Cation Current Research Articles

Silybin induces endothelium-dependent vasodilation via TRPV4 channels in mouse mesenteric arteries.

Silybin is a flavonolignan extracted from the seeds of Silybum marianum that has been used as a dietary supplement for treating hepatic diseases and components of metabolic syndrome such as diabetes, obesity and hypertension. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels that regulate vascular endothelial function and blood flow. However, the relationship between silybin and TRPV4 channels in small mesenteric arteries remains unknown. In our study, we carried out a molecular docking experiment by using Discovery Studio v3.5 to predict the binding of silybin to TRPV4. Activation of TRPV4 with silybin was detected via intracellular Ca2+ concentration ([Ca2+]i) measurement and patch clamp experiments. The molecular docking results showed that silybin was likely to bind to the ankyrin repeat domain of TPRV4. [Ca2+]i measurements in mesenteric arterial endothelial cells (MAECs) and TRPV4-overexpressing HEK293 (TRPV4-HEK293) cells demonstrated that silybin induced Ca2+ influx by activating TRPV4 channels. The patch clamp experiments indicated that in TRPV4-HEK293 cells, silybin induced TRPV4-mediated cation currents. In addition, in high-salt-induced hypertensive mice, oral administration of silybin decreased systolic blood pressure (SBP) and significantly improved the arterial dilatory response to acetylcholine. Our findings provide the first evidence that silybin could induce mesenteric endothelium-dependent vasodilation and reduce blood pressure in high-salt-induced hypertensive mice via TRPV4 channels, thereby revealing the potential effect of silybin on preventing endothelial dysfunction-related cardiovascular diseases.

The Modulation of Ubiquinone, a Lipid Antioxidant, on Neuronal Voltage-Gated Sodium Current.

Ubiquinone, composed of a 1,4-benzoquinone and naturally produced in the body, actively participates in the mitochondrial redox reaction and functions as an endogenous lipid antioxidant, protecting against peroxidation in the pituitary-dependent hormonal system. However, the questions of if and how ubiquinone directly affects neuronal ionic currents remain largely unsettled. We investigated its effects on ionic currents in pituitary neurons (GH3 and MMQ cells) with the aid of patch-clamp technology. Ubiquinone decreased the peak amplitude of the voltage-gated Na+ current (INa) with a slowing of the inactivation rate. Neither menadione nor superoxide dismutase modified the ubiquinone-induced INa inhibition. In response to an isosceles-triangular ramp pulse, the persistent INa (INa(P)) at high- and low- threshold potentials occurred concurrently with a figure-eight hysteresis loop. With ubiquinone, the INa(P) increased with no change in the intersection voltage, and the magnitude of the voltage-dependent hysteresis of the current was enhanced. Ubiquinone was ineffective in modifying the gating of hyperpolarization-activated cation currents. In MMQ lactotrophs, ubiquinone effectively decreased the amplitude of the INa and the current inactivation rate. In sum, the effects of ubiquinone demonstrated herein occur upstream of its effects on mitochondrial redox processes, involved in its modulation of sodium channels and neuronal excitability.

Mechanosensitive cation currents through TRPC6 and Piezo1 channels in human pulmonary arterial endothelial cells.

Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension.

Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study.

Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBD's activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current.

K2P1 leak cation channels contribute to ventricular ectopic beats and sudden death under hypokalemia.

Hypokalemia causes ectopic heartbeats, but the mechanisms underlying such cardiac arrhythmias are not understood. In reduced serum K+ concentrations that occur under hypokalemia, K2P1 two-pore domain K+ channels change ion selectivity and switch to conduct inward leak cation currents, which cause aberrant depolarization of resting potential and induce spontaneous action potential of human cardiomyocytes. K2P1 is expressed in the human heart but not in mouse hearts. We test the hypothesis that K2P1 leak cation channels contribute to ectopic heartbeats under hypokalemia, by analysis of transgenic mice, which conditionally express induced K2P1 specifically in hearts, mimicking K2P1 channels in the human heart. Conditional expression of induced K2P1 specifically in the heart of hypokalemic mice results in multiple types of ventricular ectopic beats including single and multiple ventricular premature beats as well as ventricular tachycardia and causes sudden death. In isolated mouse hearts that express induced K2P1, sustained ventricular fibrillation occurs rapidly after perfusion with low K+ concentration solutions that mimic hypokalemic conditions. These observed phenotypes occur rarely in control mice or in the hearts that lack K2P1 expression. K2P1-expressing mouse cardiomyocytes of transgenic mice much more frequently fire abnormal single and/or rhythmic spontaneous action potential in hypokalemic conditions, compared to wild type mouse cardiomyocytes without K2P1 expression. These findings confirm that K2P1 leak cation channels induce ventricular ectopic beats and sudden death of transgenic mice with hypokalemia and imply that K2P1 leak cation channels may play a critical role in human ectopic heartbeats under hypokalemia.

Differential effects of TRPM4 channel inhibitors on Guinea pig urinary bladder smooth muscle excitability and contractility: Novel 4-chloro-2-[2-(2-chloro-phenoxy)-acetylamino]-benzoic acid (CBA) versus classical 9-phenanthrol.

Non‐selective cation channels in urinary bladder smooth muscle (UBSM) are thought to mediate increases in cellular excitability and contractility. For transient receptor potential melastatin type‐4 (TRPM4) channels, the evidence primarily relies on the inhibitor 9‐phenanthrol, which exhibits pharmacological limitations. Recently, 4‐chloro‐2‐[2‐(2‐chloro‐phenoxy)‐acetylamino]‐benzoic acid (CBA) has been discovered as a novel TRPM4 channel blocker. We examined how, in comparison to 9‐phenanthrol, CBA affects the excitability of freshly isolated guinea pig UBSM cells and the contractility of UBSM strips. Additionally, non‐selective TRPM4 channel inhibitor flufenamic acid (FFA) and potentiator BTP2 (also known as YM‐58483) were studied in UBSM cells. Unlike robust inhibition for 9‐phenanthrol already known, CBA (up to 100 μM) displayed either no or a very weak reduction (<20%) in spontaneous phasic, 20 mM KCl‐induced, and electrical field stimulated contractions. For 300 μM CBA, reductions were higher except for an increase in the frequency of KCl‐induced contractions. In UBSM cells, examined under amphotericin B‐perforated patch‐clamp, CBA (30 μM) did not affect the membrane potential (I = 0) or voltage step‐induced whole‐cell cation currents, sensitive to 9‐phenanthrol. The currents were not inhibited by FFA (100 μM), increased by BTP2 (10 μM), nor enhanced under a strongly depolarizing holding voltage of −16 or + 6 mV (vs. −74 mV). None of the three compounds affected the cell capacitance, unlike 9‐phenanthrol. In summary, the novel inhibitor CBA and nonselective FFA did not mimic the inhibitory properties of 9‐phenanthrol on UBSM function. These results suggest that TRPM4 channels, although expressed in UBSM, play a distinct role rather than direct regulation of excitability and contractility.

Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator.

Previously our computational modeling studies (Phillips et al., 2019) proposed that neuronal persistent sodium current (INaP) and calcium-activated non-selective cation current (ICAN) are key biophysical factors that, respectively, generate inspiratory rhythm and burst pattern in the mammalian preBötzinger complex (preBötC) respiratory oscillator isolated in vitro. Here, we experimentally tested and confirmed three predictions of the model from new simulations concerning the roles of INaP and ICAN: (1) INaP and ICAN blockade have opposite effects on the relationship between network excitability and preBötC rhythmic activity; (2) INaP is essential for preBötC rhythmogenesis; and (3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. These predictions were confirmed via optogenetic manipulations of preBötC network excitability during graded INaP or ICAN blockade by pharmacological manipulations in slices in vitro containing the rhythmically active preBötC from the medulla oblongata of neonatal mice. Our results support and advance the hypothesis that INaP and ICAN mechanistically underlie rhythm and inspiratory burst pattern generation, respectively, in the isolated preBötC.

Characterization of Inhibitory Capability on Hyperpolarization-Activated Cation Current Caused by Lutein (β,ε-Carotene-3,3'-Diol), a Dietary Xanthophyll Carotenoid.

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, Ih [or funny current, If]). In the present study, GH3-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in Ih amplitude with an IC50 value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of Ih in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked Ih, respectively. The voltage-dependent anti-clockwise hysteresis of Ih responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K+ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of Ih would be an ionic mechanism underlying its changes in membrane excitability.

Inspiratory rhythm generation is stabilized by Ih.

Cellular and network properties must be capable of generating rhythmic activity that is both flexible and stable. This is particularly important for breathing, a rhythmic behavior that dynamically adapts to environmental, behavioral, and metabolic changes from the first to the last breath. The pre-Bötzinger complex (preBötC), located within the ventral medulla, is responsible for producing rhythmic inspiration. Its cellular properties must be tunable, flexible as well as stabilizing. Here, we explore the role of the hyperpolarization-activated, nonselective cation current (Ih) for stabilizing PreBötC activity during opioid exposure and reduced excitatory synaptic transmission. Introducing Ih into an in silico preBötC network predicts that loss of this depolarizing current should significantly slow the inspiratory rhythm. By contrast, in vitro and in vivo experiments revealed that the loss of Ih minimally affected breathing frequency, but destabilized rhythmogenesis through the generation of incompletely synchronized bursts (burstlets). Associated with the loss of Ih was an increased susceptibility of breathing to opioid-induced respiratory depression or weakened excitatory synaptic interactions, a paradoxical depolarization at the cellular level, and the suppression of tonic spiking. Tonic spiking activity is generated by nonrhythmic excitatory and inhibitory preBötC neurons, of which a large percentage express Ih. Together, our results suggest that Ih is important for maintaining tonic spiking, stabilizing inspiratory rhythmogenesis, and protecting breathing against perturbations or changes in network state.NEW & NOTEWORTHY The Ih current plays multiple roles within the preBötC. This current is important for promoting intrinsic tonic spiking activity in excitatory and inhibitory neurons and for preserving rhythmic function during conditions that dampen network excitability, such as in the context of opioid-induced respiratory depression. We therefore propose that the Ih current expands the dynamic range of rhythmogenesis, buffers the preBötC against network perturbations, and stabilizes rhythmogenesis by preventing the generation of unsynchronized bursts.

The Role of the Ih Current in Rhythmic Stability of the PreBötzinger Complex

The generation of rhythmicity is a fundamental property of the nervous system. Common amongst all rhythmogenic networks is the ability to reconfigure and quickly adapt to changes in metabolic, environmental, and behavioral demands. Yet, this flexibility, and plasticity must be counter‐balanced by the need to maintain stability. A group of neurons collectively known as the preBötzinger complex (preBötC) within the ventral respiratory column of the medulla assemble in a network that is both necessary and sufficient to maintain inspiratory rhythm generation. Multiple ionic conductances contribute to rhythm generation. One known for its role in the generation of rhythmic activity across multiple neural networks is the hyperpolarization activated cation current (Ih). The Ih­ current is a voltage‐dependent mixed cationic current that is activated upon phasic hyperpolarization of neurons. The role of Ih in rhythmogenesis within the preBötC is inconclusive. Some reports find that blocking Ih has minimal effect, while others conclude that this increases the rate of rhythmic bursting throughout the network. To date, Ih was specifically studied in rhythmically active neurons with unknown transmitter phenotype. Herein we sought to determine the distribution of Ih amongst the population of tonically and rhythmically active excitatory and inhibitory neurons throughout the preBötC. Additionally, we tested whether removing Ih­ in‐vitro and in‐vivo renders the network more susceptible to perturbations. Using in‐vitro patch clamping we found that Ih showed greatest expression amongst excitatory (87% of DBX1+), and inhibitory (89% of VGAT+) tonically active cell populations. Removing Ih using ZD7288 silenced nearly all tonically spiking neurons. This led to a minimal effect on the rhythmic activity of fully synchronized population bursts, but significantly increased the incidence of less synchronized burstlet activity. Moreover, removing Ih and silencing tonic activity within the network rendered the preBötC more susceptible to suppression following opioid (DAMGO) and CNQX administration. This effect was recapitulated in‐vivo with microdialysis of ZD7288 into the preBötC, which caused normally well‐tolerated doses of morphine to become lethal via terminal apnea. In summary, Ihplays a complex role within the rhythm generating network of the preBötC. Under optimal conditions, Ih is not necessary for maintaining fully synchronized bursting within the network. Yet, Ih becomes increasingly important during perturbations that challenge network function, suggesting this ionic conductance contributes to maintaining the robustness of breathing during everchanging physiological and environmental demands.

Hyperpolarization‐Activated Current Shows Excitatory Effect on Fictive Locomotion in the Neonatal Mouse Under Hyperdopaminergic Conditions

Restless Leg Syndrome (RLS) is a chronic sensorimotor disorder that has recently been linked to a downregulation in adenosine A1 receptors (A1R), leading to decreased formation of adenosine‐dopamine heteromers and a disinhibited or hyperdopaminergic transmission pathway within the central nervous system. Dopamine D1 receptor (D1R) activation has been shown to modulate HCN1 channels via its stimulatory effect on cAMP production to increase the hyperpolarization‐activated cation current (Ih). Ih has been related to the regulation of bursting activity in rhythmic motor networks, specifically in controlling the firing frequency of the system. Our study aims to establish the importance of Ih in mammalian locomotion under hyperdopaminergic conditions mimicking those seen in RLS patients. We hypothesized that blocking Ih would increase the cycle period and destabilize the rhythm.MethodsWe isolated the spinal cords of neonatal (P0‐P5; n = 6) Swiss Webster mice via ventral laminectomy and recorded the extracellular activity of ventral roots L2 and L5 using suction electrodes to assess burst amplitude, burst duration, and cycle period. Fictive locomotion was induced using serotonin (5HT; 9‐15μM) and N‐methyl‐D‐aspartate (NMDA; 6 μM) before adding dopamine (DA; 50μM) to determine the effect of DA on the system. We then used ZD7288 (1μM) block HCN1 channels to investigate the role of Ih in motor‐like activity.ResultsBlocking Ih caused a significant increase in all three parameters relative to the 5HT/NMDA control in the L2 roots, but only cycle period was significantly increased relative to the high dopamine condition. There were no significant changes in any of the parameters in the L5 roots.ConclusionsOur results suggest that Ih primarily plays an excitatory role in locomotor regulation under hyperdopaminergic conditions, such as those seen in RLS. The differential effects between roots may suggest that one is more susceptible to the blockade of Ih by ZD7288; however, given the large degree of variability in the L5 data, a larger sample size is needed to confirm this hypothesis. Future experiments include collecting more data on L5 to confirm if there are differential effects, as well as pharmacological approaches to confirm that D1R is upregulating Ih via the cAMP pathway.

A plasma membrane‐localized polycystin‐1/polycystin‐2 complex in endothelial cells elicits vasodilation

Polycystin‐1 (PC‐1, PKD1), a receptor‐like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms and physiological functions are poorly understood. Here, by studying tamoxifen‐inducible, endothelial cell (EC)‐specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC‐1‐mediated, Ca2+‐dependent cation currents in ECs. EC‐specific PC‐1 knockout attenuates flow‐mediated arterial hyperpolarization and vasodilation. PC‐1‐dependent vasodilation occurs over the entire functional shear stress range and via the activation of nitric oxide synthase (NOS) and small‐and intermediate‐conductance Ca2+‐activated K+ (SK/IK) channels. EC‐specific PC‐1 knockout increases systemic blood pressure without altering kidney anatomy. PC‐1 coimmunoprecipitates with polycystin‐2 (PC‐2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC‐1 or PC‐2 (Pkd2 ecKO mice) abolishes surface clusters of both PC‐1 and PC‐2 in ECs. Single knockout of PC‐1 or PC‐2 or double knockout of PC‐1 and PC‐2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow‐mediated vasodilation. Flow stimulates non‐selective cation currents in ECs that are similarly inhibited by either PC‐1 or PC‐2 knockout or by interference peptides corresponding to the C‐terminus coiled‐coil domains present in PC‐1 or PC‐2. In summary, we show that PC‐1 regulates arterial contractility and demonstrate that this occurs through the formation of an interdependent signaling complex with PC‐2 in endothelial cells. Flow stimulates PC‐1/PC‐2 clusters in the EC plasma membrane, leading to Ca2+ influx, NOS and SK/IK channel activation, vasodilation and a reduction in blood pressure.

Role of the Dopamine Subtype 1 Receptor (D1R) Modulation of the Ih Current in Rhythmic Spinal Mammalian Motor Networks

Restless Legs Syndrome (RLS) is a chronic sensorimotor disorder that has been recently associated with decreased expression of inhibitory Gi‐coupled adenosine A1 receptors (A1Rs), leading to increased dopamine release (hyperdopaminergic signaling) within the central nervous system. Previous studies have shown that A1Rs specifically inhibit the Gs‐coupled dopamine D1 receptor (D1R) subtype through a functional A1R‐D1R heteromer receptor complex. Furthermore, D1Rs control spinal motor neuron (MN) excitability by increasing cyclic adenosine monophosphate (cAMP) concentration and opening hyperpolarization‐activated cyclic nucleotide‐gated non‐selective cation channel (HCN). This finding raises the possibility of increased HCN activity underlying some of the motor dysfunctions associated with periodic leg movements (PLMS) during sleep in RLS. Thus, this study aims to elucidate the importance of the D1 receptor subtype on modulating the hyperpolarization‐activated cation current (Ih) in spinal mammalian motor networks. I hypothesized that blocking Ih in the presence of a D1R agonist (enhanced dopaminergic modulation) would slow locomotor activity and disrupt MN bursting stability. Neonatal (P0‐P5; n = 7) mouse spinal cord preparations were used to measure MN bursting activity patterns including amplitude, duration, and cycle period of extracellular ventral root (L2 and L5) recordings via suction electrodes. Treatment conditions include serotonin (9‐15 μM 5‐HT) and N‐methyl‐D‐aspartate (6 μM NMDA) with and without a D1R agonist (10 μM SKF38393) to assess the effect of D1R on rhythmic bursting. Subsequently, an Ih blocker (1 μM ZD7288) was added to observe the importance of Ih in modulating MN burst activity. For both L2 and L5 roots, blocking the Ih current led to increases in burst amplitude, duration, and cycle period in respect to the control 5‐HT/NMDA condition. Our results propose that D1Rs play a significant role in modulating Ih by increasing cAMP levels and opening HCN1 channels at a more depolarized membrane potential. Future experiments involve: 1) using TM5 transmembrane disrupting peptide to disrupt A1R‐D1R heteromers in spinal MNs (and use TM7 as a negative control) to confirm if dopamine modulation of Ih activity is linked to the A1R‐D1R heteromer complexes and 2) applying adenyl cyclase activators and blockers to verify that the modulation of the Ih current is through the D1R‐mediated Gs second messenger pathway.

Effects of hyperpolarization-active cation current (Ih) on sublinear dendritic integration under applied electric fields

Effects of hyperpolarization-active cation current (Ih) on sublinear dendritic integration under applied electric fields

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of −70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near −3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1–6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

Putting the theory into 'burstlet theory' with a biophysical model of burstlets and bursts in the respiratory preBötzinger complex.

Inspiratory breathing rhythms arise from synchronized neuronal activity in a bilaterally distributed brainstem structure known as the preBötzinger complex (preBötC). In in vitro slice preparations containing the preBötC, extracellular potassium must be elevated above physiological levels (to 7-9 mM) to observe regular rhythmic respiratory motor output in the hypoglossal nerve to which the preBötC projects. Reexamination of how extracellular K+ affects preBötC neuronal activity has revealed that low-amplitude oscillations persist at physiological levels. These oscillatory events are subthreshold from the standpoint of transmission to motor output and are dubbed burstlets. Burstlets arise from synchronized neural activity in a rhythmogenic neuronal subpopulation within the preBötC that in some instances may fail to recruit the larger network events, or bursts, required to generate motor output. The fraction of subthreshold preBötC oscillatory events (burstlet fraction) decreases sigmoidally with increasing extracellular potassium. These observations underlie the burstlet theory of respiratory rhythm generation. Experimental and computational studies have suggested that recruitment of the non-rhythmogenic component of the preBötC population requires intracellular Ca2+ dynamics and activation of a calcium-activated nonselective cationic current. In this computational study, we show how intracellular calcium dynamics driven by synaptically triggered Ca2+ influx as well as Ca2+ release/uptake by the endoplasmic reticulum in conjunction with a calcium-activated nonselective cationic current can reproduce and offer an explanation for many of the key properties associated with the burstlet theory of respiratory rhythm generation. Altogether, our modeling work provides a mechanistic basis that can unify a wide range of experimental findings on rhythm generation and motor output recruitment in the preBötC.

The role of activation of two different sGC binding sites by NO-dependent and NO-independent mechanisms in the regulation of SACs in rat ventricular cardiomyocytes.

The mechanoelectrical feedback (MEF) mechanism in the heart that plays a significant role in the occurrence of arrhythmias, involves cation flux through cation nonselective stretch‐activated channels (SACs). It is well known that nitric oxide (NO) can act as a regulator of MEF. Here we addressed the possibility of SAC’s regulation along NO‐dependent and NO‐independent pathways, as well as the possibility of S‐nitrosylation of SACs. In freshly isolated rat ventricular cardiomyocytes, using the patch‐clamp method in whole‐cell configuration, inward nonselective stretch‐activated cation current ISAC was recorded through SACs, which occurs during dosed cell stretching. NO donor SNAP, α1‐subunit of sGC activator BAY41‐2272, sGC blocker ODQ, PKG blocker KT5823, PKG activator 8Br‐cGMP, and S‐nitrosylation blocker ascorbic acid, were employed. We concluded that the physiological concentration of NO in the cell is a necessary condition for the functioning of SACs. An increase in NO due to SNAP in an unstretched cell causes the appearance of a Gd3+‐sensitive nonselective cation current, an analog of ISAC , while in a stretched cell it eliminates ISAC . The NO‐independent pathway of sGC activation of α subunit, triggered by BAY41‐2272, is also important for the regulation of SACs. Since S‐nitrosylation inhibitor completely abolishes ISAC , this mechanism occurs. The application of BAY41‐2272 cannot induce ISAC in a nonstretched cell; however, the addition of SNAP on its background activates SACs, rather due to S‐nitrosylation.ODQ eliminates ISAC , but SNAP added on the background of stretch increases ISAC in addition to ODQ. This may be a result of the lack of NO as a result of inhibition of NOS by metabolically modified ODQ. KT5823 reduces PKG activity and reduces SACs phosphorylation, leading to an increase in ISAC . 8Br‐cGMP reduces ISAC by activating PKG and its phosphorylation. These results demonstrate a significant contribution of S‐nitrosylation to the regulation of SACs.

Emergent Temporal Signaling in Human Trabecular Meshwork Cells: Role of TRPV4-TRPM4 Interactions.

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

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European Journal of NeuroscienceVolume 55, Issue 7 p. 1887-1889 ERRATUMFree Access ERRATUM This article corrects the following: Cyclic nucleotide-dependent ionic currents in olfactory receptor neurons of the hawkmoth Manduca sexta suggest pull–push sensitivity modulation Jan Dolzer, Katrin Schröder, Monika Stengl, Volume 54Issue 3European Journal of Neuroscience pages: 4804-4826 First Published online: July 8, 2021 First published: 28 March 2022 https://doi.org/10.1111/ejn.15638AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Dolzer et al. (2021), some reference citations in Table 3 were missing. Table 3 should be presented as below: The Publisher apologises for this error. TABLE 3. Currents and current components in ORNs of Manduca sexta Name Abbreviation I-V curve Ion channel conductance/reversal potential (RP) Characteristics Activation Inactivation/block No effects References Potassium currents delayed rectifier IK ~ 30 pS outward rectification delayed onset depol. TEA, Cs+ mM Ni2+ Dolzer 2002 Zufall et al. 1991b cGMP-inactivted outward rectifier IK(cGMP-) 30 pS cNMP block depol. TEA, cAMP, cGMP, ATP Dolzer 2002 Zufall et al. 1991b Stengl et al. 1992 non-TEA-blockable delayed rectifier IK(noTEA) 40 pS? delayed onset no TEA block ~10−6 Ca2+ active? depol. PKC? cGMP?, Zn3+? TEA, cAMP Dolzer 2002 Dolzer et al. 2008 rapidly inactivating outward rectifier IK(A) 12–18 pA not tested depol. 4-AP 5 mM Zufall et al. 1991b slowly inactivating delayed rectifier IK(cGMP+) 70 pS? slow inactivation after depol. cGMP cAMP TEA (slow, incomplete), Ni2+, Cs+? Dolzer 2002 Dolzer et al. 2008 calcium-activated outward rectifier IK(Ca2+) 66 pS I-V flattened in positive Vm >10−6 M Ca2+ cGMP TEA, Cs+, CTX cAMP Dolzer 2002 Zufall et al. 1991b Nonspecific cation currents calcium-dependent Icat(Ca2+) RP = 0 I-V steeper in positive and negative Vm >10−6 M Ca2+ mM Ca2+, TEA, Ni2+, cGMP? Dolzer 2002 Stengl 1993, 1994 fast, steady, non-inactivating Icat(nRP) RP < 0 g (K) > g (Ca2+) clearly negative reversal potential TEA, Ni2+ cGMP? cAMP Dolzer 2002 late, large, long-lasting ILL RP = 0 slow activation after depol., slow inactivation after hyperpol. long, large depol. cAMP long, large depol. Ca2+?? TEA (partly) cGMP? Dolzer 2002 fast Unknown cation current Icat(PKC?) RP = 0 linear I-V TEA block Ca2+? PKC? TEA cAMP cGMP? Dolzer 2002 PKC-dependent Icat+(PKC) not tested PKC TEA Stengl 1993 Unknown currents non-specific cat- or anion I? RP = 0 linear I-V no TEA block TEA, Ni2+, cAMP?, cGMP?, Ca2+?? TEA, SIST, (DIDS), 9 AC, PTX, ZN2+, niflumic acid Dolzer 2002 inward rectifier Iir RP ~ 0 I-V steeper in negative Vm TEA Ni2+ Dolzer 2002 Abbreviations: cNMP, cyclic nucleotides; CTX, charybdotoxin; depol, depolarization; DIDS, 4,4′-diisothiocyanostilbene-2,20-disulfonic acid; hyperpol, hyperpolarization; I–V, current–voltage relation; PKC, protein kinase C; PTX, picrotoxin; SITS, 4-acetamido-4-thiocyanostilbene-2,2′-disulfonic acid; Vm, membrane potential; 4-AP, 4-aminopyridine; 9 AC, anthracene-9-carboxylic acid. Note: Currents that were not tested in this study, (IK(A) and Icat + (PKC)), are on grey background. Newly observed properties are underlined, and findings that are only described in the literature are in bold. REFERENCE Dolzer, J., Schröder, K., & Stengl, M. (2021). Cyclic nucleotide-dependent ionic currents in olfactory receptor neurons of the hawkmoth Manduca sexta suggest pull–push sensitivity modulation. European Journal of Neuroscience, 54(3), 4804– 4826. https://doi.org/10.1111/ejn.15346Wiley Online LibraryPubMedWeb of Science®Google Scholar Volume55, Issue7April 2022Pages 1887-1889 ReferencesRelatedInformation

A plasma membrane-localized polycystin-1/polycystin-2 complex in endothelial cells elicits vasodilation.

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms, and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates nonselective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in ECs. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel, and SK channel activation, vasodilation, and a reduction in blood pressure.