IK Channels Research Articles

The acute toxic effects of platinum nanoparticles on ion channels, transmembrane potentials of cardiomyocytes in vitro and heart rhythm in vivo in mice.

BackgroundPlatinum nanoparticles (PtNPs) have been considered a nontoxic nanomaterial and been clinically used in cancer chemotherapy. PtNPs can also be vehicle exhausts and environmental pollutants. These situations increase the possibility of human exposure to PtNPs. However, the potential biotoxicities of PtNPs including that on cardiac electrophysiology have been poorly understood.MethodsIon channel currents of cardiomyocytes were recorded by patch clamp. Heart rhythm was monitored by electrocardiogram recording. Morphology and characteristics of PtNPs were examined by transmission electron microscopy, dynamic light scattering and electrophoretic light scattering analyses.ResultsIn cultured neonatal mice ventricular cardiomyocytes, PtNPs with diameters 5 nm (PtNP-5) and 70 nm (PtNP-70) concentration-dependently (10–9 – 10–5 g/mL) depolarized the resting potentials, suppressed the depolarization of action potentials and delayed the repolarization of action potentials. At the ion channel level, PtNPs decreased the current densities of INa, IK1 and Ito channels, but did not affect the channel activity kinetics. In vivo, PtNP-5 and PtNP-70 dose-dependently (3–10 mg/kg, i.v.) decreased the heart rate and induced complete atrioventricular conduction block (AVB) at higher doses. Both PtNP-5 and PtNP-70 (10–9 – 10–5 g/mL) did not significantly increase the generation of ROS and leak of lactate dehydrogenase (LDH) from cardiomyocytes within 5 mins after exposure except that only very high PtNP-5 (10–5 g/mL) slightly increased LDH leak. The internalization of PtNP-5 and PtNP-70 did not occur within 5 mins but occurred 1 hr after exposure.ConclusionPtNP-5 and PtNP-70 have similar acute toxic effects on cardiac electrophysiology and can induce threatening cardiac conduction block. These acute electrophysiological toxicities of PtNPs are most likely caused by a nanoscale interference of PtNPs on ion channels at the extracellular side, rather than by oxidative damage or other slower biological processes.

Pharmacotherapeutic Effects of Quinidine on Short QT Syndrome by Using Purkinje-Ventricle Model: A Simulation Study.

Short QT syndrome (SQTS) arises due to gene mutations leading to accelerated ventricular repolarization, and increased risk of cardiac arrhythmias and sudden cardiac death (SCD). The SQT1, SQT2 and SQT3 variants of the SQTS result from inherited gain-of-function mutations (e.g. N588K, V307L and D172N, respectively) to potassium channels. However, the effective management of SQTS remains a challenge, and is incompletely understood. In this study, computational modelling was used to investigate pharmacotherapeutic effects of selected class I drug quinidine on SQT1, SQT2 and SQT3 variants. The biophysically-detailed Stewart et al. model of Purkinje fibre cell action potentials and the ten Tusscher et al. model of ventricular cell action potentials were coupled together into a heterogeneous two-dimensional (2D) tissue model. Previously validated IKr, IKs and IK1 channel formulations for SQT1, SQT2 and SQT3 were incorporated in ventricular cell and tissue models. The channel-blocking effects of quinidine on ionic currents were modelled by using Hill coefficient and IC50 values from the literature. At the 10 μM concentration tested in this study, quinidine effectively prolonged the action potential duration (APD) under all the SQT1, SQT2 and SQT3 conditions. In 2D simulations, quinidine prolonged the ventricular repolarization process and prolonged the QT intervals under all SQTS variants conditions. Our findings provide a rational basis for the pursuit of pharmacotherapeutic agent quinidine in the treatment of all SQTS variants.

IKs ion-channel pore conductance can result from individual voltage sensor movements

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance-voltage (G-V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

CP1 Is a Potent IKs Channel Activator Which Acts by Substituting Phosphatidylinositol 4,5 Bisphosphate

CP1 Is a Potent IKs Channel Activator Which Acts by Substituting Phosphatidylinositol 4,5 Bisphosphate

KCNE1 Subunit Phosphorylation Leads to IKs Internalization in Response to Chronic Calcium-Dependent PKC Activation

Decrease of the cardiac slowly activating delayed rectified K+ current (IKs), formed by KCNQ1 and KCNE1 subunits, is observed in a number of diseases states such as heart failure and diabetes and is associated with increased risk for cardiac arrhythmias and sudden death. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in many pathological conditions. Recently, we found that sustained cPKC stimulation leads to KCNQ1 subunit internalization and decrease of IKs channel activity. Here, we show that KCNE1 is internalized together with KCNQ1 in response to sustained cPKC stimulus using immunostaining and confocal imaging. In addition, we show that KCNE1 is necessary for channel internalization independently of subunit stoichiometry both in heterologous expression systems and cardiomyocytes. Mutation of a PKC phosphorylation site shown to be important to cPKC acute regulation of the channel (KCNE1-S102A) was necessary to channel internalization. Altogether, our results suggest that KCNE1(S102) phosphorylation by cPKC leads to KCNQ1/KCNE1 internalization in response to sustained cPKC stimulus. Our results suggest that KCNE1(S102) is an important target for anti-arrhythmic drugs that would prevent IKs pathological remodeling leading to cardiac arrhythmias.

A Novel PITX2c Gain-of-Function Mutation, p.Met207Val, in Patients With Familial Atrial Fibrillation

Genome-wide studies have associated several genetic variants upstream of PITX2 on chromosome 4q25 with atrial fibrillation (AF), suggesting a potential role of PITX2 in AF. Our study aimed at identifying rare coding variants in PITX2 predisposing to AF. The Polymerase chain reaction sequencing of PITX2c was performed in 60 unrelated patients with idiopathic AF. The p.Met207Val variant was identified in 1 of 60 French patients with early onset AF and in none of 389 French referents. This variant, located in the inhibitory domain 1 distal to the homeodomain, was evaluated by the software MutationTaster as a disease-causing mutation with a probability of 0.999. Reporter gene assays demonstrated that p.Met207Val caused a 3.1-fold increase in transactivation activity of PITX2c in HeLa cells in comparison with its wild-type counterpart. When the variant was coexpressed with wild-type PITX2c in the HL-1 immortalized mouse atrial cell line, this gain-of-function caused an increase in the mRNA level of KCNH2 (2.6-fold), SCN1B (1.9-fold), GJA5 (3.1-fold), GJA1 (2.1-fold), and KCNQ1 in the homozygous form (1.8-fold). These genes encode for the IKr channel α subunit, the β-1 Na+ channel subunit, connexin 40, connexin 43 and the IKs channel α subunit, respectively. These conditions may contribute to the propensity to AF found in patients carrying the p.Met207Val variant. In conclusion, the present report is the first to associate a gain-of-function mutation of PITX2c with increased vulnerability to AF, therefore, restoration of normal PITX2c function may be a potential therapeutic target in AF patients.

Using Light to Endow Stem-Cell-Derived Cardiomyocytes With Virtual IK1 Conductances

Using Light to Endow Stem-Cell-Derived Cardiomyocytes With Virtual IK1 Conductances

Doxorubicin induces caspase-mediated proteolysis of KV7.1

Kv7.1 (KCNQ1) coassembles with KCNE1 to generate the cardiac IKs-channel. Gain- and loss-of-function mutations in KCNQ1 are associated with cardiac arrhthymias, highlighting the importance of modulating IKs activity for cardiac function. Here, we report proteolysis of Kv7.1 as an irreversible posttranslational modification. The identification of two C-terminal fragments of Kv7.1 led us to identify an aspartate critical for the generation of one of the fragments and caspases as responsible for mediating proteolysis. Activating caspases reduces Kv7.1/KCNE1 currents, which is abrogated in cells expressing caspase-resistant channels. Enhanced cleavage of Kv7.1 can be detected for the LQT mutation G460S, which is located adjacent to the cleavage site, whereas a calmodulin-binding-deficient mutation impairs cleavage. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provokes caspase-mediated cleavage of endogenous IKs in human cardiomyocytes. In summary, caspases are novel regulatory components of IKs channels that may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity.

The IKs Channel Response to cAMP Is Modulated by the KCNE1:KCNQ1 Stoichiometry

The delayed potassium rectifier current, IKs, is assembled from tetramers of KCNQ1 and varying numbers of KCNE1 accessory subunits in addition to calmodulin. This channel complex is important in the response of the cardiac action potential to sympathetic stimulation, during which IKs is enhanced. This is likely due to channels opening more quickly, more often, and to greater sublevel amplitudes during adrenergic stimulation. KCNQ1 alone is unresponsive to cyclic adenosine monophosphate (cAMP), and thus KCNE1 is required for a functional effect of protein kinase A phosphorylation. Here, we investigate the effect that KCNE1 has on the response to 8-4-chlorophenylthio (CPT)-cAMP, a membrane-permeable cAMP analog, by varying the number of KCNE1 subunits present using fusion constructs of IKs with either one (EQQQQ) or two (EQQ) KCNE1 subunits in the channel complex with KCNQ1. These experiments use both whole-cell and single-channel recording techniques. EQQ (2:4, E1:Q1) shows a significant shift in V1/2 of activation from 10.4 mV ± 2.2 in control to −2.7 mV ± 1.2 (p-value: 0.0024). EQQQQ (1:4, E1:Q1) shows a smaller change in response to 8-CPT-cAMP, 6.3 mV ± 2.3 to −3.2 mV ± 3.0 (p-value: 0.0435). As the number of KCNE1 subunits is reduced, the shift in the V1/2 of activation becomes smaller. At the single-channel level, a similar graded change in subconductance occupancy and channel activity is seen in response to 8-CPT-cAMP: the less E1, the smaller the response. However, both constructs show a significant reduction of a similar magnitude in the first latency to opening (EQQ control: 0.90 s ± 0.07 to 0.71 s ± 0.06, p-value: 0.0032 and EQQQQ control: 0.94 s ± 0.09 to 0.56 s ± 0.07, p-value < 0.0001). This suggests that there are both E1-dependent and E1-independent effects of 8-CPT-cAMP on the channel.

Endotoxin modulates the electrophysiological characteristics of human embryonic stem cell‐differentiated cardiomyocytes

Gram‐negative bacteria‐induced infections result in fever, arrhythmia, and even death. Lipopolysaccharide (LPS), a constituent of bacteria, leads to an inflammatory response under sepsis and increase arrhythmogenesis. This study analyzed the effects on human embryonic stem cell‐differentiated cardiomyocytes (HIPSC‐CMs) exposed to LPS. A whole cell patch clamp was used to record the action potential (AP) and ionic currents with or without different doses of LPS in HIPSC‐CMs. Compared with the control, a different dose (0.04, 0.2, 1, and 5 µg/ml) of LPS‐treated HIPSC‐CMs resulted in a longer AP duration. The IC50 of sodium channel current was 1.254 µg/ml, L‐type calcium channel current was 5 µg/ml, and Ik channel currents were 1.254 µg/ml. LPS‐treated HIPSC‐CMs showed a lower sodium channel current, L‐type calcium channel current, and Ik channel currents. Furthermore, the expressions of Nav1.5 were decreased, and L‐Ca, Kv11.1, and Kv7.1 were increased in LPS‐treated HIPSC‐CMs. LPS‐induced arrhythmogenesis was related to the electrophysiological characteristics of sodium channel current, L‐type calcium channel current, and Ik channel currents. These results suggest a potential mechanism of cardiomyocyte injury in endotoxemia.

Integrating dynamic clamping with automated patch clamping devices: Adding virtual IK1 channels to iPSC-CM

Integrating dynamic clamping with automated patch clamping devices: Adding virtual IK1 channels to iPSC-CM

Regulation of inward rectifier potassium current ionic channel remodeling by AT1 -Calcineurin-NFAT signaling pathway in stretch-induced hypertrophic atrial myocytes.

Previous studies have shown that the activation of angiotensin II receptor type I (AT1 ) is attributed to cardiac remodeling stimulated by increased heart load, and that it is followed by the activation of the calcineurin-nuclear factor of activated T-cells (NFAT) signaling pathway. Additionally, AT1 has been found to be a regulator of cardiocyte ionic channel remodeling, and calcineurin-NFAT signals participate in the regulation of cardiocyte ionic channel expression. A hypothesis therefore follows that stretch stimulation may regulate cardiocyte ionic channel remodeling by activating the AT1 -calcineurin-NFAT pathway. Here, we investigated the role of the AT1 -calcineurin-NFAT pathway in the remodeling of inward rectifier potassium (Ik1 ) channel, in addition to its role in changing action potential, in stretch-induced hypertrophic atrial myocytes of neonatal rats. Our results showed that increased stretch significantly led to atrial myocytes hypertrophy; it also increased the activity of calcineurin enzymatic activity, which was subsequently attenuated by telmisartan or cyclosporine-A. The level of NFAT3 protein in nuclear extracts, the mRNA and protein expression of Kir2.1 in whole cell extracts, and the density of Ik1 were noticeably increased in stretched samples. Stretch stimulation significantly shortened the action potential duration (APD) of repolarization at the 50% and 90% level. Telmisartan, cyclosporine-A, and 11R-VIVIT attenuated stretch-induced alterations in the levels of NFAT3 , mRNA and protein expression of Kir2.1, the density of Ik1 , and the APD. Our findings suggest that the AT1 -calcineurin-NFAT signaling pathway played an important role in regulating Ik1 channel remodeling and APD change in stretch-induced hypertrophic atrial myocytes of neonatal rats.

KCNE1 tunes the sensitivity of KV7.1 to polyunsaturated fatty acids by moving turret residues close to the binding site.

The voltage-gated potassium channel KV7.1 and the auxiliary subunit KCNE1 together form the cardiac IKs channel, which is a proposed target for future anti-arrhythmic drugs. We previously showed that polyunsaturated fatty acids (PUFAs) activate KV7.1 via an electrostatic mechanism. The activating effect was abolished when KV7.1 was co-expressed with KCNE1, as KCNE1 renders PUFAs ineffective by promoting PUFA protonation. PUFA protonation reduces the potential of PUFAs as anti-arrhythmic compounds. It is unknown how KCNE1 promotes PUFA protonation. Here, we found that neutralization of negatively charged residues in the S5-P-helix loop of KV7.1 restored PUFA effects on KV7.1 co-expressed with KCNE1 in Xenopus oocytes. We propose that KCNE1 moves the S5-P-helix loop of KV7.1 towards the PUFA-binding site, which indirectly causes PUFA protonation, thereby reducing the effect of PUFAs on KV7.1. This mechanistic understanding of how KCNE1 alters KV7.1 pharmacology is essential for development of drugs targeting the IKs channel.

Modeling the Hidden Pathways of IKs Channel Activation

Modeling the Hidden Pathways of IKs Channel Activation

The effect of myocardial action potential duration on cardiac pumping efficacy: a computational study

Background and aimsAlthough studies on the relation between arrhythmias and the action potential duration (APD) have been carried out, most of them are based only on electrophysiological factors of the heart and lack experiments that consider cardiac mechanical and electromechanical characteristics. Therefore, we conducted this study to clarify the relevance of the shortening of APD of a cell in relation to the mechanical contraction activity of the heart and the associated risk of arrhythmia.MethodsThe human ventricular model used in this study has two dynamic characteristics: electrophysiological conduction and mechanical contraction. The model simulating electrophysiological characteristics was consisted of lumped parameter circuit that can mimic the phenomenon of ion exchange through the cell membrane of myocyte and consisted of 214,319 tetrahedral finite elements. In contrast, the model simulating mechanical contraction characteristics was constructed to mimic cardiac contraction by means of the crossbridge of a myofilament and consisted of 14,720 hermite-based finite elements to represent a natural 3D curve of the cardiac surface. First, we performed a single cell simulation and the electrophysiological simulation according to the change of the APD by changing the electrical conductivity of the IKs channel. Thus, we confirmed the correlation between APD and intracellular Ca2+ concentration. Then, we compared mechanical response through mechanical simulation using Ca2+ data from electrical simulation.ResultsThe APD and the sum of the intracellular Ca2+ concentrations showed a positive correlation. The shortened APD reduced the conduction wavelength of ventricular cells by shortening the plateau and early repolarization in myocardial cells. The decrease in APD reduced ventricular pumping efficiency by more than 60% as compared with the normal group (normal conditions). This change is caused by the decline of ventricular output owing to reduced ATP consumption during the crossbridge of myofilaments and decreased tension.ConclusionThe shortening of APD owing to increased electrical conductivity of a protein channel on myocardial cells likely decreases the wavelength and the pumping efficiency of the ventricles. Additionally, it may increase tissue sensitivity to ventricular fibrillation, including reentry, and cause symptoms such as dyspnea and dizziness.

Relationship between KCNQ1 (LQT1) and KCNH2 (LQT2) gene mutations and sudden death during illegal drug use

Long QT syndrome (LQTS), a congenital genetic disorder, can cause torsades de pointes (TdP), and lethal cardiac arrhythmia may result from ingestion of cardiotoxic drugs. Methamphetamine (MP) and new psychoactive substances (NPSs) can trigger TdP due to QT prolongation, leading to sudden death. We therefore analysed variations in the LQTS-associated genes KCNQ1 (LQT1) and KCNH2 (LQT2) using cardiac blood and myocardial tissue from subjects having died suddenly during MP or NPS use to investigate the relationship between congenital genetic abnormalities and sudden death during illegal drug use. We amplified and sequenced all exons of these genes using samples from 20 subjects, half of whom had died taking MP and half after using NPSs. G643S, a KCNQ1 missense polymorphism, was significantly more common among sudden deaths involving NPSs (6 subjects) than those involving MP (1 subject) and healthy Japanese subjects (P = 0.001). Notably, synthetic cathinones were detected in 2 of 3 cases involving G643S carriers. Previous functional analyses have indicated that the G643S polymorphism in the KCNQ1 potassium channel gene causes mild IKs channel dysfunction. Our data suggest that use of NPSs, particularly synthetic cathinones, is associated with elevated risk of serious cardiac arrhythmia and sudden death for subjects carrying KCNQ1 G643S.

Activation of TRPV4 stimulates transepithelial ion flux in a porcine choroid plexus cell line.

The choroid plexus (CP) epithelium plays a major role in the production of cerebrospinal fluid (CSF). A polarized cell line, the porcine CP-Riems (PCP-R) line, which exhibits many of the characteristics of the native epithelium, was used to study the effect of activation of the transient receptor potential vanilloid 4 (TRPV4) cation channel found in the PCP-R cells as well as in the native epithelium. Ussing-style electrophysiological experiments showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux and conductance. These changes were inhibited by either of two distinct antagonists, HC067047 or RN1734. The change in conductance was reversible and did not involve disruption of epithelial junctional complexes. Activation of TRPV4 results in Ca2+ influx, therefore, we examined whether the electrophysiological changes were the result of secondary activation of Ca2+-sensitive channels. PCP-R cells contain two Ca2+-activated K+ channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes whereas one of the three isoforms of the IK channel (KCNN4c) may play a role in the apical secretion of K+. Blocking the activity of this IK isoform with TRAM34 inhibited the TRPV4-mediated change in net transepithelial ion flux and the increased conductance. These studies implicate TRPV4 as a hub protein in the control of CSF production through stimulation by multiple effectors resulting in transepithelial ion and subsequent water movement.

Cyproheptadine Regulates Pyramidal Neuron Excitability in Mouse Medial Prefrontal Cortex.

Cyproheptadine (CPH), a first-generation antihistamine, enhances the delayed rectifier outward K+ current (IK) in mouse cortical neurons through a sigma-1 receptor-mediated protein kinase A pathway. In this study, we aimed to determine the effects of CPH on neuronal excitability in current-clamped pyramidal neurons in mouse medial prefrontal cortex slices. CPH (10 µmol/L) significantly reduced the current density required to generate action potentials (APs) and increased the instantaneous frequency evoked by a depolarizing current. CPH also depolarized the resting membrane potential (RMP), decreased the delay time to elicit an AP, and reduced the spike threshold potential. This effect of CPH was mimicked by a sigma-1 receptor agonist and eliminated by an antagonist. Application of tetraethylammonium (TEA) to block IK channels hyperpolarized the RMP and reduced the instantaneous frequency of APs. TEA eliminated the effects of CPH on AP frequency and delay time, but had no effect on spike threshold or RMP. The current-voltage relationship showed that CPH increased the membrane depolarization in response to positive current pulses and hyperpolarization in response to negative current pulses, suggesting that other types of membrane ion channels might also be affected by CPH. These results suggest that CPH increases the excitability of medial prefrontal cortex neurons by regulating TEA-sensitive IK channels as well as other TEA-insensitive K+ channels, probably ID and inward-rectifier Kir channels. This effect of CPH may explain its apparent clinical efficacy as an antidepressant and antipsychotic.

Cerebral Capillary TRPA1 Channels Mediate Functional Hyperemia via Retrograde Conducted Vasodilation

Cerebral capillaries are the sites of nutrient exchange and removal of metabolic by‐products within the brain parenchyma. Recent studies showed that cerebral capillary endothelial cells (ECs) are central players in matching augmented neuronal metabolism to an increase in local blood perfusion, a process known as functional hyperemia. However, little is known about the molecular sensors present in capillary EC that orchestrate dilation of upstream parenchymal arterioles (PAs). We previously showed that the transient receptor potential ankyrin 1 (TRPA1) channel, a Ca2+‐permeable non‐selective cation channel, is present in ECs from cerebral arteries, but not in other peripheral vascular beds. We tested the hypothesis that TRPA1 is also present in cerebral capillary EC and its activation induces dilation of upstream PAs through a Ca2+‐dependent mechanism. Immunofluorescence staining of freshly isolated cerebral capillaries showed the presence of TRPA1, which was absent in EC‐specific TRPA1 knockout mice (eTRPA1−/−). Further, currents evoked by the TRPA1 agonist allyl isothyocyanate (AITC, 30 μM) in whole‐cell patch clamp electrophysiology experiments were blocked by the selective TRPA1 antagonist HC‐030031 (10 μM) and were absent from eTRPA1−/− mice. Using a recently‐described ex vivo capillary‐parenchymal arteriole preparation in which the capillary bed remains attached to pressurized PAs, we observed that picospritzing capillaries with AITC induced dilation of the upstream PA, an effect inhibited by HC‐030031 and absent in eTRPA1−/− mice. We then investigated the mechanisms underlying propagation of the vasodilatory signal from capillary to arteriolar EC. Using mice expressing the genetically‐encoded Ca2+ biosensor GCaMP6f in EC, we observed that picospritzing AITC onto capillary beds generated an intercellular Ca2+ wave that propagated from capillaries to the upstream PA. Pre‐incubation of preparations with the TRPA1 inhibitor HC‐030031 abolished the generation of Ca2+ waves. Moreover, the frequency of Ca2+ transients in arteriolar EC was increased immediately after dissipation of the Ca2+ wave, a process blocked by HC‐030031. Increases in arteriolar EC intracellular Ca2+ can activate intermediate and small conductance Ca2+‐activated K+ channels (IK and SK, respectively), leading to vasodilation. To test if this mechanism was responsible for PA dilation, preparations were incubated with the IK and SK channels inhibitors TRAM‐34 (1 μM) and apamin (1 μM), which blunted arteriolar dilation induced by picospritzing AITC onto capillaries. Somatosensory stimulation of the whisker barrel cortex, a model of functional hyperemia, caused a smaller increase in perfusion in eTRPA1−/− mice than in wildtype littermates. Taken together, these data suggest that functional TRPA1 channels are present in cerebral capillary EC and that activation of the channel elicits intercellular Ca2+ waves that propagate to upstream PAs, where it activates IK and SK channels to cause hyperpolarization and dilation, thus mediating functional hyperemia responses.Support or Funding InformationSupported by the National Institutes of Health (R01HL091095 to SE) and the American Heart Association (15POST2472002 to PWP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acid Sensing Ion Channel 1 Contributes to Endothelium‐Derived Hyperpolarizing Factor Induced Vasodilation in Small Mesenteric Arteries.

Acid sensing ion channel 1 (ASIC1) belongs to the amiloride‐sensitive degenerin/epithelial sodium channel superfamily, and is permeable to both Na+ and Ca2+. ASIC channels have been found to regulate blood pressure through mechanotransduction in arterial baroreceptors and are also pH sensors in carotid body chemoreceptors. ASIC1 is additionally expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds; yet little is known regarding the role of ASIC1 in the regulation of local vascular resistance. Although extracellular H+ is the recognized activator of ASIC1, we have previously shown this channel can be activated following G protein‐coupled receptor stimulation. In small mesenteric arteries, we found that ASIC1 contributes to acetylcholine (ACh)‐induced endothelial cell Ca2+ entry and vasodilation. However, the mechanisms involved in activating ASIC1 and its downstream effects on vasodilation are unknown. We hypothesized that ACh activates ASIC1‐mediated endothelial cell Ca2+ entry to mediate endothelium‐derived hyperpolarizing factor (EDHF)‐type vasodilation through activation of small (SK) and intermittent (IK) conductance Ca2+‐activated K+ channels. To test this hypothesis, we used pressure myography to study the effects of ASIC1 on ACh‐induced vasodilation (10−9 – 10−5 M) of freshly isolated mesenteric arteries (100–200 μm inner diameter), in the presence of Nω‐nitro‐L‐arginine (nitric oxide synthase inhibitor) and indomethacin (cyclooxygenase inhibitor). ACh‐induced vasodilation was abolished by specific inhibitors of SK and IK channels (apamin and TRAM‐34, respectively), confirming that the ACh‐induced dilation is EDHF‐dependent. The specific ASIC1 antagonist, psalmotoxin 1 (PcTX1; 20 nM) similarly blocks the EDHF‐mediated vasodilatory response, suggesting ASIC1 contributes to ACh‐induced vasodilation through an EDHF‐mediated pathway. Future studies will examine the upstream mechanisms involved in activation of endothelial ASIC1 and subsequent downstream effects to mediate EDHF‐dependent vasodilation. These studies suggest a role for ASIC1 as a novel contributor to agonist‐induced mesenteric vasodilation and cardiovascular homeostasis.Support or Funding InformationSupported by National Heart, Lung, and Blood Institute Grants R01 HL‐111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta), and R01 HL132883 (to T.C. Resta).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.