fluorescence size exclusion chromatography (FSEC). We found that dodecylmaltoside or undecylmaltoside are suitable detergents to solubilize bacterial HCN channels in a tetrameric monodisperse state.In parallel, we investigated the functional properties of the bacterial HCN channel homologs using expression of C-terminal GFP fusions in Xenopus oocytes. Using cell-attached patch recordings we observed in a minority of patches a current that may closely resemble the kinetics of the invertebrate spHCN channel as clear current inactivation is observed at most hyperpolarizing potentials. Confocal microscopy demonstrates marked fluorescence just below the oocyte membrane, indicating that trafficking to the oocyte cell membrane may be compromised and limits our success in obtaining patches for detailed functional characterization.Together, our biochemical characterization paves the way for large-scale production and crystallization screening.

Modulation of Sarcolemmal ATP-Sensitive Potassium Channels by Atrial Natriuretic Peptide in Ventricular Cardiomyocytes

ATP-sensitive potassium (KATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation in the heart. Natriuretic peptides (NPs) produced by the heart, the vasculature and the kidney exert physiologically important actions such as natriuresis and inhibition of sympathetic tone and cardiomyocyte hypertrophy. Our recent study reveals that the gaseous messenger nitric oxide evokes cardiac KATP channel stimulation via activation of soluble guanylyl cyclase and subsequently a cGMP-dependent signaling pathway. Binding of NPs like atrial natriuretic peptide (ANP) to the type 1 NP receptor (NPR-A) also increases intracellular cGMP level, through activating guanylyl cyclase intrinsic to the receptor; however, how ANP modulates the function of cardiac KATP channels remains to be established. By performing cell-attached patch recordings in ventricular cardiomyocytes acutely isolated from adult rabbits, we found that the single-channel activity of sarcolemmal KATP (sarcKATP) channels induced by sodium azide through metabolic inhibition was potentiated by addition of ANP (100 nM) (n=16), whereas the potentiation by ANP was abolished when the NPR-A antagonist anantin (1 μM) was coapplied (n=5); these findings suggest that the stimulatory action of ANP is mediated by NPR-A activation, which may depend on generation of diffusible cGMP. Indeed, sarcKATP channel stimulation by ANP in intact ventricular myocytes was significantly abated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823 (1 μM) (n=5). Moreover, the ANP effect was prevented by selective suppression of extracellular signal-regulated protein kinase (ERK)1/2 (n=6) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (n=6). Collectively, here we demonstrate that the polypeptide hormone ANP positively modulates ventricular KATP channel function via activation of NPR-A, PKG, ERK1/2 and CaMKII. By opening myocardial KATP channels, this signaling pathway may regulate cardiac excitability and contribute to cytoprotection against ischemia-reperfusion injury.

Functional expression of a Kir2.1-like inwardly rectifying potassium channel in mouse mammary secretory cells

K(+) channels in mammary secretory (MS) cells are believed to play a role in transcellular electrolyte transport and thus determining ionic composition of the aqueous phase of milk. However, direct evidence for specific K(+) channel activity in native MS cells is lacking at the single-cell level. Here, we show for the first time that an inwardly rectifying K(+) (Kir) channel is functionally expressed in fully differentiated MS cells that were freshly isolated from the mammary gland of lactating mice. Using the standard whole cell patch-clamp technique, we found that mouse MS cells consistently displayed a K(+) current, whose electrophysiological properties are similar to those previously reported for Kir2.x channels, particularly Kir2.1: 1) current-voltage relationship with strong inward rectification, 2) slope conductance approximately proportional to the square root of external K(+) concentration, 3) voltage- and time-dependent and high-affinity block by external Ba(2+), and 4) voltage-dependent inhibition by external Cs(+). Accordingly, RT-PCR analysis revealed the gene expression of Kir2.1, but not Kir2.2, Kir2.3, and Kir2.4, in lactating mouse mammary gland, and immunohistochemical staining showed Kir2.1 protein expression in the secretory cells. Cell-attached patch recordings from MS cells revealed that a 31-pS K(+) channel with strong inward rectification was likely active at the resting membrane potential. Collectively, the present work demonstrates that a functional Kir2.1-like channel is expressed in lactating mouse MS cells. We propose that the channel might be involved, at least in part, in secretion and/or preservation of ionic components of milk stored into the lumen of these cells.

Slower Piezo1 Inactivation in Dehydrated Hereditary Stomatocytosis (Xerocytosis)

Slower Piezo1 Inactivation in Dehydrated Hereditary Stomatocytosis (Xerocytosis)

Functional Modulation of Cardiac ATP-Sensitive Potassium Channels by Nitric Oxide via Intracellular Signaling

ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. Nitric oxide (NO) has been shown to stimulate cardiac KATP channels; however, the mechanistic details remain poorly understood. Here we sought to delineate the intracellular mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in combination with pharmacological, genetic and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the cardiac-type KATP) channels in transfected HEK293 cells, which was abolished by selective suppression of cGMP-dependent protein kinase (PKG), extracellular signal-regulated protein kinase (ERK)1/2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and reactive oxygen species (ROS) (hydrogen peroxide H2O2 in particular), respectively. Importantly, NO donors potentiated function of sarcKATP channels preactivated by the channel opener pinacidil in adult rabbit ventricular myocytes, through destabilizing the longest closed state and facilitating opening transitions, and the potentiation was nullified when PKG, calmodulin, CaMKII or ERK1/2 was inhibited. Exogenous H2O2 also stimulated ventricular sarcKATP channels in intact cells in an ERK1/2- and CaMKII-dependent manner. Genetic ablation of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished PKG stimulation of mouse ventricular sarcKATP channels (compared with wild-type controls). Kinase activity and Western blot assays further supported that NO-PKG activation augmented CaMKII activity in ventricular myocytes, which was mediated by ERK1/2. Collectively, we demonstrate that NO stimulates ventricular sarcKATP channels via a cGMP/PKG/ROS(H2O2)/ERK/calmodulin/CaMKII signaling cascade that alters channel gating. This novel signaling pathway may control cardiac excitability and mediate, in part, cytoprotection against ischemia-reperfusion injury, by opening myocardial KATP channels.

Effects of exogenous hydrogen sulphide on calcium signalling, background (TASK) K channel activity and mitochondrial function in chemoreceptor cells

It has been proposed that endogenous H2S mediates oxygen sensing in chemoreceptors; this study investigates the mechanisms by which H2S excites carotid body type 1 cells. H2S caused a rapid reversible increase in intracellular calcium with EC50 ≈ 6 μM. This [Ca2+]i response was abolished in Ca-free Tyrode. In perforated patch current clamp recordings, H2S depolarised type 1 cells from −59 to −35 mV; this was accompanied by a robust increase in [Ca2+]i. Voltage clamping at the resting membrane potential abolished the H2S-induced rise in [Ca2+]i. H2S inhibited background K+ current in whole cell perforated patch and reduced background K+ channel activity in cell-attached patch recordings. It is concluded that H2S excites type 1 cells through the inhibition of background (TASK) potassium channels leading to membrane depolarisation and voltage-gated Ca2+ entry. These effects mimic those of hypoxia. H2S also inhibited mitochondrial function over a similar concentration range as assessed by NADH autofluorescence and measurement of intracellular magnesium (an index of decline in MgATP). Cyanide inhibited background K channels to a similar extent to H2S and prevented H2S exerting any further influence over channel activity. These data indicate that the effects of H2S on background K channels are a consequence of inhibition of oxidative phosphorylation. Whilst this does not preclude a role for endogenous H2S in oxygen sensing via the inhibition of cytochrome oxidase, the levels of H2S required raise questions as to the viability of such a mechanism.

Piezo1 Gating: Comparison Between Whole Cell Currents and the Patch

Piezo1 channels gate with mechanical stress in the membrane and gating involves both activation and inactivation. In HEK293 cells transfected with Piezo1 and subjected to pressure stimuli, cell-attached patch recordings showed that the inactivation rate slowed as extracellular divalent ions were reduced. With >1mM Mg+2, activation had no measurable latency and the inactivation rate was rapid but stress dependent, suggesting that Mg+2 may act as an open channel blocker (the effects of Ca+2 are in progress). Without divalents there was no inactivation, but surprisingly, activation now had a pronounced latency (∼500 ms). Inactivation may actually represent adaptation of the local stimulus by the cytoskeleton and not overt channel closure. To disrupt the cytoskeleton we treated cells with cytochalasin D before patching and found inactivation was unaffected suggesting cytoskeletal adaption was not the cause. Attempting the inverse experiment, we increased cytoskeletal stress by swelling the cells osmotically, but that too didn't affect the inactivation rate.For analogy close to the in situ situation, we evoked whole cell Piezo1 currents by indenting cells with a glass probe. Like the patch, removing extracellular divalent ions reversibly reduced the inactivation rate. However, in contrast to patch recordings, Cytochalasin D caused a loss of whole cell current and cell swelling increased the evoked currents. These results suggest that the forces that gate Piezo1 in whole cell mode propagate through the cytoskeleton, and that divalent ion block may be responsible for inactivation.Supported by the NIH and CDMRP

CAMP Activates TRPC6 Channels via the Phosphatidylinositol 3-Kinase (PI3K)-Protein Kinase B (PKB)-Mitogen-activated Protein Kinase Kinase (MEK)-ERK1/2 Signaling Pathway

cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 μm 8-Br-cAMP, a cell-permeable analog of cAMP, elicited [Ca(2+)](i) increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced [Ca(2+)](i) increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.