乙醇对大鼠心肌动作电位及人Kv1.5通道的影响

为了研究乙醇对心肌动作电位的作用及其机制,本实验采用标准玻璃微电极细胞内记录技术记录离体大鼠心肌细胞的动作电位(action potential,AP),采用全细胞膜片钳技术记录HEK293细胞上表达的人Kv1.5(human Kv1.5,hKv1.5)通道电流,观察6.25、12.5、25.0、50.0、100.0及...     

Influence of permeating ions on Kv1.5 channel block by nifedipine

Nifedipine can block K(+) currents through Kv1.5 channels in an open-channel manner (32). Replacement of internal and external K(+) with equimolar Rb(+) or Cs(+) reduced the potency of nifedipine block of Kv1.5 from an IC(50) of 7.3 microM (K(+)) to 16.0 microM (Rb(+)) and 26.9 microM (Cs(+)). The voltage dependence of block was unaffected, and a single binding site block model was used to describe block for all three ions. By varying ion species at the intra- and extracellular mouth of the channel and by using a nonconducting W472F-Kv1.5 mutant, we demonstrated that block was conditioned by the ion permeating the pore and, to a lesser extent, by the extracellular ion species alone. In Kv1.5, the outer pore mutations R487V and R487Y reduced nifedipine potency close to that of Kv4.2 and other Kv channels with an equivalent valine. Although changing this residue can affect C-type inactivation of Kv channels, the normalized reduction and time course of currents blocked by nifedipine in 5, 135, and 300 mM extracellular K(+) concentration was the same. Similarly, a mean recovery time constant from nifedipine block of 316 ms was unchanged (332 ms) after 5-s prepulses to allow C-type inactivation. This is consistent with the conclusion that nifedipine block and C-type inactivation in the Kv1.5 channel can coexist but are mediated by distinct mechanisms coordinated by outer pore conformation.     

Computational modelling of the open-state Kv 1.5 ion channel block by bupivacaine

Binding of R(+)-bupivacaine to open-state homology models of the mammalian K(v)1.5 membrane ion channel is studied using automated docking and molecular dynamics (MD) methods. Homology models of K(v)1.5 are built using the 3D structures of the KcsA and MthK channels as a template. The packing of transmembrane (TM) alpha-helices in the KcsA structure corresponds to a closed channel state. Opening of the channel may be reached by a conformational transition yielding a bent structure of the internal S6 helices. Our first model of the K(v) open state involves a PVP-type of bending hinge in the internal helices, while the second model corresponds to a Gly-type of bending hinge as found in the MthK channel. Ligand binding to these models is probed using the common local anaesthetic bupivacaine, where blocker binding from the intracellular side of the channel is considered. Conformational properties and partial atomic charges of bupivacaine are determined from quantum mechanical HF/6-31G* calculations with inclusion of solvent effects. The automated docking and MD calculations for the PVP-bend model predict that bupivacaine could bind either in the central cavity or in the PVP region of the channel pore. Linear interaction energy (LIE) estimates of the binding free energies for bupivacaine predict strongest binding to the PVP region. Surprisingly, no binding is predicted for the Gly-bend model. These results are discussed in light of electrophysiological data which show that the K(v)1.5 channel is unable to close when bupivacaine is bound.     

Localization of Kv1.5 channels in rat and canine myocyte sarcolemma

Voltage-gated potassium (Kv) channel subtypes localize to the plasma membrane of a number of cell types, and the sarcolemma in myocytes. Because many signaling molecules concentrate in subdomains of the plasma membrane, the localization of Kv channels to these sites may have important implications for channel function and regulation. In this study, the association of the voltage-gated potassium channel Kv1.5 with a specific subtype of lipid rafts, caveolae, in rat and canine cardiac myocytes has been investigated. Interactions between caveolin-3 and beta-dystroglycan or eNOS, as well as between Kv1.5 and alpha-actinin were readily detected in co-immunoprecipitation experiments, whereas no association between Kv1.5 and caveolin-3 was evident. Wide-field microscopy and deconvolution techniques revealed that the percent co-localization of Kv1.5 with caveolin-3 was extremely low in atrial myocytes from rat and canine hearts (8+/-1% and 12.2+/-2%, respectively), and limited in ventricular myocytes (11+/-4% and 20+/-3% in rat and canine, respectively). Immunoelectron microscopic imaging of rat atrial and ventricular tissues showed that Kv1.5 and caveolin-3 labeling generally did not overlap. In HEK293 cells stably expressing the channel, Kv1.5 did not target to the low buoyant density raft fraction along with flotillin but instead fractionated along with the non-raft associated transferrin receptor. Taken together, these results suggest that Kv1.5 is not present in caveolae of rat and canine heart.     

Over-expression of Kv1.5 in rat cardiomyocytes extremely shortens the duration of the action potential and causes rapid excitation

BACKGROUND: Genetically abnormal action potential duration (APD) can be a cause of arrhythmias that include long and short QT interval syndrome. PURPOSE: The aim of this study was to evaluate the arrhythmogenic effect of short QT syndrome induced by the over-expression of Kv1.5 in rat. METHODS: From Sprague-Dawley rats on fetal days 18-19, cardiomyocytes were excised and cultured with and without transfection with the Kv-1.5 gene using an adenovirus vector. The expression of Kv1.5 was proven by immunohistochemistry and Western blot analysis. In the culture dish and in the whole cells, the electrical activities were recorded using the whole-cell patch-clamp technique and the effects of 4-AP and verapamil were tested. RESULTS: After transfection with Kv1.5 for 12h, immunohistochemical staining and Western blot analysis were positive for Kv1.5 while they were negative in the control transfected with only Lac-Z. In the culture dish, the myocytes showed spontaneous beating at 115beats/min (bpm) just prior to the transfection with Kv1.5 and increased to 367bpm at 24h. The control myocytes showed stable beating rates during culturing. 4-AP at 200microM slowed down the rate and verapamil abolished the beating. In the whole cells, the maximal resting membrane potential was slightly depolarized and APD was extremely abbreviated both at 50% and 90% of repolarization compared with those of the control. Rapid spontaneous activities were found in a single myocyte with Kv1.5 transfection and 4-AP slowed down the frequency of the activities with a reversal of the shortened APD. CONCLUSION: The over-expression of Kv1.5 induced short APD and triggered activities in rat cardiomyocytes. This model can be used to study the arrhythmogenic substrate of short QT syndrome.     

Homology modeling of Kv1.5 channel block by cationic and electroneutral ligands

The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic “blocking particle”. A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.     

Transmembrane action potential heterogeneity in the canine isolated arterially perfused right atrium: effect of IKr and IKur/Ito block

The role of electrical heterogeneity in development of cardiac arrhythmias is well recognized. The extent to which transmembrane action potential (TAP) heterogeneity contributes to the normal electrophysiology of well-oxygenated atria is not well defined. The principal objective of the present study was to define regional and transmural differences in characteristics of the TAP in isolated superfused and arterially perfused canine right atrial (RA) preparations under baseline, rapidly activating delayed rectifier K(+) current (I(Kr)) block, and combined block of ultrarapid delayed rectifier and transient outward K(+) current (I(Kur)/I(to) block). Superfused preparations that survived generally displayed a triangle-shaped TAP. Exceptions included cells from the crista terminalis, where TAPs with a normal plateau could be recorded. In contrast, most TAPs recorded from throughout the perfused RA displayed a spike-and-dome and/or plateau morphology. The perfused RA displayed a heterogeneous distribution of repolarization, V(max), and spike-and-dome morphology along the epicardial and endocardial surfaces as well as transmurally, in the region of the upper crista terminalis. I(Kr) block with E-4031 prolonged repolarization homogeneously in the perfused RA, whereas I(Kur)/I(to) block using low concentrations of 4-aminopyridine abbreviated action potential duration at 90% repolarization heterogeneously, leading to a reduction in dispersion of repolarization. Our data indicate that the electrical heterogeneities, previously described for the canine ventricle, also exist within the atria and that I(Kr) block does not accentuate and I(Kur)/I(to) block reduces RA dispersion of repolarization. Our study also points to major differences in the transmembrane activity recorded using superfused vs. arterially perfused atrial preparations.     

Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels

We characterized the effects of intracellular Mg²⁺ (Mg²⁺_i) on potassium currents mediated by the Kv1.5 and Kv2.1 channels expressed in Xenopus oocytes. Increase in Mg²⁺_i caused a voltage-dependent block of the current amplitude, apparent acceleration of the current kinetics (explained by a corresponding shift in the steady-state activation) and leftward shifts in activation and inactivation dependencies for both channels. The voltage-dependent block was more potent for Kv2.1 [dissociation constant at 0 mV, K_d(0), was ~70 mM and the electric distance of the Mg²⁺ binding site, , was 0.2] than for the Kv1.5 channel [K_d(0)~40 mM and =0.1]. Similar shifts in the voltage-dependent parameters for both channels were described by the Gouy-Chapman formalism with the negative charge density of 1 e^–/100 Ų. Additionally, Mg²⁺_i selectively reduced a non-inactivating current and increased the accumulation of inactivation of the Kv1.5, but not the Kv2.1 channel. A potential functional role of the differential effects of Mg²⁺_i on the Kv channels is discussed.     

Distinct mechanisms of block of Kv1.5 channels by tertiary and quaternary amine clofilium compounds

The quaternary ammonium compound clofilium and its tertiary amine derivative LY97241 were used to analyze mechanisms of block in a voltage-gated potassium channel. Wild-type and mutant Kv1.5 channels expressed in Xenopus oocytes were recorded by two-electrode voltage clamp. Open-channel block to 20% of the control current amplitude was induced reversibly by 50 microM clofilium or 200 microM LY97241, and was seen as an acceleration of the macroscopic current decay. Although blockers remained present after application, channels recovered from block during each interpulse interval. The optimum voltage for recovery (-45 mV at pH 7.3) at the threshold for channel activation indicated that clofilium block and recovery occurred principally through the open channel state. In contrast, LY97241 appeared to exit from the closed state and the open state. In an acid-tolerant Kv1.5 mutant channel (H452Q), external pH was used to titrate LY97241. At low pH, which protonates the LY97241 amine group, recovery from block at hyperpolarized potentials was impaired in a manner similar to that seen with clofilium. Recovery from clofilium block was reduced at negative potentials independent of pH, an effect attributed to trapping of the permanently charged compound within the closed channels.     

In vivo gene transfer of Kv1.5 normalizes action potential duration and shortens QT interval in mice with long QT phenotype

Mutations in cardiac voltage-gated K+ channels cause long QT syndrome (LQTS) and sudden death. We created a transgenic mouse with a long QT phenotype (Kv1DN) by overexpression of a truncated K+ channel in the heart and investigated whether the dominant negative effect of the transgene would be overcome by the direct injection of adenoviral vectors expressing wild-type Kv1.5 (AV-Kv1.5) into the myocardium. End points at 3-10 days included electrophysiology in isolated cardiomyocytes, surface ECG, programmed stimulation of the right ventricle, and in vivo optical mapping of action potentials and repolarization gradients in Langendorff-perfused hearts. Overexpression of Kv1.5 reconstituted a 4-aminopyridine-sensitive outward K+ current, shortened the action potential duration, eliminated early afterdepolarizations, shortened the QT interval, decreased dispersion of repolarization, and increased the heart rate. Each of these changes is consistent with a physiologically significant primary effect of adenoviral expression of Kv1.5 on ventricular repolarization of Kv1DN mice.     

Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers.

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of external pH on activation of the Kv1.5 potassium channel

We studied the mechanism by which external acidification from pH 7.3 to 6.8 reduced current magnitude in the Kv1.5 potassium channel. At physiological external [K(+)], a shift in the voltage-dependence of activation was entirely responsible for the acidification-induced decrease in Kv1.5 current magnitude (pK = 7.15). Elevation of external [Ca(2+)] or [Mg(2+)] identically shifted activation curves to the right and identically shifted the pH-sensitivity of the activation curves to more acidic values. Similar observations were made with the Kv2.1 K(+) channel, except that the pK for the activation shift was out of the physiological range. These data are consistent with a mechanism by which acidification shifted activation via modification of a local surface potential. Elimination of eight positive charges within the outer vestibule of the conduction pathway had no effect on the voltage-dependence of activation at pH 7.3 or higher, which suggested that sites exposed to the conduction pathway within the outer vestibule did not directly contribute to the relevant local surface potential. However, mutations at position 487 (within the conduction pathway) displaced the pK of the pH-sensitive shift in activation, such that the sensitivity of Kv1.5 current to physiologically relevant changes in pH was reduced or eliminated. These results suggest that, among voltage-gated K(+) channels, activation in Kv1.5 is uniquely sensitive to physiologically relevant changes in pH because the pK for the sites that contribute to the local surface potential effect is near pH 7. Moreover, the pK for the activation shift depends not only on the nature of the sites involved but also on structural orientation conferred, in part, by at least one residue within the conduction pathway.     

Discovery of triarylethanolamine inhibitors of the Kv1.5 potassium channel

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure–activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.     

Aryl sulfonamido indane inhibitors of the Kv1.5 ion channel

A collection of aryl sulfonamido indanes based on the lead compound 1 was synthesized and evaluated for Kv1.5 inhibitory activity. Kv1.5 inhibitors have the potential to be atrium-selective agents for treatment of atrial fibrillation. (1R,2R)-1 has an IC(50) of 0.033microM against Kv1.5 and is selective against other cardiac ion channels, including hERG.     

Aryl sulfonamido tetralin inhibitors of the Kv1.5 ion channel

Aryl sulfonamido tetralins based on lead compound 2a were synthesized and evaluated for Kv1.5 inhibitory activity. Several compounds having IC₅₀ values less then 0.1 μM were identified. Kv1.5 inhibitors have the potential to be atrium-selective agents for the treatment of atrial fibrillation.     

Molecular basis for Kv1.5 channel block: conservation of drug binding sites among voltage-gated K+ channels

Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) that contributes to action potential repolarization of human atrial myocytes. Block of these channels has been proposed as a treatment for atrial arrhythmias. Here we report a novel and potent inhibitor of Kv1.5 potassium channels, N-benzyl-N-pyridin-3-yl-methyl-2-(toluene-4-sulfonylamino)-benzamide hydrochloride (S0100176), which exhibits features consistent with preferential block of the open state. The IC50 of S0100176 for Kv1.5 expressed in Xenopus oocytes was 0.7 microm. Ala-scanning mutagenesis within the pore helix and the S6 segment, regions that form the walls of the central cavity, was combined with voltage clamp analysis to identify point mutations that altered drug affinity. This approach identified Thr-479, Thr-480, Val-505, Ile-508, and Val-512 as the most important residues for block by S0100176. Mutations of these key residues to Ala or other amino acids caused marked changes in the IC50 of S0100176 (p<0.01). For example, the IC50 of S0100176 increased 362-fold for T480A, 26-fold for V505A, 150-fold for I508A, and 99-fold for V512A. We used modeling to dock S0100176 into the inner cavity of a Kv1.5 pore homology model that was generated based on the crystal structure of KcsA. The docking predicted that the five residues identified by the Ala scan were positioned less than 4.5 A from the compound. Based on the homology models, the positions of the five amino acids identified to interact with S0100176 face toward the central cavity and overlap with putative binding sites for other blockers and voltage-gated potassium channels.     

Rab-GTPase-dependent endocytic recycling of Kv1.5 in atrial myocytes

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. Maintaining a balance between anterograde and retrograde trafficking of channel proteins is vital in regulating steady-state cell surface expression. Kv1.5 is an important voltage-gated K(+) channel in the cardiovascular system underlying the ultra-rapid rectifying potassium current (Ik(ur)), a major repolarizing current in atrial myocytes, and regulating the resting membrane potential and excitability of smooth muscle cells. Defects in the expression of Kv1.5 are associated with pathological states such as chronic atrial fibrillation and hypoxic pulmonary hypertension. There is, thus, substantial interest in understanding the mechanisms regulating cell surface channel levels. Here, we investigated the internalization and recycling of Kv1.5 in the HL-1 immortalized mouse atrial myocytes. Kinetic studies indicate that Kv1.5 is rapidly internalized to a perinuclear region where it co-localizes with the early endosomal marker, EEA1. Importantly, we identified that a population of Kv1.5, originating on the cell surface, internalized and recycled back to the plasma membrane. Notably, Kv1.5 recycling processes are driven by specific Rab-dependent endosomal compartments. Thus, co-expression of GDP-locked Rab4S22N and Rab11S25N dominant-negative mutants decreased the steady-state Kv1.5 surface levels, whereas GTPase-deficient Rab4Q67L and Rab11Q70L mutants increased steady-state Kv1.5 surface levels. These data reveal an unexpected dynamic trafficking of Kv1.5 at the myocyte plasma membrane and demonstrate a role for recycling in the maintenance of steady-state ion channel surface levels.     

PKC and AMPK regulation of Kv1.5 potassium channels

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K⁺ current (I_Kur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.     

PKC and AMPK regulation of Kv1.5 potassium channels

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K⁺ current (I_Kur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.     

Evidence for proteasomal degradation of Kv1.5 channel protein

BACKGROUND: The voltage-gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential. While protein degradation is one of the major mechanisms for the regulation of channel functions, little is known on the degradation mechanism of Kv1.5. METHODS AND RESULTS: Kv1.5 was expressed in COS cells and its degradation, intracellular localization, and channel activities were assessed by pulse-chase analysis, immunofluorescence, and patch clamp techniques, respectively. Expressed Kv1.5 had a half-life time of approximately 6.7 h, which was prolonged by the proteasome inhibitors of MG132, ALLN, proteasomal inhibitor 1, or lactacystine, but not by a lysosomal inhibitor chloroquine. MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner. Similar effects of MG132 on endogenous Kv1.5 were seen in cultured rat atrial cells. Within a cell, Kv1.5 was mainly localized in both the endoplasmic reticulum and Golgi apparatus. MG132 increased the immunoreactivity of Kv1.5 in these compartments and also increased Ik(ur) currents through the cell-surface Kv1.5. Pretreatment with either brefeldin A or colchicine abolished MG132-induced increase in Ik(ur) currents. CONCLUSION: Kv1.5 is degraded by the proteasome. The inhibition of the proteasome increased Ik(ur) currents secondary to stabilization of the channel protein in the endoplasmic reticulum/Golgi apparatus.