Selective inhibition of physiological late Na+ current stabilizes ventricular repolarization.

The physiological role of cardiac late Na+ current ( INa) has not been well described. In this study, we tested the hypothesis that selective inhibition of physiological late INa abbreviates the normal action potential (AP) duration (APD) and counteracts the prolongation of APD and arrhythmic activities caused by inhibition of the delayed rectifier K+ current ( IKr). The effects of GS-458967 (GS967) on the physiological late INa and APs in rabbit isolated ventricular myocytes and on the monophasic APs and arrhythmias in rabbit isolated perfused hearts were determined. In ventricular myocytes, GS967 and, for comparison, tetrodotoxin concentration dependently decreased the physiological late INa with IC50 values of 0.5 and 1.9 µM, respectively, and significantly shortened the APD measured at 90% repolarization (APD90). A strong correlation between inhibition of the physiological late INa and shortening of APD by GS967 or tetrodotoxin ( R2 of 0.96 and 0.97, respectively) was observed. Pretreatment of isolated myocytes or hearts with GS967 (1 µM) significantly shortened APD90 and monophasic APD90 and prevented the prolongation and associated arrhythmias caused by the IKr inhibitor E4031 (1 µM). In conclusion, selective inhibition of physiological late INa shortens the APD, stabilizes ventricular repolarization, and decreases the proarrhythmic potential of pharmacological agents that slow ventricular repolarization. Thus, selective inhibition of late INa may constitute a generalizable approach to stabilize ventricular repolarization and suppress arrhythmogenicity associated with conditions whereby AP or QT intervals are prolonged. NEW & NOTEWORTHY The contribution of physiological late Na+ current in action potential duration (APD) of rabbit cardiac myocytes was estimated. The inhibition of this current prevented the prolongation of APD in rabbit cardiac myocytes, the prolongation of monophasic APD, and generation of arrhythmias in rabbit isolated hearts caused by delayed rectifier K+ current inhibition.

Overexpression of SCN5A in mouse heart mimics human syndrome of enhanced atrioventricular nodal conduction.

BACKGROUND: In enhanced atrioventricular (A-V) nodal conduction (EAVNC) syndrome, patients have short A-V conduction times. Multiple mechanisms have been proposed to explain EAVNC; however, the electrophysiological or molecular substrate responsible for it remains unclear. OBJECTIVE: The purpose of this study was to test the hypothesis that overexpression of SCN5A in the mouse heart may provide an animal model mimicking EAVNC. METHODS: Electrocardiogram, atrial, His bundle, and ventricular electrograms were recorded from wild-type (WT) and transgenic (TG) mice overexpressing human SCN5A. The sodium current and NaV1.5 expression were measured using patch-clamp and immunohistochemistry techniques. RESULTS: The P-R interval in TG mice (13.6 ± 1.2 ms) was much shorter than that in WT mice (40.2 ± 0.59 ms). In TG isolated hearts, the A-V conduction time (14.4 ± 0.81 ms) during right atrial pacing was also shorter than that in WT hearts (39.5 ± 0.62 ms). Records of His bundle electrograms revealed that atrial-to-His and His-to-ventricular intervals were shorter in TG than in WT hearts. In addition, TG hearts had a shorter Wenckebach cycle length and A-V effective refractory period. The sodium current was 2-fold greater in TG ventricular myocytes than in WT ventricular myocytes. Flecainide prolonged the A-V conduction time in TG hearts to a value close to that in WT hearts. Nifedipine prolonged the atrial-to-His interval in WT hearts but not in TG hearts. Immunohistochemistry studies revealed increased NaV1.5 labeling in TG atrial and ventricular tissues, and NaV1.5 expression in A-V junction and A-V ring regions in TG hearts. CONCLUSION: Enhanced A-V conduction in mice overexpressing SCN5A in the heart mimics the human syndrome of EAVNC. Thus, variants in sodium channel expression in the A-V nodal region may be an electrophysiological substrate responsible for EAVNC.

Estradiol promotes sudden cardiac death in transgenic long QT type 2 rabbits while progesterone is protective.

BACKGROUND: Postpubertal women with inherited long QT syndrome type 2 (LQT2) are at increased risk for polymorphic ventricular tachycardia (pVT) and sudden cardiac death (SCD), particularly during the postpartum period. OBJECTIVE: To investigate whether sex hormones directly modulate the arrhythmogenic risk in LQTS. METHODS: Prepubertal ovariectomized transgenic LQT2 rabbits were treated with estradiol (EST), progesterone (PROG), dihydrotestosterone (DHT), or placebo (OVX). RESULTS: During 8 weeks of treatment, major cardiac events-spontaneous pVT or SCD-occurred in 5 of the 7 EST rabbits and in 2 of the 9 OVX rabbits (P <.05); in contrast, no events occurred in 9 PROG rabbits and 6 DHT rabbits (P <.01 vs PROG; P <.05 vs DHT). Moreover, EST increased the incidence of pVT (P <.05 vs OVX), while PROG reduced premature ventricular contractions, bigeminy, couplets, triplets, and pVT (P <.01 vs OVX; P <.001 vs EST). In vivo electrocardiographic monitoring, in vivo electrophysiological studies, and ex vivo optical mapping studies revealed that EST promoted SCD by steepening the QT/RR slope (P <.05), by prolonging cardiac refractoriness (P <.05), and by altering the spatial pattern of action potential duration dispersion. Isoproterenol-induced Ca(2+) oscillations resulted in early afterdepolarizations in EST-treated hearts (4 of 4), while PROG prevented SCD by eliminating this early afterdepolarization formation in 4 of the 7 hearts (P = .058 vs EST; P <.05 vs OVX). Analyses of ion currents demonstrated that EST increased the density of I(Ca,L) as compared with OVX (P <.05) while PROG decreased it (P <.05). CONCLUSION: This study reveals the proarrhythmic effect of EST and the antiarrhythmic effect of PROG in LQT2 in vivo, outlining a new potential antiarrhythmic therapy for LQTS.

Differential conditions for early after-depolarizations and triggered activity in cardiomyocytes derived from transgenic LQT1 and LQT2 rabbits.

Early after-depolarization (EAD), or abnormal depolarization during the plateau phase of action potentials, is a hallmark of long-QT syndrome (LQTS). More than 13 genes have been identified as responsible for LQTS, and elevated risks for EADs may depend on genotypes, such as exercise in LQT1 vs. sudden arousal in LQT2 patients. We investigated mechanisms underlying different high-risk conditions that trigger EADs using transgenic rabbit models of LQT1 and LQT2, which lack I(Ks) and I(Kr) (slow and fast components of delayed rectifying K(+) current), respectively. Single-cell patch-clamp studies show that prolongation of action potential duration (APD) can be further enhanced by lowering extracellular potassium concentration ([K(+)](o)) from 5.4 to 3.6 mm. However, only LQT2 myocytes developed spontaneous EADs following perfusion with lower [K(+)](o), while there was no EAD formation in littermate control (LMC) or LQT1 myocytes, although APDs were also prolonged in LMC myocytes and LQT1 myocytes. Isoprenaline (ISO) prolonged APDs and triggered EADs in LQT1 myocytes in the presence of lower [K(+)](o). In contrast, continuous ISO perfusion diminished APD prolongation and reduced the incidence of EADs in LQT2 myocytes. These different effects of ISO on LQT1 and LQT2 were verified by optical mapping of the whole heart, suggesting that ISO-induced EADs are genotype specific. Further voltage-clamp studies revealed that ISO increases L-type calcium current (I(Ca)) faster than I(Ks) (time constant 9.2 s for I(Ca) and 43.6 s for I(Ks)), and computer simulation demonstrated a high-risk window of EADs in LQT2 during ISO perfusion owing to mismatch in the time courses of I(Ca) and I(Ks), which may explain why a sympathetic surge rather than high sympathetic tone can be an effective trigger of EADs in LQT2 perfused hearts. In summary, EAD formation is genotype specific, such that EADs can be elicited in LQT2 myocytes simply by lowering [K(+)](o), while LQT1 myocytes require sympathetic stimulation. Slower activation of I(Ks) than of I(Ca) by ISO may explain why different sympathetic modes, i.e. sympathetic surge vs. high sympathetic tone, are associated with polymorphic ventricular tachycardia in LQTS patients.

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines.

We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.

Electrophysiological studies of transgenic long QT type 1 and type 2 rabbits reveal genotype-specific differences in ventricular refractoriness and His conduction.

We have generated transgenic rabbits lacking cardiac slow delayed-rectifier K(+) current [I(Ks); long QT syndrome type 1 (LQT1)] or rapidly activating delayed-rectifier K(+) current [I(Kr); long QT syndrome type 2 (LQT2)]. Rabbits with either genotype have prolonged action potential duration and QT intervals; however, only LQT2 rabbits develop atrioventricular (AV) blocks and polymorphic ventricular tachycardia. We therefore sought to characterize the genotype-specific differences in AV conduction and ventricular refractoriness in LQT1 and LQT2 rabbits. We carried out in vivo electrophysiological studies in LQT1, LQT2, and littermate control (LMC) rabbits at baseline, during isoproterenol infusion, and after a bolus of dofetilide and ex vivo optical mapping studies of the AV node/His-region at baseline and during dofetilide perfusion. Under isoflurane anesthesia, LQT2 rabbits developed infra-His blocks, decremental His conduction, and prolongation of the Wenckebach cycle length. In LQT1 rabbits, dofetilide altered the His morphology and slowed His conduction, resulting in intra-His block, and additionally prolonged the ventricular refractoriness, leading to pseudo-AV block. The ventricular effective refractory period (VERP) in right ventricular apex and base was significantly longer in LQT2 than LQT1 (P < 0.05) or LMC (P < 0.01), with a greater VERP dispersion in LQT2 than LQT1 rabbits. Isoproterenol reduced the VERP dispersion in LQT2 rabbits by shortening the VERP in the base more than in the apex but had no effect on VERP in LQT1. EPS and optical mapping experiments demonstrated genotype-specific differences in AV conduction and ventricular refractoriness. The occurrence of infra-His blocks in LQT2 rabbits under isoflurane and intra-His block in LQT1 rabbits after dofetilide suggest differential regional sensitivities of the rabbit His-Purkinje system to drugs blocking I(Kr) and I(Ks).

5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches.

BACKGROUND: 5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5HD + CoA + ATP --> 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K(ATP) (sarcK(ATP)) channel activity. METHODS: Single sarcK(ATP) channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK(ATP) channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A. RESULTS: 5-HD inhibited (IC(50) approximately 30 microM) K(ATP) channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 microM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 microM coenzyme A (CoA) inhibited sarcK(ATP) channels. Using giant inside-out patches, which are weakly sensitive to "contaminating" CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK(ATP) channel activation by either metabolic inhibition or rilmakalim. GENERAL SIGNIFICANCE: SarcK(ATP) channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at "directly" inhibiting sarcK(ATP) and mitoK(ATP) channels. However, in intact cells, 5-HD fails to inhibit sarcK(ATP) channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.

cANF causes endothelial cell hyperpolarization by activation of chloride channels.

OBJECTIVES: Natriuretic peptides bind with natriuretic peptide receptor (NPR)-C, which can alter cellular function through its interaction with the G(i) protein complex. NPR-C has been found to mediate the activation of K(+) channels and non-selective cation channels in vascular smooth muscle and cardiac fibroblast cells, respectively. However, the electrophysiological effect of NPR-C activation on endothelial cells (EC) has not been previously examined. In this study we sought to elucidate the effect of cANF(4-23), a selective NPR-C ligand, on EC membrane potential (E(m)). METHODS/RESULTS: Changes in EC E(m) was measured through non-invasive fluorescence imaging. EC were preincubated in the potentiometric dye, DiBAC(4)(3) and subsequently exposed to cANF(4-23), in the presence of selective inhibitors of ion-channels or second messengers. NPR-C expression in rat lung microvascular endothelial cells was assessed by RT-PCR. cANF(4-23) induced a sustained decrease in EC cellular fluorescence, indicating endothelial cell hyperpolarization. The cANF-induced hyperpolarization could not be attenuated by TEA, barium, ouabain or by the reduction of extracellular Ca(2+). Further, the cANF-induced hyperpolarization was insensitive to inhibition of G(i) and protein kinase G (PKG), downstream messengers of NPRs. However, the Cl(-) channel inhibitors, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, niflumic acid, and hypertonic saline attenuated the cANF-induced hyperpolarization. Perforated patch clamp recordings confirmed the cANF-induced current was carried by Cl(-) and could be inhibited by niflumic acid. RT-PCR confirmed expression of NPR-C in vascular smooth muscle cells but not in EC. CONCLUSIONS: cANF causes hyperpolarization that is most likely mediated via activation of Cl(-) channels by a PKG and G(i) independent mechanism.

Mechanism of C-type natriuretic peptide-induced endothelial cell hyperpolarization.

C-type natriuretic peptide (CNP) has a demonstrated hyperpolarizing effect on vascular smooth muscle cells. However, its autocrine function, including its electrophysiological effect on endothelial cells, is not known. Here, we report the effect of CNP on the membrane potential (E(m)) of pulmonary microvascular endothelial cells and describe its target receptors, second messengers, and ion channels. We measured changes in E(m) using fluorescence imaging and perforated patch-clamping techniques. In imaging experiments, samples were preincubated in the potentiometric dye DiBAC(4)(3), and subsequently exposed to CNP in the presence of selective inhibitors of ion channels or second messengers. CNP exposure induced a dose-dependent decrease in fluorescence, indicating that CNP induces endothelial cell hyperpolarization. CNP-induced hyperpolarization was inhibited by the K(+) channel blockers, tetraethylammonium or iberiotoxin, the nonspecific cation channel blocker, La(3+), or by depletion or repletion of extracellular Ca(2+) or K(+), respectively. CNP-induced hyperpolarization was also blocked by pharmacological inhibition of PKG or by small interfering RNA (siRNA)-mediated knockdown of natriuretic peptide receptor-B (NPR-B). CNP-induced hyperpolarization was mimicked by the PKG agonist, 8-bromo-cGMP, and attenuated by both the endothelial nitric oxide synthase (eNOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (l-NAME), and the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Presence of iberiotoxin-sensitive, CNP-induced outward current was confirmed by perforated patch-clamping experiments. We conclude that CNP hyperpolarizes pulmonary microvascular endothelial cells by activating large-conductance calcium-activated potassium channels mediated by the activation of NPR-B, PKG, eNOS, and sGC.

Mechanisms of cardiac arrhythmias and sudden death in transgenic rabbits with long QT syndrome.

Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K+ channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of IKs and IKr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary IKr and IKs without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type alpha subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties.

The physiological function of ion channels is affected by protein-protein and protein-membrane interactions that modulate their activity and/or localization. Palmitoylation modulates protein function by facilitating targeted membrane association, interaction with other proteins, and determining subcellular localization. In this study, we demonstrate that the voltage-dependent potassium (Kv) channel Kv1.5 is palmitoylated and that the mutation of COOH-terminal cysteines is sufficient to abolish the palmitoylation of the Kv1.5 polypeptide in Chinese hamster ovary (CHO) cells. The labeling represented the thioester linkage of the labeled palmitic acid to cysteine rather than amide and oxygen ester linkages as judged by the release of the palmitic acid upon the treatment of the gel with hydroxylamine at a neutral pH. Site-directed mutagenesis and radiolabeling studies revealed that C593 was the sole site of palmitoylation. The elucidation of the biological function of palmitoylation revealed that the expression of the FLAG-Kv1.5 palmitoylation-deficient mutant (FL-Kv1.5(Palm-)) in stable CHO cells increased membrane expression as determined by the biotinylation of surface proteins and quantitative immunofluorescence analyses of these cells, in turn enhancing the outward potassium current. This enhanced surface expression and the currents were consequential to the slower rate of internalization, causing an increased localization of FL-Kv1.5(Palm-) in the plasma membrane compared with the wild-type FL-Kv1.5 channels. We conclude that the Kv1.5 channel is palmitoylated and that its palmitoylation modulates its biological functions and, therefore, might provide a physiological link between the metabolic state and the expression of Kv1.5 on the plasma membrane.

Single-channel properties of I K,slow1 and I K,slow2 in mouse ventricular myocytes.

I K,slow1 and I K,slow2 are two important voltage-gated potassium (K+) currents expressed in mouse ventricular myocytes. However, their properties at the single-channel level have not been characterized. In this paper, we report two new single K+ channels, mK1 and mK2, in myocytes isolated from mouse ventricles and their possible correlation with the macroscopic currents I K,slow1 and I K,slow2. The conductance of mK1 and mK2 was 24 and 17 pS, respectively. Ensemble-averaged current demonstrated an inactivation time constant of 400 to 500 ms for mK1 compared with 1,300 to 2,000 ms for mK2. The mK1 channel was more sensitive than the MK2 channel to the K channel blocker 4-AP. In myocytes isolated from Kv1DN mice with functional knock out of the Kv1.5 channel, mK1 was not detectable but mK2 was present. Our data suggest that the newly characterized K+ channels, mK1 and mK2, likely correspond to the macroscopic currents of I K,slow1 and I K,slow2, respectively.

Modulation of human cardiovascular outward rectifying chloride channel by intra- and extracellular ATP.

The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl(-) channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out single-channel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl(-) solutions, exhibited a slope conductance of approximately 90-100 pS at positive potentials and approximately 22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, "extracellular" ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATPgammaS > ATP = UTP > ADP > AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions.

Effects of divalent cations and spermine on the K+ channel TASK-3 and on the outward current in thalamic neurons.

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca2+ or Mg2+. Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia.

Caveolin-3 and SAP97 form a scaffolding protein complex that regulates the voltage-gated potassium channel Kv1.5.

The targeting of ion channels to particular membrane microdomains and their organization in macromolecular complexes allow excitable cells to respond efficiently to extracellular signals. In this study, we describe the formation of a complex that contains two scaffolding proteins: caveolin-3 (Cav-3) and a membrane-associated guanylate kinase (MAGUK), SAP97. Complex formation involves the association of Cav-3 with a segment of SAP97 localized between its PDZ2 and PDZ3 domains. In heterologous expression systems, this scaffolding complex can recruit Kv1.5 to form a tripartite complex in which each of the three components interacts with the other two. These interactions regulate the expression of currents encoded by a glycosylation-deficient mutant of Kv1.5. We conclude that the association of Cav-3 with SAP97 may constitute the nucleation site for the assembly of macromolecular complexes containing potassium channels.

Single-channel recordings of a rapid delayed rectifier current in adult mouse ventricular myocytes: basic properties and effects of divalent cations.

The rapidly delayed rectifier current (I(Kr)) has been described in ventricular myocytes isolated from many species, as well as from neonatal mice. However, whether I(Kr) is present in the adult mouse heart remains controversial. We used cell-attached patch-clamp recording in symmetrical K(+) solutions to assess the presence and behaviour of single I(Kr) channels in adult mouse cardiomyocytes (mI(Kr)). Of 314 patches, 158 (50.1%) demonstrated mI(Kr) currents as compared with 131 (42.3%) for the I(K1) channel. Single mI(Kr) channel activity was rarely observed at potentials positive to -10 mV. The slope conductance at negative potentials was 12 pS. Upon repolarization, ensemble-averaged mI(Kr) showed slow deactivation with a biexponential time course. A selective I(Kr) blocker, E-4031 (1 microm), completely blocked mI(Kr) channel activity. Extracellular Ca(2+) and Mg(2+) at physiological concentrations shifted the activation by approximately 30 mV, accelerated deactivation kinetics, prolonged long-closed time, and reduced open probability without affecting single-channel conductance, suggesting a direct channel-blocking effect in addition to well-recognized voltage shifts. HERG subunits expressed in Chinese hamster ovary cells produced channels with properties similar to those of mI(Kr), except for the more-negative activation of the HERG channels. Despite the abundant expression of mI(Kr), single-channel events were rarely observed during action-potential clamp and 5 microm E-4031 had no detectable effect on the action potential parameters, confirming that mI(Kr) plays at best a minor role in repolarization of adult mouse cardiomyocytes, probably because the modulatory effects of divalent cations prevent significant mI(Kr) opening under physiological conditions.

"Sleepy" inward rectifier channels in guinea-pig cardiomyocytes are activated only during strong hyperpolarization.

K(+) channels of isolated guinea-pig cardiomyocytes were studied using the patch-clamp technique. At transmembrane potentials between -120 and -220 mV we observed inward currents through an apparently novel channel. The novel channel was strongly rectifying, no outward currents could be recorded. Between -200 and -160 mV it had a slope conductance of 42.8 +/- 3.0 pS (S.D.; n = 96). The open probability (P(o)) showed a sigmoid voltage dependence and reached a maximum of 0.93 at -200 mV, half-maximal activation was approximately -150 mV. The voltage dependence of P(o) was not affected by application of 50 microM isoproterenol. The open-time distribution could be described by a single exponential function, the mean open time ranged between 73.5 ms at -220 mV and 1.4 ms at -160 mV. At least two exponential components were required to fit the closed time distribution. Experiments with different external Na(+), K(+) and Cl(-) concentrations suggested that the novel channel is K(+) selective. Extracellular Ba(2+) ions gave rise to a voltage-dependent reduction in P(o) by inducing long closed states; Cs(+) markedly reduced mean open time at -200 mV. In cell-attached recordings the novel channel frequently converted to a classical inward rectifier channel, and vice versa. This conversion was not voltage dependent. After excision of the patch, the novel channel always converted to a classical inward rectifier channel within 0-3 min. This conversion was not affected by intracellular Mg(2+), phosphatidylinositol (4,5)-bisphosphate or spermine. Taken together, our findings suggest that the novel K(+) channel represents a different "mode" of the classical inward rectifier channel in which opening occurs only at very negative potentials.