A molecular complex of Cav1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation-transcription coupling.

Elevation of intracellular Ca2+ concentration ([Ca2+]i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as excitation­transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts [Ca2+]i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.

Caveolin-1 forms a complex with P2X7 receptor and tunes P2X7-mediated ATP signaling in mouse bone marrow-derived macrophages.

The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger proinflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca2+] and sustained decrease in cytosolic [K+]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca2+] increase and cytosolic [K+] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca2+ influx and K+ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macropore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1ß secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these datasets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1ß secretion and pyroptosis.NEW & NOTEWORTHY In bone marrow-derived macrophages, Cav-1 suppresses the macropore formation of P2X7 receptors through their direct or indirect interactions, resulting in reduced membrane permeability of cations (Ca2+ and K+) and large cell-impermeable dye (TO-PRO3) induced by ATP. Cav-1 also inhibits ATP-induced IL-1ß secretion, ROS production, GSDMD cleavage, and pyroptosis. Cav-1 contributes to the maintenance of proper immune responses by finely tuning IL-1ß secretion and cell death in macrophages.

KCa3.1 regulates cell cycle progression by modulating Ca2+ signaling in murine preosteoblasts.

Osteoblasts synthesize and deposit essential components of the extracellular bone matrix and collagen scaffolds, leading to mineralized bone formation. Therefore, the proliferation of preosteoblasts (precursors of mature osteoblasts) helps in regulating skeletal homeostasis. This study demonstrated that the functional expression of KCa3.1, an intermediate-conductance Ca2+-activated K+ channel, is markedly upregulated in murine preosteoblastic MC3T3-E1 cells in the G0/G1 phase. The enhancement of KCa3.1 is involved in the establishment of more negative membrane potentials in MC3T3-E1 cells. This hyperpolarization can promote intracellular Ca2+ signaling because store-operated Ca2+ channels are activated. Treatment with TRAM-34, a specific KCa3.1 inhibitor, attenuated the cell cycle progression from the G0/G1 phase to the S/G2/M phases. In MC3T3-E1 cells, KCa3.1 significantly promoted the transition from the G1 phase to the S phase. KCa3.1 inhibition also caused G0 phase cell accumulation. Furthermore, TRAM-34 decreased the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin, osteoblast differentiation markers in MC3T3-E1 cells, and inhibited the endochondral ossification of murine metatarsals. These results reveal novel ways by which KCa3.1 activity can strongly modulate osteoblast maturation during bone formation.

A computational method for identifying an optimal combination of existing drugs to repair the action potentials of SQT1 ventricular myocytes.

Mutations are known to cause perturbations in essential functional features of integral membrane proteins, including ion channels. Even restricted or point mutations can result in substantially changed properties of ion currents. The additive effect of these alterations for a specific ion channel can result in significantly changed properties of the action potential (AP). Both AP shortening and AP prolongation can result from known mutations, and the consequences can be life-threatening. Here, we present a computational method for identifying new drugs utilizing combinations of existing drugs. Based on the knowledge of theoretical effects of existing drugs on individual ion currents, our aim is to compute optimal combinations that can 'repair' the mutant AP waveforms so that the baseline AP-properties are restored. More specifically, we compute optimal, combined, drug concentrations such that the waveforms of the transmembrane potential and the cytosolic calcium concentration of the mutant cardiomyocytes (CMs) becomes as similar as possible to their wild type counterparts after the drug has been applied. In order to demonstrate the utility of this method, we address the question of computing an optimal drug for the short QT syndrome type 1 (SQT1). For the SQT1 mutation N588K, there are available data sets that describe the effect of various drugs on the mutated K+ channel. These published findings are the basis for our computational analysis which can identify optimal compounds in the sense that the AP of the mutant CMs resembles essential biomarkers of the wild type CMs. Using recently developed insights regarding electrophysiological properties among myocytes from different species, we compute optimal drug combinations for hiPSC-CMs, rabbit ventricular CMs and adult human ventricular CMs with the SQT1 mutation. Since the 'composition' of ion channels that form the AP is different for the three types of myocytes under consideration, so is the composition of the optimal drug.

Frequency-Dependent Properties of the Hyperpolarization-Activated Cation Current, If, in Adult Mouse Heart Primary Pacemaker Myocytes.

A number of distinct electrophysiological mechanisms that modulate the myogenic spontaneous pacemaker activity in the sinoatrial node (SAN) of the mammalian heart have been investigated extensively. There is agreement that several (3 or 4) different transmembrane ionic current changes (referred to as the voltage clock) are involved; and that the resulting net current interacts with direct and indirect effects of changes in intracellular Ca2+ (the calcium clock). However, significant uncertainties, and important knowledge gaps, remain concerning the functional roles in SAN spontaneous pacing of many of the individual ion channel- or exchanger-mediated transmembrane current changes. We report results from patch clamp studies and mathematical modeling of the hyperpolarization-activated current, If, in the generation/modulation of the diastolic depolarization, or pacemaker potential, produced by individual myocytes that were enzymatically isolated from the adult mouse sinoatrial node (SAN). Amphotericin-mediated patch microelectrode recordings at 35 °C were made under control conditions and in the presence of 5 or 10 nM isoproterenol (ISO). These sets of results were complemented and integrated with mathematical modeling of the current changes that take place in the range of membrane potentials (-70 to -50 mV), which corresponds to the 'pacemaker depolarization' in the adult mouse SAN. Our results reveal a very small, but functionally important, approximately steady-state or time-independent current generated by residual activation of If channels that are expressed in these pacemaker myocytes. Recordings of the pacemaker depolarization and action potential, combined with measurements of changes in If, and the well-known increases in the L-type Ca2+ current, ICaL, demonstrated that ICaL activation, is essential for myogenic pacing. Moreover, after being enhanced (approximately 3-fold) by 5 or 10 nM ISO, ICaL contributes significantly to the positive chronotropic effect. Our mathematical model has been developed in an attempt to better understand the underlying mechanisms for the pacemaker depolarization and action potential in adult mouse SAN myocytes. After being updated with our new experimental data describing If, our simulations reveal a novel functional component of If in adult mouse SAN. Computational work carried out with this model also confirms that in the presence of ISO the residual activation of If and opening of ICaL channels combine to generate a net current change during the slow diastolic depolarization phase that is essential for the observed accelerated pacemaking rate of these SAN myocytes.

Mechanisms of flecainide induced negative inotropy: An in silico study.

It is imperative to develop better approaches to predict how antiarrhythmic drugs with multiple interactions and targets may alter the overall electrical and/or mechanical function of the heart. Safety Pharmacology studies have provided new insights into the multi-target effects of many different classes of drugs and have been aided by the addition of robust new in vitro and in silico technology. The primary focus of Safety Pharmacology studies has been to determine the risk profile of drugs and drug candidates by assessing their effects on repolarization of the cardiac action potential. However, for decades experimental and clinical studies have described substantial and potentially detrimental effects of Na+ channel blockers in addition to their well-known conduction slowing effects. One such side effect, associated with administration of some Na+ channel blocking drugs is negative inotropy. This reduces the pumping function of the heart, thereby resulting in hypotension. Flecainide is a well-known example of a Na+ channel blocking drug, that exhibits strong rate-dependent block of INa and may cause negative cardiac inotropy. While the phenomenon of Na+ channel suppression and resulting negative inotropy is well described, the mechanism(s) underlying this effect are not. Here, we set out to use a modeling and simulation approach to reveal plausible mechanisms that could explain the negative inotropic effect of flecainide. We utilized the Grandi-Bers model [1] of the cardiac ventricular myocyte because of its robust descriptions of ion homeostasis in order to characterize and resolve the relative effects of QRS widening, flecainide off-target effects and changes in intracellular Ca2+ and Na+ homeostasis. The results of our investigations and predictions reconcile multiple data sets and illustrate how multiple mechanisms may play a contributing role in the flecainide induced negative cardiac inotropic effect.

Swelling-activated ClC-3 activity regulates prostaglandin E2 release in human OUMS-27 chondrocytes.

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl- currents (ICl,swell) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, ICl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl- channel blockers, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca2+ influx and subsequent release of prostaglandin E2 (PGE2) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for ICl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE2 release.

Physiological Roles of the Rapidly Activated Delayed Rectifier K+ Current in Adult Mouse Heart Primary Pacemaker Activity.

Robust, spontaneous pacemaker activity originating in the sinoatrial node (SAN) of the heart is essential for cardiovascular function. Anatomical, electrophysiological, and molecular methods as well as mathematical modeling approaches have quite thoroughly characterized the transmembrane fluxes of Na+, K+ and Ca2+ that produce SAN action potentials (AP) and 'pacemaker depolarizations' in a number of different in vitro adult mammalian heart preparations. Possible ionic mechanisms that are responsible for SAN primary pacemaker activity are described in terms of: (i) a Ca2+-regulated mechanism based on a requirement for phasic release of Ca2+ from intracellular stores and activation of an inward current-mediated by Na+/Ca2+ exchange; (ii) time- and voltage-dependent activation of Na+ or Ca2+ currents, as well as a cyclic nucleotide-activated current, If; and/or (iii) a combination of (i) and (ii). Electrophysiological studies of single spontaneously active SAN myocytes in both adult mouse and rabbit hearts consistently reveal significant expression of a rapidly activating time- and voltage-dependent K+ current, often denoted IKr, that is selectively expressed in the leading or primary pacemaker region of the adult mouse SAN. The main goal of the present study was to examine by combined experimental and simulation approaches the functional or physiological roles of this K+ current in the pacemaker activity. Our patch clamp data of mouse SAN myocytes on the effects of a pharmacological blocker, E4031, revealed that a rapidly activating K+ current is essential for action potential (AP) repolarization, and its deactivation during the pacemaker potential contributes a small but significant component to the pacemaker depolarization. Mathematical simulations using a murine SAN AP model confirm that well known biophysical properties of a delayed rectifier K+ current can contribute to its role in generating spontaneous myogenic activity.

Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells.

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and ß-subunit (BKß1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.

Transient outward K+ current can strongly modulate action potential duration and initiate alternans in the human atrium.

Efforts to identify the mechanisms for the initiation and maintenance of human atrial fibrillation (AF) often focus on changes in specific elements of the atrial "substrate," i.e., its electrophysiological properties and/or structural components. We used experimentally validated mathematical models of the human atrial myocyte action potential (AP), both at baseline in sinus rhythm (SR) and in the setting of chronic AF, to identify significant contributions of the Ca2+-independent transient outward K+ current ( Ito) to electrophysiological instability and arrhythmia initiation. First, we explored whether changes in the recovery or restitution of the AP duration (APD) and/or its dynamic stability (alternans) can be modulated by Ito. Recent reports have identified disease-dependent spatial differences in expression levels of the specific K+ channel α-subunits that underlie Ito in the left atrium. Therefore, we studied the functional consequences of this by deletion of 50% of native Ito (Kv4.3) and its replacement with Kv1.4. Interestingly, significant changes in the short-term stability of the human atrial AP waveform were revealed. Specifically, this K+ channel isoform switch produced discontinuities in the initial slope of the APD restitution curve and appearance of APD alternans. This pattern of in silico results resembles some of the changes observed in high-resolution clinical electrophysiological recordings. Important insights into mechanisms for these changes emerged from known biophysical properties (reactivation kinetics) of Kv1.4 versus those of Kv4.3. These results suggest new approaches for pharmacological management of AF, based on molecular properties of specific K+ isoforms and their changed expression during progressive disease. NEW & NOTEWORTHY Clinical studies identify oscillations (alternans) in action potential (AP) duration as a predictor for atrial fibrillation (AF). The abbreviated AP in AF also involves changes in K+ currents and early repolarization of the AP. Our simulations illustrate how substitution of Kv1.4 for the native current, Kv4.3, alters the AP waveform and enhances alternans. Knowledge of this "isoform switch" and related dynamics in the AF substrate may guide new approaches for detection and management of AF.

ATP increases [Ca2+ ]i and activates a Ca2+ -dependent Cl- current in rat ventricular fibroblasts.

NEW FINDINGS: What is the central question of this study? Although electrophysiological and biophysical characteristics of heart fibroblasts have been studied in detail, their responses to prominent paracrine agents in the myocardium have not been addressed adequately. Our experiments characterize changes in cellular electrophysiology and intracellular calcium in response to ATP. What is the main finding and its importance? In rat ventricular fibroblasts maintained in cell culture, we find that ATP activates a specific subset of Ca2+ -activated Cl- channels as a consequence of binding to P2Y purinoceptors and then activating phospholipase C. This response is not dependent on [Ca2+ ]o but requires an increase in [Ca2+ ]i and is modulated by the type of nucleotide that is the purinergic agonist. ABSTRACT: Effects of ATP on enzymatically isolated rat ventricular fibroblasts maintained in short-term (36-72 h) cell culture were examined. Immunocytochemical staining of these cells revealed that a fibroblast, as opposed to a myofibroblast, phenotype was predominant. ATP, ADP or uridine 5'-triphosphate (UTP) all produced large increases in [Ca2+ ]i . Voltage-clamp studies (amphotericin-perforated patch) showed that ATP (1-100 µm) activated an outwardly rectifying current, with a reversal potential very close to the Nernst potential for Cl- . In contrast, ADP was much less effective, and UTP produced no detectable current. The non-selective Cl- channel blockers niflumic acid, DIDS and NPPB (each at 100 µm), blocked the responses to 100 µm ATP. An agonist for P2Y purinoceptors, 2-MTATP, activated a very similar outwardly rectifying C1- current. The P2Y receptor antagonists, suramin and PPADS (100 µm each), significantly inhibited the Cl- current produced by 100 µm ATP. ATP was able to activate this Cl- current when [Ca2+ ]o was removed, but not when [Ca2+ ]i was buffered with BAPTA-AM. In the presence of the phospholipase C inhibitor U73122, this Cl- current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors and for the Ca2+ -activated Cl- channel, TMEM16F (also denoted ANO6). In summary, these results demonstrate that activation of P2Y receptors by ATP causes a phospholipase C-dependent increase in [Ca2+ ]i , followed by activation of a Ca2+ -dependent Cl- current in rat ventricular fibroblasts.

A New Splice Variant of Large Conductance Ca2+-activated K+ (BK) Channel α Subunit Alters Human Chondrocyte Function.

Large conductance Ca2+-activated K+ (BK) channels play essential roles in both excitable and non-excitable cells. For example, in chondrocytes, agonist-induced Ca2+ release from intracellular store activates BK channels, and this hyperpolarizes these cells, augments Ca2+ entry, and forms a positive feed-back mechanism for Ca2+ signaling and stimulation-secretion coupling. In the present study, functional roles of a newly identified splice variant in the BK channel α subunit (BKαΔe2) were examined in a human chondrocyte cell line, OUMS-27, and in a HEK293 expression system. Although BKαΔe2 lacks exon2, which codes the intracellular S0-S1 linker (Glu-127-Leu-180), significant expression was detected in several tissues from humans and mice. Molecular image analyses revealed that BKαΔe2 channels are not expressed on plasma membrane but can traffic to the plasma membrane after forming hetero-tetramer units with wild-type BKα (BKαWT). Single-channel current analyses demonstrated that BKα hetero-tetramers containing one, two, or three BKαΔe2 subunits are functional. These hetero-tetramers have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo-tetramers in a stoichiometry-dependent manner. Site-directed mutagenesis of residues in exon2 identified Helix2 and the linker to S1 (Trp-158-Leu-180, particularly Arg-178) as an essential segment for channel function including voltage dependence and trafficking. BKαΔe2 knockdown in OUMS-27 chondrocytes increased BK current density and augmented the responsiveness to histamine assayed as cyclooxygenase-2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as osteoarthritis.

A computational model predicts adjunctive pharmacotherapy for cardiac safety via selective inhibition of the late cardiac Na current.

BACKGROUND: The QT interval is a phase of the cardiac cycle that corresponds to action potential duration (APD) including cellular repolarization (T-wave). In both clinical and experimental settings, prolongation of the QT interval of the electrocardiogram (ECG) and related proarrhythmia have been so strongly associated that a prolonged QT interval is largely accepted as surrogate marker for proarrhythmia. Accordingly, drugs that prolong the QT interval are not considered for further preclinical development resulting in removal of many promising drugs from development. While reduction of drug interactions with hERG is an important goal, there are promising means to mitigate hERG block. Here, we examine one possibility and test the hypothesis that selective inhibition of the cardiac late Na current (INaL) by the novel compound GS-458967 can suppress proarrhythmic markers. METHODS AND RESULTS: New experimental data has been used to calibrate INaL in the Soltis-Saucerman computationally based model of the rabbit ventricular action potential to study effects of GS-458967 on INaL during the rabbit ventricular AP. We have also carried out systematic in silico tests to determine if targeted block of INaL would suppress proarrhythmia markers in ventricular myocytes described by TRIaD: Triangulation, Reverse use dependence, beat-to-beat Instability of action potential duration, and temporal and spatial action potential duration Dispersion. CONCLUSIONS: Our computer modeling approach based on experimental data, yields results that suggest that selective inhibition of INaL modifies all TRIaD related parameters arising from acquired Long-QT Syndrome, and thereby reduced arrhythmia risk. This study reveals the potential for adjunctive pharmacotherapy via targeted block of INaL to mitigate proarrhythmia risk for drugs with significant but unintended off-target hERG blocking effects.

The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes.

Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl(-) and an associated decrease of cell volume. We have previously reported that a Cl(-) conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca(2+) concentration ([Ca(2+)]i) in human chondrocytes. The molecular identity and pathologic function of these Cl(-) channels, however, remained to be determined. Here, we show that the ClC-7 Cl(-) channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl(-) currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; IC50 = 13 µM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca(2+)]i. ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca(2+)]i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

A computational modelling approach combined with cellular electrophysiology data provides insights into the therapeutic benefit of targeting the late Na+ current.

KEY POINTS: The ventricular action potential plateau is a phase of high resistance, which makes ventricular myocytes vulnerable to small electrical perturbations. We developed a computationally based model of GS-458967 interaction with the cardiac Na+ channel, informed by experimental data recorded from guinea pig isolated single ventricular myocytes. The model predicts that the therapeutic potential of GS-458967 derives largely from the designed property of significant potent selectivity for INaL. ABSTRACT: Selective inhibition of the slowly inactivating or late Na(+) current (INaL) in patients with inherited or acquired arrhythmia syndrome may confer therapeutic benefit by reducing the incidence of triggers for arrhythmia and suppressing one component of arrhythmia-promoting cardiac substrates (e.g. prolonged refractoriness and spatiotemporal dispersion of action potential duration). Recently, a novel compound that preferentially and potently reduces INaL, GS-458967 (IC50 for block of INaL = 130 nM) has been studied. Experimental measurements of the effects of GS-458967 on endogenous INaL in guinea pig ventricular myocytes demonstrate a robust concentration-dependent reduction in action potential duration (APD). Using experimental data to calibrate INaL and the rapidly activating delayed rectifier K(+) current, IKr, in the Faber-Rudy computationally based model of the guinea pig ventricular action potential, we simulated effects of GS-458967 on guinea pig ventricular APD. GS-458967 (0.1 µM) caused a 28.67% block of INaL and 12.57% APD shortening in experiments, while the model predicted 10.06% APD shortening with 29.33% block of INaL. An additional effect of INaL block is to reduce the time during which the membrane potential is in a high resistance state (i.e. the action potential plateau). To test the hypothesis that targeted block of INaL would make ventricular myocytes less susceptible to small electrical perturbations, we used the computational model to test the degree of APD prolongation induced by small electrical perturbations in normal cells and in cells with simulated long QT syndrome. The model predicted a substantial dose-dependent reduction in sensitivity to small electrical perturbations as evidenced by action potential duration at 90% repolarization variability in the presence of GS-458967-induced INaL block. This effect was especially potent in the 'disease setting' of inherited long QT syndrome. Using a combined experimental and theoretical approach, our results suggest that INaL block is a potent therapeutic strategy. This is because reduction of INaL stabilizes the action potential waveform by reducing depolarizing current during the plateau phase of the action potential. This reduces the most vulnerable phase of the action potential with high membrane resistance. In summary, by reducing the sensitivity of the myocardial substrate to small electrical perturbations that promote arrhythmia triggers, agents such as GS-458967 may constitute an effective antiarrhythmic pharmacological strategy.

Ion channels in pediatric CNS Atypical Teratoid/Rhabdoid Tumor (AT/RT) cells: potential targets for novel therapeutic agents.

The central nervous system Atypical Teratoid/Rhabdoid Tumor (CNS AT/RT) is a highly malignant neoplasm that commonly affects infants and young children, and has an extremely poor prognosis. Recently, a small subset of ion channels have been found to be over-expressed in a variety of malignant cells, thus emerging as potential therapeutic targets for difficult to treat tumors. We have studied the electrophysiological properties of AT/RT cell lines with particular attention to cell volume sensitive ion channels (VSC). This class of membrane proteins can play a fundamental role in cellular processes relevant to tumor development. We have found that chloride selective VSCs are particularly active in AT/RT cell lines, compared to non-tumor cells. We evaluated specific inhibitors for activity against chloride selective VSCs and consequently for their ability to inhibit the growth and survival of AT/RT cells in vitro. The results demonstrated that the extent of volume sensitive membrane current inhibition by these agents was correlated with their potency in AT/RT cell growth inhibition in vitro. In addition, we showed that ion channel inhibition enhanced the activity of certain anti-neoplastic agents, suggesting its value in effective drug combination protocols. Results presented provide preliminary in vitro data for possible evaluation of distinct ion channels as plausible therapeutic targets in the treatment of AT/RT.

Arrhythmogenic influence of mutations in a myocyte-based computational model of the pulmonary vein sleeve.

In the heart, electrophysiological dysregulation arises from defects at many biological levels (from point mutations in ion channel proteins to gross structural abnormalities). These defects disrupt the normal pattern of electrical activation, producing ectopic activity and reentrant arrhythmia. To interrogate mechanisms that link these primary biological defects to macroscopic electrophysiologic dysregulation most prior computational studies have utilized either (i) detailed models of myocyte ion channel dynamics at limited spatial scales, or (ii) homogenized models of action potential conduction that reproduce arrhythmic activity at tissue and organ levels. Here we apply our recent model (EMI), which integrates electrical activation and propagation across these scales, to study human atrial arrhythmias originating in the pulmonary vein (PV) sleeves. These small structures initiate most supraventricular arrhythmias and include pronounced myocyte-to-myocyte heterogeneities in ion channel expression and intercellular coupling. To test EMI's cell-based architecture in this physiological context we asked whether ion channel mutations known to underlie atrial fibrillation are capable of initiating arrhythmogenic behavior via increased excitability or reentry in a schematic PV sleeve geometry. Our results illustrate that EMI's improved spatial resolution can directly interrogate how electrophysiological changes at the individual myocyte level manifest in tissue and as arrhythmia in the PV sleeve.

The T-type Calcium Channel Cav3.1 in Y79 Retinoblastoma Cells is Regulated by the Epidermal Growth Factor Receptor via the MAPK Signaling Pathway.

PURPOSE: Retinoblastoma is the most frequent intraocular cancer in children. It is also one of the most common causes for enucleation and carries a significant morbidity rate in affected individuals. Hence, studies on its pathophysiological and growth regulatory mechanisms are urgently needed to identify more effective novel therapeutics. METHODS: Using the Y79 retinoblastoma cell line, we investigated the electrophysiological and functional activities of the T-type voltage-gated calcium channel Cav3.1, that is constitutively expressed in these cells. We also analyzed the Akt and MAPK signaling pathways downstream of the epidermal growth factor receptor (EGFR) to understand the mechanism responsible for the inhibition of Cav3.1. RESULTS: We demonstrate that the EGFR inhibitor Afatinib significantly reduced cell viability and Cav3.1 mRNA expression and electrophysiological activity. At low concentrations (1 µM), Afatinib reduced the amplitude of Cav3.1 current density, whereas at a high concentration (10 µM), it completely abolished the voltage-gated calcium current. Our results show that inhibition of the MAPK pathway by a specific inhibitor VX-11e affected the Cav3.1 current in a dose-dependent manner. VX-11e (50 nM-1 µM) treatment reduced Cav3.1 current densities in Y79 cells, with complete abolishment of Cav3.1 current at higher concentrations (5 µM). We also demonstrate that the specific inhibition of the Akt kinase (using MK-2206) had no effect on the Cav3.1 currents. CONCLUSION: Our study provides a functional relationship between the MAPK pathway and EGFR signaling and indicates that the MAPK signaling pathway mediates the control of Cav3.1 by EGFR in retinoblastoma.

Two-pore domain K⁺ channels regulate membrane potential of isolated human articular chondrocytes.

Potassium channels that regulate resting membrane potential (RMP) of human articular chondrocytes (HACs) of the tibial joint maintained in short-term (0-3 days) non-confluent cell culture were studied using patch-clamp techniques. Quantitative PCR showed that transcripts of genes for two-pore domain K(+) channels (KCNK1, KCNK5 and KCNK6), and 'BK' Ca(2+)-activated K(+) channels (KCNMA1) were abundantly expressed. Immunocytological methods detected α-subunits for BK and K(2p)5.1 (TASK-2) K(+) channels. Electrophysiological recordings identified three distinct K(+) currents in isolated HACs: (i) a voltage- and time-dependent 'delayed rectifier', blocked by 100 nM α-dendrotoxin, (ii) a large 'noisy' voltage-dependent current that was blocked by low concentrations of tetraethylammonium (TEA; 50% blocking dose = 0.15 mM) and iberiotoxin (52% block, 100 nM) and (iii) a voltage-independent 'background' K(+) current that was blocked by acidic pH (5.5-6), was increased by alkaline pH (8.5), and was not blocked by TEA, but was blocked by the local anaesthetic bupivacaine (0.25 mM). The RMP of isolated HACs was very slightly affected by 5 mM TEA, which was sufficient to block both voltage-dependent K(+) currents, suggesting that these currents probably contributed little to maintaining RMP under 'resting' conditions (i.e. low internal [Ca(2+)]). Increases in external K(+) concentration depolarized HACs by 30 mV in response to a 10-fold increase in [K(+)], indicating a significant but not exclusive role for K(+) current in determining RMP. Increases in external [K(+)] in voltage-clamped HACs revealed a voltage-independent K(+) current whose inward current magnitude increased with external [K(+)]. Block of this current by bupivacaine (0.25-1 mM) in 5 and 25 mM external [K(+)] resulted in a large (8-25 mV) depolarization of RMP. The biophysical and pharmacological properties of the background K(+) current, together with expression of mRNA and α-subunit protein for TASK-2, strongly suggest that these two-pore domain K(+) channels contribute significantly to stabilizing the RMP of HACs.