Dominant-negative control of cAMP-dependent IKs upregulation in human long-QT syndrome type 1.

RATIONALE: The mutation A341V in the S6 transmembrane segment of KCNQ1, the α-subunit of the slowly activating delayed-rectifier K(+) (I(Ks)) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death. OBJECTIVE: Several genetic risk modifiers have been identified in A341V patients, but the molecular mechanisms underlying the pronounced repolarization phenotype, particularly during ß-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMP-dependent modulation of I(Ks). METHODS AND RESULTS: We characterized the effects of A341V on the I(Ks) macromolecular channel complex in transfected Chinese hamster ovary cells and found a dominant-negative suppression of cAMP-dependent Yotiao-mediated I(Ks) upregulation on top of a dominant-negative reduction in basal current. Phosphomimetic substitution of the N-terminal position S27 with aspartic acid rescued this loss of upregulation. Western blot analysis showed reduced phosphorylation of KCNQ1 at S27, even for heterozygous A341V, suggesting that phosphorylation defects in some (mutant) KCNQ1 subunits can completely suppress I(Ks) upregulation. Functional analyses of heterozygous KCNQ1 WT:G589D and heterozygous KCNQ1 WT:S27A, a phosphorylation-inert substitution, also showed such suppression. Immunoprecipitation of Yotiao with KCNQ1-A341V (in the presence of KCNE1) was not different from wild-type. CONCLUSIONS: Our results indicate the involvement of the KCNQ1-S6 region at/or around A341 in cAMP-dependent stimulation of I(Ks), a process that is under strong dominant-negative control, suggesting that tetrameric KCNQ1 phosphorylation is required. Specific long-QT1 mutations, including heterozygous A341V, disable this regulation.

Protein kinase C (PKC) activity regulates functional effects of Kvß1.3 subunit on KV1.5 channels: identification of a cardiac Kv1.5 channelosome.

K(v)1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (I(Kur)). The regulatory K(v)ß1.3 subunit converts K(v)1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by K(v)ß1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack K(v)ß subunits) transiently cotransfected with K(v)1.5+K(v)ß1.3 and also rat ventricular and atrial tissue to study native α-ß subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that K(v)1.5 and K(v)ß1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, K(v)ß1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed K(v)ß1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of K(v)1.5 and K(v)ß1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between K(v)1.5, K(v)ß1.3, the receptor for activated C kinase (RACK1), PKCßI, PKCßII, and PKCθ in HEK293 cells. A very similar K(v)1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.

Ceramide inhibits Kv currents and contributes to TP-receptor-induced vasoconstriction in rat and human pulmonary arteries.

Neutral sphingomyelinase (nSMase)-derived ceramide has been proposed as a mediator of hypoxic pulmonary vasoconstriction (HPV), a specific response of the pulmonary circulation. Voltage-gated K(+) (K(v)) channels are modulated by numerous vasoactive factors, including hypoxia, and their inhibition has been involved in HPV. Herein, we have analyzed the effects of ceramide on K(v) currents and contractility in rat pulmonary arteries (PA) and in mesenteric arteries (MA). The ceramide analog C6-ceramide inhibited K(v) currents in PA smooth muscle cells (PASMC). Similar effects were obtained after the addition of bacterial sphingomyelinase (SMase), indicating a role for endogenous ceramide in K(v) channel regulation. K(v) current was reduced by stromatoxin and diphenylphosphine oxide-1 (DPO-1), selective inhibitors of K(v)2.1 and K(v)1.5 channels, respectively. The inhibitory effect of ceramide was still present in the presence of stromatoxin or DPO-1, suggesting that this sphingolipid inhibited both components of the native K(v) current. Accordingly, ceramide inhibited K(v)1.5 and K(v)2.1 channels expressed in Ltk(-) cells. Ceramide-induced effects were reduced in human embryonic kidney 293 cells expressing K(v)1.5 channels but not the regulatory subunit K(v)ß2.1. The nSMase inhibitor GW4869 reduced the thromboxane-endoperoxide receptor agonist U46619-induced, but not endothelin-1-induced pulmonary vasoconstriction that was partly restored after addition of exogenous ceramide. The PKC-ζ pseudosubstrate inhibitor (PKCζ-PI) inhibited the K(v) inhibitory and contractile effects of ceramide. In MA ceramide had no effect on K(v) currents and GW4869 did not affect U46619-induced contraction. The effects of SMase were also observed in human PA. These results suggest that ceramide represents a crucial signaling mediator in the pulmonary vasculature.

Celecoxib blocks cardiac Kv1.5, Kv4.3 and Kv7.1 (KCNQ1) channels: effects on cardiac action potentials.

Celecoxib is a COX-2 inhibitor that has been related to an increased cardiovascular risk and that exerts several actions on different targets. The aim of this study was to analyze the effects of this drug on human cardiac voltage-gated potassium channels (Kv) involved on cardiac repolarization Kv1.5 (I(Kur)), Kv4.3+KChIP2 (I(to1)) and Kv7.1+KCNE1 (I(Ks)) and to compare with another COX-2 inhibitor, rofecoxib. Currents were recorded in transfected mammalian cells by whole-cell patch-clamp. Celecoxib blocked all the Kv channels analyzed and rofecoxib was always less potent, except on Kv4.3+KChIP2 channels. Kv1.5 block increased in the voltage range of channel activation, decreasing at potentials positive to 0 mV. The drug modified the activation curve of the channels that became biphasic. Block was frequency-dependent, increasing at fastest frequencies. Celecoxib effects were not altered by TEA(out) in R487Y mutant Kv1.5 channels but the kinetics of block were slower and the degree of block was smaller with TEA(in), indicating that celecoxib acts from the cytosolic side. We confirmed the blocking properties of celecoxib on native Kv currents from rat vascular cells, where Kv1.5 are the main contributors (IC(50)≈ 7 µM). Finally, we demonstrate that celecoxib prolongs the action potential duration in mouse cardiac myocytes and shortens it in guinea pig cardiac myocytes, suggesting that Kv block induced by celecoxib may be of clinical relevance.

Cell cycle-dependent expression of Kv1.5 is involved in myoblast proliferation.

Voltage-dependent K(+) channels (Kv) are involved in the proliferation of many types of cells, but the mechanisms by which their activity is related to cell growth remain unclear. Kv antagonists inhibit the proliferation of mammalian cells, which is of physiological relevance in skeletal muscle. Although myofibres are terminally differentiated, some resident myoblasts may re-enter the cell cycle and proliferate. Here we report that the expression of Kv1.5 is cell-cycle dependent during myoblast proliferation. In addition to Kv1.5 other Kv, such as Kv1.3, are also up-regulated. However, pharmacological evidence mainly implicates Kv1.5 in myoblast growth. Thus, the presence of S0100176, a Kv antagonist, but not margatoxin and dendrotoxin, led to cell cycle arrest during the G(1)-phase. The use of selective cell cycle blockers showed that Kv1.5 was transiently accumulated during the early G(1)-phase. Furthermore, while myoblasts treated with S0100176 expressed low levels of cyclin A and D(1), the expression of p21(cip-1) and p27(kip1), two cyclin-dependent kinase inhibitors, increased. Our results indicate that the cell cycle-dependent expression of Kv1.5 is involved in skeletal muscle cell proliferation.

Modulation of the atrial specific Kv1.5 channel by the n-3 polyunsaturated fatty acid, alpha-linolenic acid.

Epidemiological, clinical and experimental studies suggest that the cardioprotective effect of fish intake is mainly due to the antiarrhythmic properties of marine n-3 polyunsaturated fatty acids (PUFA), which modulate ion currents. Emerging evidences point to similar effects of alpha-linolenic acid (ALA), a vegetable n-3 PUFA, but much less is known about its effects on the specific cardiac ion channels. Using electrophysiology, protein biochemistry and fluorescence anisotropy measurements, we tested the effects of ALA on the atrial specific Kv1.5 channel. In stably transfected Ltk(-) cells, ALA blocked Kv1.5 channels in a time- and voltage-dependent manner with an IC(50) value of 3.7+/-0.3 microM. ALA at 2.5 microM inhibited the Kv1.5 current, shifted the midpoint of the activation curve by -8.8+/-4.3 mV (p<0.05), accelerated the activation kinetics of Kv1.5 due to a negative shift in its voltage dependency and slowed its deactivation process. Marine n-3 PUFA eicosapentaenoic and docosahexaenoic (EPA and DHA) acids, but not ALA, reduced the steady-state levels of Kv1.5 protein. DHA, but not ALA, increased the cell membrane order parameter. These results demonstrate that ALA directly blocks atria-specific Kv1.5 channels without modifying their expression or the bilayer order. Together, these effects suggest that the antiarrhythmic potential of diets enriched with plant-derived n-3 PUFA result, in part, from direct effects on cardiac ion channels.

Differential regulation of Na(v)beta subunits during myogenesis.

Voltage-gated sodium channels (Na(v)) consist of a pore-forming alpha subunit (Na(v)alpha) associated with beta regulatory subunits (Na(v)beta). Adult skeletal myocytes primarily express Na(v)1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na(v)1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na(v)1.5 currents than those recorded from myoblasts. Unlike myoblasts, I(Na) recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na(v)beta subunits modulate channel gating and pharmacology, the goal of the present work was to study Na(v)beta subunits during myogenesis. All four Na(v)beta (Na(v)beta1-4) isoforms were present in L6E9 myocytes. While Na(v)beta1-3 subunits were up-regulated by myogenesis, Na(v)beta4 subunits were not. These results show that Na(v)beta genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na(+) channels during development and myotube formation.

Irvalec inserts into the plasma membrane causing rapid loss of integrity and necrotic cell death in tumor cells.

Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca(2+) influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn(2+). Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.

Kv1.5-Kv beta interactions: molecular determinants and pharmacological consequences.

Kv1.5 channels are homotetramers of alpha-pore subunits mainly present in human atrium and pulmonary vasculature. Thus, Kv1.5 is a pharmacological target for cardiovascular diseases. Kv beta 1.3 assemblies with Kv alpha 1.5 and modifies its gating and pharmacology. A further knowledge of alpha-beta interactions and pharmacology will lead a better design of new drugs.

Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels.

Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages. While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in macrophages this K(+) current is generated by the association of Kv1.3 and Kv1.5. Patients with autoimmune diseases show a high number of effector memory T cells that are characterized by a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca(2+)-activated K(+) channels (K(Ca)3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.

Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences.

Voltage-dependent potassium (K(v)) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K(v) channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K(+) currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K(v)1.3/K(v)1.5 channels, which generate the main K(v) in macrophages. An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K(v)1.3 subunits in the K(v)1.3/K(v)1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression. Neither of these treatments apparently altered the expression of K(v)1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.

Kvbeta1.3 reduces the degree of stereoselective bupivacaine block of Kv1.5 channels.

BACKGROUND: Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. METHODS: Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. RESULTS: Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). CONCLUSIONS: The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.

Voltage-dependent Na+ channel phenotype changes in myoblasts. Consequences for cardiac repair.

OBJECTIVE: Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na(+) channels (Na(v)) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Na(v) during proliferation and differentiation in skeletal myocytes. METHODS AND RESULTS: We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Na(v)1.4 and Na(v)1.5, although neither protein has an apparent role in cell growth. During myogenesis, Na(v)1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Na(v)1.5 accounts for almost 90% of the Na(+) current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Na(v)1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Na(v)1.5 in skeletal myocytes. CONCLUSION: Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Na(v)1.5 acts as an underlying molecular mechanism.

{Omega}-3 and {omega}-6 polyunsaturated fatty acids block HERG channels.

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na(+), Ca(2+), and several K(+) channels. However, their effects on human ether-a-go-go-related gene (HERG) channels are unknown. In this study we have analyzed the effects of arachidonic acid (AA, omega-6) and docosahexaenoic acid (DHA, omega-3) on HERG channels stably expressed in Chinese hamster ovary cells by using the whole cell patch-clamp technique. At 10 microM, AA and DHA blocked HERG channels, at the end of 5-s pulses to -10 mV, to a similar extent (37.7 +/- 2.4% vs. 50.2 +/- 8.1%, n = 7-10, P > 0.05). 5,6,11,14-Eicosatetrayenoic acid, a nonmetabolizable AA analog, induced effects similar to those of AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by -5.1 +/- 1.8 mV (n = 10, P < 0.05) and -11.2 +/- 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 +/- 3.9 mV (n = 4; P < 0.05) and +15.8 +/- 4.3 mV (n = 4; P < 0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to -120 mV (I-->O). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time, voltage, and use dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA.