Outward Current Research Articles

Dynamical properties of the improved FK3V heart cell model

The main aim of this paper is to study the evolution of the transmembrane potential on the cardiac cell under different rates and amplitudes of stimulation. For modeling this potential, the modification of the Fenton‐Karma model was applied. It is a phenomenological model with 3 degrees of freedom that corresponds to nondimensional transmembrane potential and gating variables for regulation of inward and outward ion currents that can better reproduce the shape of the transmembrane potential than the original Fenton‐Karma model. The model was newly forced by stimulus with the shape of the half‐sine period. As the main goal of the paper is to show that this model is showing regular as well as irregular motion; periodic and chaotic patterns are detected using bifurcation diagrams, the Fourier spectra, Poincaré sections, and 0‐1 test for chaos.

Probing the B-fields of AGN jets on kiloparsec scales - NGC 6251

AbstractBy constructing images of the Faraday rotation measure (RM) of large scale astrophysical jets, the line-of-sight magnetic field component and electron density in the region of Farady rotation can be investigated. A significant gradient in the RM transverse to the jet direction may indicate a corresponding gradient in the line-of-sight magnetic field, implying a toroidal or helical magnetic field, which would, in turn, imply the presence of an associated electrical current in the jet. The detection of such large scale gradients can reliably demonstrate that helical or toroidal fields can persist to large distances from the central AGN. We present a kiloparsec-scale Faraday rotation map of NGC 6251 that shows statistically significant transverse RM gradients across its kiloparsec scale jet structure that imply an outward current.

The endogenous agonist, β-alanine, activates glycine receptors in rat spinal dorsal neurons

β-alanine is a structural analog of glycine and γ-aminobutyric acid (GABA) and is thought to be involved in the modulation of nociceptive information at the spinal cord. However, it is not known whether β-alanine exerts its effect in substantia gelatinosa (SG) neurons of the spinal dorsal horn, where glycine and GABA play an important role in regulating nociceptive transmission from the periphery. Here, we investigated the effects of β-alanine on inhibitory synaptic transmission in adult rat SG neurons using whole-cell patch-clamp. β-alanine dose-dependently induced outward currents in SG neurons. Current-voltage plots revealed a reversal potential at approximately −70 mV, which was close to the equilibrium potential of Cl−. Pharmacological analysis revealed that β-alanine activates glycine receptors, but not GABAA receptors. These results suggest that β-alanine hyperpolarizes the membrane potential of SG neurons by activating Cl− channels through glycine receptors. Our findings raise the possibility that β-alanine may modulate pain sensation through glycine receptors.

Cleavage of potassium channel Kv2.1 by BACE2 reduces neuronal apoptosis

Potassium channel Kv2.1 regulates potassium current in cortical neurons and potassium efflux is necessary for cell apoptosis. As a major component of delayed rectifier current potassium channels, Kv2.1 forms clusters in the membrane of hippocampal neurons. BACE2 is an aspartyl protease to cleave APP to prevent the generation of Aβ, a central component of neuritic plaques in Alzheimer's brain. We now identified Kv2.1 as a novel substrate of BACE2. We found that BACE2 cleaved Kv2.1 at Thr376, Ala717, and Ser769 sites and disrupted Kv2.1 clustering on cell membrane, resulting in decreased Ik of Kv2.1 and a hyperpolarizing shift in primary neurons. Furthermore, we discovered that the BACE2-cleaved Kv2.1 forms, Kv2.1-1-375, Kv2.1-1-716, and Kv2.1-1-768, depressed the delayed rectifier Ik surge and reduced neuronal apoptosis. Our study suggests that BACE2 plays a neuroprotective role by cleavage of Kv2.1 to prevent the outward potassium currents, a potential new target for Alzheimer's treatment.

Calcium- and ATP-dependent regulation of Na/Ca exchange function in BHK cells: Comparison of NCX1 and NCX3 exchangers

Na+/Ca2+ exchangers (NCX) mediate bidirectional Ca2+ fluxes across cell membranes and contribute to Ca2+ homeostasis in many cell types. Exchangers are regulated by gating reactions that depend on Na+ and Ca2+ binding to transport and regulatory sites. A Na+i-dependent inactivation is prominent in all isoforms, whereas Ca2+i-dependent regulation varies among isoforms. Here we characterize new details of NCX operation and describe differences and similarities between NCX3 and NCX1 regulation by intracellular Ca2+ and ATP. To compare isoforms, we employed BHK cells expressing NCX3 or NCX1 constitutively and exchange activity was analysed in whole-cell and excised patch recordings under “zero-trans” conditions (i.e., with only one transported ion species on each side). Using BHK cells with low cytoplasmic Ca2+ buffering, outward (reverse) currents, reflecting Ca2+ influx, are activated by applying extracellular Ca2+ (Cao) in the presence of Na+ on the cytoplasmic side. When firstly activated, peak outward NCX3 currents rapidly decay over seconds and then typically develop a secondary transient peak with slower kinetics, until Cao removal abolishes all outward current. The delayed rise of outward current is the signature of an activating process since peak outward NCX3 currents elicited at subsequent Cao bouts remain stimulated for minutes and slower decline towards a non-zero level during continued Cao application. Secondary transient peaks and current stimulation are suppressed by increasing the intracellular Ca2+ buffer capacity or by replacing cytoplasmic ATP with the analogues AMP-PNP or ATPγS. In BHK cells expressing NCX1, outward currents activated under identical settings decay to a steady-state level during single Cao application and are significantly larger, causing strong and long-lived run down of subsequent outward currents. NCX1 current run down is not prevented by increasing cytoplasmic Ca2+ buffering but secondary transient peaks in the outward current profile can be resolved in the presence of ATP. Finally, inward currents recorded in patches excised from NCX3-expressing cells reveal a proteolysis-sensitive, Ca-dependent inactivation process that is unusual for NCX1 forward activity. Together, our results suggest that NCX function is regulated more richly than appreciated heretofore, possibly including processes that are lost in excised membrane patches.

Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells.

The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR) is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1) depolarization responses induced by paxilline (PAX), a specific KCa1.1 blocker and (2) PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory β and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in breast cancer cells.

New Targets for Old Drugs: Cardiac Glycosides Inhibit Atrial-Specific K2P3.1 (TASK-1) Channels.

Cardiac glycosides have been used in the treatment of arrhythmias for more than 200 years. Two-pore-domain (K2P) potassium channels regulate cardiac action potential repolarization. Recently, K2P3.1 [tandem of P domains in a weak inward rectifying K+ channel (TWIK)-related acid-sensitive K+ channel (TASK)-1] has been implicated in atrial fibrillation pathophysiology and was suggested as an atrial-selective antiarrhythmic drug target. We hypothesized that blockade of cardiac K2P channels contributes to the mechanism of action of digitoxin and digoxin. All functional human K2P channels were screened for interactions with cardiac glycosides. Human K2P channel subunits were expressed in Xenopus laevis oocytes, and voltage clamp electrophysiology was used to record K+ currents. Digitoxin significantly inhibited K2P3.1 and K2P16.1 channels. By contrast, digoxin displayed isolated inhibitory effects on K2P3.1. K2P3.1 outward currents were reduced by 80% (digitoxin, 1 Hz) and 78% (digoxin, 1 Hz). Digitoxin inhibited K2P3.1 currents with an IC50 value of 7.4 µM. Outward rectification properties of the channel were not affected. Mutagenesis studies revealed that amino acid residues located at the cytoplasmic site of the K2P3.1 channel pore form parts of a molecular binding site for cardiac glycosides. In conclusion, cardiac glycosides target human K2P channels. The antiarrhythmic significance of repolarizing atrial K2P3.1 current block by digoxin and digitoxin requires validation in translational and clinical studies.

Inhibition of A-Type K+ Channels by Urotensin-II Induces Sensory Neuronal Hyperexcitability Through the PKCα-ERK Pathway.

Previous studies have implicated urotensin-II in the nociception of sensory neurons. However, to date the relevant mechanisms remain unknown. In the current study we determined the role of urotensin-II in the regulation of transient outward A-type potassium currents (IA) and neuronal excitability in trigeminal ganglion (TG) neurons. We found that application of urotensin-II to small-diameter TG neurons decreased IA in a dose-dependent manner, whereas the delayed rectifier potassium current was unaffected. The IA decrease induced by urotensin-II depended on the urotensin-II receptor (UT-R) and was associated with a hyperpolarizing shift in the steady-state inactivation curve. Exposure of TG cells to urotensin-II markedly increased protein kinase C (PKC) activity, and PKC inhibition eliminated the UT-R-mediated IA decrease. Antagonism of PKCα, either pharmacologically or genetically, but not of PKCβ prevented the decrease in IA induced by urotensin-II. Analysis of phospho-extracellular signal-regulated kinase (p-ERK) revealed that urotensin-II significantly increased the expression level of p-ERK, whereas p-p38 and p-c-Jun N-terminal kinase remained unchanged. Inhibition of mitogen-activated protein kinase/ERK signaling by the kinase antagonist U0126 and PD98059 completely abolished the UT-R-mediated IA decrease. Moreover, urotensin-II significantly increased the action potential firing rate of small TG neurons; pretreatment with 4-aminopyridine prevented this effect. In summary, our findings suggest that urotensin-II selectively attenuated IA through stimulation of the PKCα-dependent ERK1/2 signaling pathway. This UT-R-dependent mechanism might contribute to neuronal hyperexcitability in TG neurons.

Interaction of a dinoflagellate neurotoxin with voltage-activated ion channels in a marine diatom.

BackgroundThe potent neurotoxins produced by the harmful algal bloom species Karenia brevis are activators of sodium voltage-gated channels (VGC) in animals, resulting in altered channel kinetics and membrane hyperexcitability. Recent biophysical and genomic evidence supports widespread presence of homologous sodium (Na+) and calcium (Ca2+) permeable VGCs in unicellular algae, including marine phytoplankton. We therefore hypothesized that VGCs of these phytoplankton may be an allelopathic target for waterborne neurotoxins produced by K. brevis blooms that could lead to ion channel dysfunction and disruption of signaling in a similar manner to animal Na+ VGCs.MethodsWe examined the interaction of brevetoxin-3 (PbTx-3), a K. brevis neurotoxin, with the Na+/Ca2+ VGC of the non-toxic diatom Odontella sinensis using electrophysiology. Single electrode current- and voltage- clamp recordings from O. sinensis in the presence of PbTx-3 were used to examine the toxin’s effect on voltage gated Na+/Ca2+ currents. In silico analysis was used to identify the putative PbTx binding site in the diatoms. We identified Na+/Ca2+ VCG homologs from the transcriptomes and genomes of 12 diatoms, including three transcripts from O. sinensis and aligned them with site-5 of Na+ VGCs, previously identified as the PbTx binding site in animals.ResultsUp to 1 µM PbTx had no effect on diatom resting membrane potential or membrane excitability. The kinetics of fast inward Na+/Ca2+ currents that underlie diatom action potentials were also unaffected. However, the peak inward current was inhibited by 33%, delayed outward current was inhibited by 25%, and reversal potential of the currents shifted positive, indicating a change in permeability of the underlying channels. Sequence analysis showed a lack of conservation of the PbTx binding site in diatom VGC homologs, many of which share molecular features more similar to single-domain bacterial Na+/Ca2+ VGCs than the 4-domain eukaryote channels.DiscussionAlthough membrane excitability and the kinetics of action potential currents were unaffected, the permeation of the channels underlying the diatom action potential was significantly altered in the presence of PbTx-3. However, at environmentally relevant concentrations the effects of PbTx- on diatom voltage activated currents and interference of cell signaling through this pathway may be limited. The relative insensitivity of phytoplankton VGCs may be due to divergence of site-5 (the putative PbTx binding site), and in some cases, such as O. sinensis, resistance to toxin effects may be because of evolutionary loss of the 4-domain eukaryote channel, while retaining a single domain bacterial-like VGC that can substitute in the generation of fast action potentials.

Oxytocin Reduction of Nodose Ganglion Neurons Potassium Currents is Enhanced in Chronic Intermittent Hypoxia (CIH)

Oxytocin (OT) from the hypothalamus is increased in several cardiorespiratory nuclei and the circulation in response to a variety of stimuli and stressors, including hypoxia. Oxytocin within the nucleus tractus solitarii (nTS) enhances synaptic transmission, action potential (AP) discharge, and cardiac baroreflex gain to lower heart rate. The baroreceptor reflex is mediated through afferent neurons whose somas originate in the nodose ganglia and project centrally to the nTS. Nodose neurons express the OT receptor, and its activation elevates intracellular calcium; the influence of OT on other ion channels, especially during hypoxia, is not known. The hypoxic stressor obstructive sleep apnea, and its CIH animal model, elevates blood pressure and alters heart rate variability. Recent studies suggest enhancing OT in the dorsal vagal complex during CIH may reduce blood pressure. This study was undertaken to determine the mechanism(s) by which OT modulates sensory afferent‐nTS mediated reflexes normally and after CIH. Nodose neurons of 5–6 wk old male Sprague‐Dawley rats were isolated after 10 d of Normoxia (21% O2, Norm) or CIH (alternating 21% & 6% O2, 8 hr/day). Two‐5 hrs after isolation, we recorded outward potassium currents (IK) and APs by the patch clamp method. To examine IK, neurons were held at −60 mV and currents were evoked by depolarizing voltages (−70 to +60 mV, 140 ms). The steady state IK‐voltage relationship was analyzed. AP discharge was evoked by injecting 50 pA depolarizing steps (−20 to 280 pA, 200 ms); the current‐discharge relationship was evaluated. IK and APs were examined during baseline and after 3 min of vehicle or OT (300, 600, or 1000 nM). Vehicle did not alter IK in Norm (n=6) and CIH (n=4) cells, confirming lack of time‐dependent IK changes. In Norm cells, OT decreased IK at voltages greater than +40 mV (300 nM, n=8) and +60 mV (600 nM, n=7). After CIH, OT (300 nM, n=5) also decreased IK at +40 mV and greater. OT at 600 nM (n=5) attenuated IK at significantly lower voltages (+20 mV) in CIH vs. Norm. OT‐sensitive currents at +60 mV were greater in CIH than Norm at 300 and 600 nM. In Norm and CIH groups (n=4, 5), OT at 1000 nM had variable IK responses which overall were not significantly different from baseline. Fitting IKs to Boltzmann functions demonstrated a reduction in half‐activation during OT in Norm cells. In Norm and CIH cells, application of the K+ channel blocker tetraethylammonium (TEA, 10 mM) decreased IK and blocked the OT‐induced reduction in IK (600 nM, n=3). Depolarizing nodose neurons with up to 280 pA injection induced APs in Norm (n=7) and CIH (n=5) neurons, and 600 nM OT did not alter the number of APs during a given current step. Yet, 600 nM OT decreased AP threshold and tended to increase AP half‐width in CIH but not Norm cells, consistent with the reduction in IK by OT in CIH. In Norm cells, vehicle and OT did not alter holding currents measured at −60 mV, but after CIH 600 nM OT induced modest depolarizing inward currents. Taken together, these data demonstrate OT induces changes in ionic currents in nodose ganglia cells that are consistent with increasing excitability in CIH, and may also shed light on the bradycardic and anti‐hypertensive action of OT.Support or Funding InformationRO1 HL 128454 & HL 098602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional molecular complexes of junctophilin‐2 and caveolin‐1 provide a structural/functional basis for Ca2+‐microdomain formation between BKCa channels and RyRs in vascular smooth muscle cells

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in plasma membrane (PM) and ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) is an essential mechanism for mechanical force regulation in smooth muscle cells (SMCs). Spontaneous Ca2+ release through RyRs (knows as Ca2+ sparks) and subsequent BKCa channels activation detected as spontaneous transient outward currents (STOCs) occur within PM-SR junctional sites, often called Ca2+ microdomains. We have previously reported that Caveolae, W-shaped invaginations of PM, accumulate BKCa channels within the structure and enhance coupling efficiency between Ca2+ sparks and BKCa channels/STOC activity in mouse mesenteric artery SMCs (mMASMCs). However, the molecular basis between caveolae and RyRs in SR membrane is unclear. In the present study, we demonstrated that both caveolin-1 (Cav1), a caveola forming protein, and junctophilin-2 (JP2), a bridging protein between PM and SR, reciprocally contribute to Ca2+microdomain formation. Co-immunoprecipitation using rat mesenteric arterial tissues and double-immunocytochemical staining analyses by total internal reflection fluorescent microscopy revealed the direct interaction between JP2 and Cav1. Mouse mesenteric arterial tissues were organ-cultured and concomitantly treated with siRNA using reversible permeabilization procedures. Knockdown of JP2 in mMASMCs significantly decreased the colocalization of Cav1 and RyRs and the amplitude of STOCs. The contraction induced by the inhibition of BKCa channels/STOC activity and subsequent membrane depolarization in siJP2-treated mMASMCs was significantly smaller than that in siControl. These results suggested that the novel interaction of JP2 with Cav1 seems to provide a structural/functional basis for Ca2+-mediated crosstalk between RyRs and BKCa channels, and likely positions the PM-SR junctional sites to convert Ca2+ spark signals into hyperpolarizing signals in SMCs lacking transverse tubular system. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The inhibitory effect of BKCa channels induced by autoantibodies against angiotensin II type 1 receptor is independent of AT1R.

Autoantibodies against angiotensin II Type 1 receptor (AT1-AA) are routinely detected in the serum of preeclampsia patients, which results in an increase in vascular tone and an elevation in intracellular calcium concentration of rat vascular smooth muscle (VSM). The big conductance calcium-activated potassium channels (BKCa channels) account for the dominant outward currents in VSMCs, contributing to membrane hyperpolarization and vasodilation. In the present study, we investigated the effect of AT1-AA on BKCa channels. A preeclampsia model was established by passively immunizing healthy pregnant BALB/c mice with AT1-AA extracted from hybridoma culture supernatant. Blood pressure, serum AT1-AA levels, and urinary protein were measured in the immunized mice. BKCa channel expression was detected using qRT-PCR and immunohistochemical technique. The patch-clamp technique was used to record the single currents of BKCa channels in the HEK293T cells that had been transfected. AT1-AA immunized mice exhibited elevated AT1-AA and urinary protein levels compared with mice of the vehicle group. Systolic blood pressure was also increased in the immunized group. BKCa channel β1-subunit expression was reduced in the mesenteric arteries of immunized mice. AT1-AA could inhibit the BKCa currents and the inhibitory effects were not completely reversed following the application of valsartan, an inhibitor of AT1 receptor. In conclusion, AT1-AA could decrease BKCa expression and inhibit BKCa activity independent of AT1R. These inhibitory effects are likely to be contributory factors in the promotion of increased vascular tone caused by AT1-AA in preeclampsia.

Calcium Spark Activity is Modulated by Perfusion Pressure in Vascular Smooth Muscle of Afferent Arterioles

Myogenic response and tubuloglomerular feedback are the two primary mechanisms for renal blood flow autoregulation by regulating the afferent arteriolar vascular resistance. However, the mechanisms of mechanotransduction in pressure induced myogenic response are not well defined in afferent arterioles. We have previously demonstrated that integrin‐mediated mechanical force increases the occurrence of Ca2+ sparks in freshly isolated renal vascular smooth muscle cells (VSMCs). To test whether generation of Ca2+ sparks is a downstream signal of mechanotransduction in pressure induced myogenic constriction, the relationship between Ca2+ spark frequency and transmural perfusion pressure was investigated in intact VSMCs of afferent arterioles isolated from Sprague‐Dawley rats. Spontaneous Ca2+ sparks were found in VSMCs when afferent arterioles were perfused at 80 mmHg. The Ca2+ spark frequency was significantly increased when perfusion pressure was increased by 40 mmHg. Similar increase of Ca2+ spark frequency was also found when arterioles were stimulated with β1‐integrin activating antibody. Spontaneous membrane current recorded with whole cell perforated‐patch in renal VSMCs indicated that there were more spontaneous transient inward currents (STICs) than spontaneous transient outward currents (STOCs) when holding potential was set close to physiological resting membrane potential. These observations indicated that Ca2+ sparks activity might couple to Ca2+‐activated Cl− (ClCa) channel and trigger pressure induced myogenic constriction via membrane depolarization. Real‐time PCR and immunofluorescence confirmed the presence of the ClCa channel TMEM16A in renal VSMCs. Inhibition of TMEM16A with the specific antagonist T16Ainh‐A01 impaired pressure induced myogenic contraction in the perfused afferent arterioles. It is known that myogenic response is enhanced in spontaneously hypertensive rat (SHR). TMEM16A abundance and spontaneous Ca2+ sparks were more prevalent in renal arterioles of SHR than those of Sprague‐Dawley rats, which are consistent with notion that Ca2+ spark activity is part of the signal transduction process in myogenic response. In conclusion, Ca2+ sparks were detected in intact VSMCs of afferent arterioles for the first time and their frequencies were positively modulated by the perfusion pressure. The coupling Ca2+ sparks to ClCa might contribute to the mechanical signal transduction process in pressure induced myogenic constriction.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic modulation of the Xenopus laevis tadpole spinal mechanosensory pathway

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in spinal functions like locomotion. In Xenopus laevis tadpoles at two days old, a model vertebrate for motor control research, we investigated the role of mAChRs in the skin mechanosensory pathway. We found that mAChR activation by carbachol did not affect the sensory Rohon-Beard neuron properties. However, carbachol could hyperpolarise sensory interneurons and decrease their voltage responses to outward currents. Carbachol could increase the threshold for the mechanosensory pathway to start swimming, preventing the initiation of swimming at higher concentrations altogether. Recording from the sensory interneurons in carbachol showed that their spiking after skin stimulation was depressed. However, the general muscarinic antagonist atropine did not have a clear effect on the swimming threshold or the modulation of sensory interneuron membrane conductance. Our results suggest the skin mechanosensory pathway may be subject to muscarinic modulation in this simple vertebrate system.

Negative regulation of cellular Ca2+ mobilization by ryanodine receptor type 3 in mouse mesenteric artery smooth muscle.

Physiological functions of type 3 ryanodine receptors (RyR3) in smooth muscle (SM) tissues are not well understood, in spite of their wide expression. However, the short isoform of RyR3 is known to be a dominant-negative variant (DN-RyR3), which may negatively regulate functions of both RyR2 and full-length (FL) RyR3 by forming hetero-tetramers. Here, functional roles of RyR3 in the regulation of Ca2+ signaling in mesenteric artery SM cells (MASMCs) were examined using RyR3 homozygous knockout mice (RyR3-/-). Quantitative PCR analyses suggested that the predominant RyR3 subtype in MASMs from wild-type mice (RyR3+/+) was DN-RyR3. In single MASMCs freshly isolated from RyR3-/-, the EC50 of caffeine to induce Ca2+ release was lower than that in RyR3+/+ myocytes. The amplitude and frequency of Ca2+ sparks and spontaneous transient outward currents in MASMCs from RyR3-/- were all larger than those from RyR3+/+. Importantly, mRNA and functional expressions of voltage-dependent Ca2+ channel and large-conductance Ca2+-activated K+ (BK) channel in MASMCs from RyR3-/- were identical to those from RyR3+/+. However, in the presence of BK channel inhibitor, paxilline, the pressure rises induced by BayK8644 in MA vascular beds of RyR3-/- were significantly larger than in those of RyR3+/+. This indicates that the negative feedback effects of BK channel activity on intracellular Ca2+ signaling was enhanced in RyR3-/-. Thus, RyR3, and, in fact, mainly DN-RyR3, via a complex with RyR2 suppresses Ca2+ release and indirectly regulated membrane potential by reducing BK channel activity in MASMCs and presumably can affect the regulation of intrinsic vascular tone.

KV4.3 Expression Modulates NaV1.5 Sodium Current.

In cardiomyocytes, the voltage-gated transient outward potassium current (Ito) is responsible for the phase-1 repolarization of the action potential (AP). Gain-of-function mutations in KCND3, the gene encoding the Ito carrying KV4.3 channel, have been associated with Brugada syndrome (BrS). While the role of Ito in the pro-arrhythmic mechanism of BrS has been debated, recent studies have suggested that an increased Ito may directly affect cardiac conduction. However, the effects of an increased Ito on AP upstroke velocity or sodium current at the cellular level remain unknown. We here investigated the consequences of KV4.3 overexpression on NaV1.5 current and consequent sodium channel availability. We found that overexpression of KV4.3 protein in HEK293 cells stably expressing NaV1.5 (HEK293-NaV1.5 cells) significantly reduced NaV1.5 current density without affecting its kinetic properties. In addition, KV4.3 overexpression decreased AP upstroke velocity in HEK293-NaV1.5 cells, as measured with the alternating voltage/current clamp technique. These effects of KV4.3 could not be explained by alterations in total NaV1.5 protein expression. Using computer simulations employing a multicellular in silico model, we furthermore demonstrate that the experimentally observed increase in KV4.3 current and concurrent decrease in NaV1.5 current may result in a loss of conduction, underlining the potential functional relevance of our findings. This study gives the first proof of concept that KV4.3 directly impacts on NaV1.5 current. Future studies employing appropriate disease models should explore the potential electrophysiological implications in (patho)physiological conditions, including BrS associated with KCND3 gain-of-function mutations.

Complex electrophysiological remodeling in postinfarction ischemic heart failure

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl- current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

Plaque-dependent morphological and electrophysiological heterogeneity of microglia in an Alzheimer's disease mouse model.

Microglia, the central nervous system resident innate immune cells, cluster around Aβ plaques in Alzheimer's disease (AD). The activation phenotype of these plaque-associated microglial cells, and their differences to microglia distant to Aβ plaques, are incompletely understood. We used novel three-dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aβ plaques. We found strong morphological changes exclusively in plaque-associated microglia, whereas plaque-distant microglia showed only minor changes. In addition, patch-clamp recordings of microglia in acute cerebral slices of TgCRND8 mice revealed increased K+ currents in plaque-associated but not plaque-distant microglia. Within the subgroup of plaque-associated microglia, two different current profiles were detected. One subset of cells displayed only increased inward currents, while a second subset showed both increased inward and outward currents, implicating that the plaque microenvironment differentially impacts microglial ion channel expression. Using pharmacological channel blockers, multiplex single-cell PCR analysis and RNA fluorescence in situ hybridization, we identified Kir and Kv channel types contributing to the in- and outward K+ conductance in plaque-associated microglia. In summary, we have identified a previously unrecognized level of morphological and electrophysiological heterogeneity of microglia in relation to amyloid plaques, suggesting that microglia may display multiple activation states in AD.

Reactive Oxygen Species Mediate the Suppression of Arterial Smooth Muscle T-type Ca2+ Channels by Angiotensin II

Vascular T-type Ca2+ channels (CaV3.1 and CaV3.2) play a key role in arterial tone development. This study investigated whether this conductance is a regulatory target of angiotensin II (Ang II), a vasoactive peptide that circulates and which is locally produced within the arterial wall. Patch clamp electrophysiology performed on rat cerebral arterial smooth muscle cells reveals that Ang II (100 nM) inhibited T-type currents through AT1 receptor activation. Blocking protein kinase C failed to eliminate channel suppression, a finding consistent with unique signaling proteins enabling this response. In this regard, inhibiting NADPH oxidase (Nox) with apocynin or ML171 (Nox1 selective) abolished channel suppression highlighting a role for reactive oxygen species (ROS). In the presence of Ni2+ (50 µM), Ang II failed to modulate the residual T-type current, an observation consistent with this peptide targeting CaV3.2. Selective channel suppression by Ang II impaired the ability of CaV3.2 to alter spontaneous transient outward currents or vessel diameter. Proximity ligation assay confirmed Nox1 colocalization with CaV3.2. In closing, Ang II targets CaV3.2 channels via a signaling pathway involving Nox1 and the generation of ROS. This unique regulatory mechanism alters BKCa mediated feedback giving rise to a “constrictive” phenotype often observed with cerebrovascular disease.

Down-regulation of Inwardly Rectifying K+ Currents in Astrocytes Derived from Patients with Monge’s Disease

Chronic mountain sickness (CMS) or Monge’s disease is a disease in highlanders. These patients have a variety of neurologic symptoms such as migraine, mental fatigue, confusion, dizziness, loss of appetite, memory loss and neuronal degeneration. The cellular and molecular mechanisms underlying CMS neuropathology is not understood. In the previous study, we demonstrated that neurons derived from CMS patients’ fibroblasts have a decreased expression and altered gating properties of voltage-gated sodium channel. In this study, we further characterize the electrophysiological properties of iPSC-derived astrocytes from CMS patients. We found that the current densities of the inwardly rectifying potassium (Kir) channels in CMS astrocytes (−5.7 ± 2.2 pA/pF at −140 mV) were significantly decreased as compared to non-CMS (−28.4 ± 3.4 pA/pF at −140 mV) and sea level subjects (−28.3 ± 5.3 pA/pF at −140 mV). We further demonstrated that the reduced Kir current densities in CMS astrocytes were caused by their decreased protein expression of Kir4.1 and Kir2.3 channels, while single channel properties (i.e., Po, conductance) of Kir channel in CMS astrocytes were not altered. In addition, we found no significant differences of outward potassium currents between CMS and non-CMS astrocytes. As compared to non-CMS and sea level subjects, the K+ uptake ability in CMS astrocytes was significantly decreased. Taken together, our results suggest that down-regulation of Kir channels and the resulting decreased K+ uptake ability in astrocytes could be one of the major molecular mechanisms underlying the neurologic manifestations in CMS patients.