Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

External nickel blocks human Kv1.5 channels stably expressed in CHO cells

We have investigated the actions of Nickel (Ni²⁺) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni²⁺ reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 μm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni²⁺. Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni²⁺, prior to channel activation, was prolonged indicating that Ni²⁺ interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni²⁺ decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni²⁺ preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni²⁺ are distinct from those of other K⁺ channels. Received: 12 October 2000/Revised: 14 May 2001     

The antibody targeting the E314 peptide of human Kv1.3 pore region serves as a novel, potent and specific channel blocker

Selective blockade of Kv1.3 channels in effector memory T (T(EM)) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca(2+) or voltage-gated Na(+) currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related K(v)1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous system (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker.     

Regulation of a mammalian Shaker-related potassium channel, hKv1.5, by extracellular potassium and pH

Using the whole-cell recording mode of the patch-clamp technique we studied the effects of removal of extracellular potassium, [K(+)](o), on a mammalian Shaker-related K(+) channel, hKv1.5. In the absence of [K(+)](o), current through hKv1.5 was similar to currents obtained in the presence of 4.5 mM [K(+)](o). This observation was not expected as earlier results had suggested that either positively charged residues or the presence of a nitrogen-containing residue at the external TEA(+) binding site (R487 in hKv1.5) caused current loss upon removal of [K(+)](o). However, the current loss in hKv1.5 was observed when the extracellular pH, pH(o), was reduced from 7.4 to 6.0, a behavior similar to that observed previously for current through mKv1.3 with a histidine at the equivalent position (H404). These observations suggested that the charge at R487 in hKv1.5 channels was influenced by other amino acids in the vicinity. Replacement of a histidine at position 463 in hKv1.5 by glycine confirmed this hypothesis making this H463G mutant channel sensitive to removal of [K(+)](o) even at pH(o) 7.4. We conclude that the protonation of H463 at pH 7.4 might induce a pK(a) shift of R487 that influences the effective charge at this position leading to a not fully protonated arginine. Furthermore, we assume that the charge at position 487 in hKv1.5 can directly or indirectly disturb the occupation of a K(+) binding site within the channel pore possibly by electrostatic interaction. This in turn might interfere with the concerted transition of K(+) ions resulting in a loss of K(+) conduction.     

hKv1.5 channels play a pivotal role in the functions of human alveolar macrophages

We examined the pharmacological properties, the molecular identity, and the functional roles of hKv1.5 channel in human alveolar macrophage. Some of outward K(+) current was inhibited by 4-aminopyridine and antisense oligodeoxynucleotides against hKv1.5 mRNA. Consistently, the protein and mRNA expressions of hKv1.5 channel were detected. Furthermore, the phagocytosis and migration of human alveolar macrophages were significantly suppressed when the protein expression of hKv1.5 channel was lowered by the antisense hKv1.5 oligodeoxynucleotides. These results suggest that hKv1.5 channel is expressed in human alveolar macrophages and it plays a role in phagocytosis and migration of the human alveolar macrophage.     

Rapid induction of P/C-type inactivation is the mechanism for acid-induced K+ current inhibition

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H(+)(o)-induced current inhibition by taking advantage of Na(+) permeation through inactivated channels. In hKv1.5, H(+)(o) inhibited open-state Na(+) current with a similar potency to K(+) current, but had little effect on the amplitude of inactivated-state Na(+) current. In support of inactivation as the mechanism for the current reduction, Na(+) current through noninactivating hKv1.5-R487V channels was not affected by [H(+)(o)]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H(+)(o). These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na(+) currents but the maintained presence of slow Na(+) tail currents, combined with changes in the Na(+) tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na(+) current at low pH.     

Evidence for multiple open and inactivated states of the hKv1.5 delayed rectifier.

The kinetic properties of hKv1.5, a Shaker-related cardiac delayed rectifier, expressed in Ltk- cells were studied. hKv1.5 currents elicited by membrane depolarizations exhibited a delay followed by biphasic activation. The biphasic activation remained after 5-s prepulses to membrane potentials between -80 and -30 mV; however, the relative amplitude of the slow component increased as the prepulse potential approached the threshold of channel activation, suggesting that the second component did not reflect activation from a hesitant state. The decay of tail currents at potentials between -80 and -30 mV was adequately described with a biexponential. The time course of deactivation slowed as the duration of the depolarizing pulse increased. This was due to a relative increase in the slowly decaying component, despite similar initial amplitudes reflecting a similar open probability after 50- and 500-ms prepulses. To further investigate transitions after the initial activated state, we examined the temperature dependence of inactivation. The time constants of slow inactivation displayed little temperature and voltage dependence, but the degree of the inactivation increased substantially with increased temperature. Recovery from inactivation proceeded with a biexponential time course, but long prepulses at depolarized potentials slowed the apparent rate of recovery from inactivation. These data strongly indicate that hKv1.5 has both multiple open states and multiple inactivated states.     

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)     

A specific N-terminal residue in Kv1.5 is required for upregulation of the channel by SAP97

We have previously reported that SAP97 enhancement of hKv1.5 currents requires an intact Kv1.5 N-terminus and is independent of the PDZ-binding motif at the C-terminus of the channel [J. Eldstrom, W.S. Choi, D.F. Steele, D. Fedida, SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism, FEBS Lett. 547 (2003) 205-211]. Here, we report that an interaction between the two proteins can be detected under certain conditions but their interaction is irrelevant to the enhancement of channel expression. Instead, a threonine residue at position 15 in the hKv1.5 N-terminus is critically important. Mutation of this residue, which lies within a consensus site for phosphorylation by protein kinase C, to an alanine, completely abrogated the effect of SAP97 on channel expression. Although we were unable to detect phosphorylation of this residue, specific inhibition of kinase C by Calphostin C eliminated the increase in wild-type hKv1.5 currents associated with SAP97 overexpression suggesting a role for this kinase in the response.     

Allitridi Inhibits Multiple Cardiac Potassium Channels Expressed in HEK 293 Cells

Allitridi (diallyl trisulfide) is an active compound (volatile oil) from garlic. The previous studies reported that allitridi had anti-arrhythmic effect. The potential ionic mechanisms are, however, not understood. The present study was designed to determine the effects of allitridi on cardiac potassium channels expressed in HEK 293 cells using a whole-cell patch voltage-clamp technique and mutagenesis. It was found that allitridi inhibited hKv4.3 channels (IC₅₀ = 11.4 µM) by binding to the open channel, shifting availability potential to hyperpolarization, and accelerating closed-state inactivation of the channel. The hKv4.3 mutants T366A, T367A, V392A, and I395A showed a reduced response to allitridi with IC₅₀s of 35.5 µM, 44.7 µM, 23.7 µM, and 42.4 µM. In addition, allitridi decreased hKv1.5, hERG, hKCNQ1/hKCNE1 channels stably expressed in HEK 293 cells with IC₅₀s of 40.2 µM, 19.6 µM and 17.7 µM. However, it slightly inhibited hKir2.1 current (100 µM, inhibited by 9.8% at −120 mV). Our results demonstrate for the first time that allitridi preferably blocks hKv4.3 current by binding to the open channel at T366 and T367 of P-loop helix, and at V392 and I395 of S6 domain. It has a weak inhibition of hKv1.5, hERG, and hKCNQ1/hKCNE1 currents. These effects may account for its anti-arrhythmic effect observed in experimental animal models.     

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

敬钊毒素-XI与突变体R3A-JZTX-XI的合成、复性及其药理活性鉴定

表达于b-胰岛细胞上的Kv2.1钾通道电流负责动作电位的复极化,从而调节胰岛素的分泌,是治疗2型糖尿病的有效作用靶点。敬钊毒素-XI(JZTX-XI) 是从敬钊缨毛蛛Chilobrachys jingzhao粗毒中分离纯化到的一种新型的肽类神经毒素,能够抑制非洲爪蟾卵母细胞上表达的Kv2.1钾通道电流。为了研究JZTX-XI的结构与功能关系,用芴甲氧羰基 (Fomc) 固相多肽合成方法合成了野生型JZTX-XI和突变体R3A-JZTX-XI,结合反相HPLC和质谱对不同条件下的氧化复性结果进行检测,从而得     

Kinetic analysis of phasic inhibition of neuronal sodium currents by lidocaine and bupivacaine.

Phasic ("use-dependent") inhibition of sodium currents by the tertiary amine local anesthetics, lidocaine and bupivacaine, was observed in voltage-clamped node of Ranvier of the toad, Bufo marinus. Local anesthetics were assumed to inhibit sodium channels through occupation of a binding site with 1:1 stoichiometry. A three-parameter empirical model for state-dependent anesthetic binding to the Na channel is presented: this model includes two discrete parameters that represent the time integrals of binding and unbinding reactions during a depolarizing pulse, and one continuous parameter that represents the rate of unbinding of drug between pulses. The change in magnitude of peak sodium current during a train of depolarizing pulses to 0 mV was used as an assay of the extent of anesthetic binding at discrete intervals; estimates of model parameters were made by applying a nonlinear least-squares algorithm to the inhibition of currents obtained at two or more depolarizing pulse rates. Increasing the concentration of drug increased the rate of binding but had little or no effect on unbinding, as expected for a simple bimolecular reaction. The dependence of the model parameters on pulse duration was assessed for both drugs: as the duration of depolarizing pulses was increased, the fraction of channels binding drug during each pulse became significantly larger, whereas the fraction of occupied channels unbinding drug remained relatively constant. The rate of recovery from block between pulses was unaffected by pulse duration or magnitude. The separate contributions of open (O) and inactivated (I) channel binding of drug to the net increase in block per pulse were assessed at 0 mV: for lidocaine, the forward binding rate ko was 1.3 x 10(5) M-1 s-1, kl was 2.4 x 10(4) M-1 s-1; for bupivacaine, ko was 2.5 x 10(5) M-1 s-1, kl was 4.4 x 10(4) M-1 s-1. These binding rates were similar to those derived from time-dependent block of maintained Na currents in nodes where inactivation was incomplete due to treatment with chloramine-T. The dependence of model parameters on the potential between pulses (holding potential) was examined. All three parameters were found to be nearly independent of holding potential from -70 to -100 mV. These results are discussed with respect to established models of dynamic local anesthetic-Na channel interactions.     

TEA prevents inactivation while blocking open K+ channels in human T lymphocytes.

The whole-cell recording mode of the patch-clamp technique was used to study the effect of external tetraethylammonium ([TEA+]o) on the inactivating, voltage-dependent K+ channels of human T lymphocytes. TEA+ reduced the peak amplitude and slowed the time course of the K+ current decay during a depolarizing pulse, resulting in a crossover of the current records in the presence and absence of TEA+. In solutions with different [TEA+]o both the peak K+ current amplitude, lKpeak, and the time constant of the decay of the K+ current, tau d, were reduced in a dose-dependent manner, both with apparent binding constants, KD, of 12 mM. The integral of K+ current during a prolonged depolarizing pulse was unaltered in solutions with different [TEA+]o. The concentration dependence of [TEA+]o on lKpeak, tau d, and the unchanged current integral can be explained with a kinetic scheme in which open channels blocked by TEA+ cannot inactivate.     

Modulation of voltage sensitivity by N-terminal cytoplasmic residues in human Kv1.2 channels

Potassium channels are now among the best understood membrane proteins and most salient functions have been mapped onto distinct portions of the protein. The detailed mechanism by which movement of the voltage sensor is transduced into channel opening is yet to be understood. We have constructed chimaeras from our collection of human voltage-gated potassium channels and expressed them in Xenopus oocytes. Here we report on a chimaeric construct, 1N/2, generated by swapping the N-terminal cytoplasmic residues of hKv1.1 onto the transmembrane body of hKv1.2. This chimaera functions as a classic outward rectifier but with a 25 mV hyperpolarizing shift in the mid-point of channel activation. The conductance of oocytes expressing this construct decreases significantly on depolarizing beyond +5 mV, unlike full-length hKv1.2. Other parameters such as ionic selectivity and charybdotoxin blockage are unaffected in making the chimaera. These observations suggest that the introduction of the "foreign" chain from hKv1.1 does not cause a large-scale perturbation of channel structure. Loss of the N-terminus from hKv1.2 is not responsible for the shift in voltage dependence, as a truncation construct, delta75N2, starting at the splice junction, has the same voltage-dependence as full-length hKv1.2. Both constructs show a maximum in their conductance-voltage curves. This decline in conductance on extensive depolarization may arise due to perturbations to the machinery that locks channels into their open state on depolarization. Taken together with our observations on other N-terminal swapped chimaeras, our data imply that N-terminal residues can interact with transmembrane regions and perturb the machinery mediating voltage-dependent channel gating.     

SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism

The functional interaction of the voltage-gated potassium channel hKv1.5 with the PDZ domain containing protein SAP97 has been investigated. In marked contrast with the known dependence of SAP97-induced Kv1 potassium current down-regulation on the channel C-termini, SAP97 increased hKv1.5 current through an indirect interaction with the Kv1.5 N-terminus. Deletion of the Kv1.5 N-terminus eliminated the SAP97-mediated increase in potassium currents whereas deletion of the channel's C-terminal PDZ binding motif had no effect. In contrast with other Kv1-SAP97 interactions, no physical interaction could be detected in vivo or in vitro between the two proteins. The proteins did not co-localize in cardiac myocytes nor did they co-immunoprecipitate from transfected HEK cells. Yeast two-hybrid experiments also failed to detect any interaction between the two proteins, but in one experiment of six, Kv1.5 co-immunoprecipitated very inefficiently with SAP97 from rat ventricular myocytes. Thus, we conclude that the influence of SAP97 on Kv1.5 potassium current levels is dependent upon a novel regulatory mechanism.     

Ranolazine inhibits shear sensitivity of endogenous Na+ current and spontaneous action potentials in HL-1 cells

Na_V1.5 is a mechanosensitive voltage-gated Na⁺ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na⁺ current and delayed rectifier (I_Kr) currents. Recently, ranolazine was also shown to be an inhibitor of Na_V1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na⁺ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na⁺ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.     

Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.     

Inhibition of Cav3.1 channel by silver ions

We have investigated the effects of AgCl and AgNO3 on the Cav3.1 calcium channels stably expressed in the HEK 293 cells. Ca2+ was used as a charge carrier. Both forms of Ag+ blocked the Cav3.1 channel and negatively shifted the I-V relations in a concentration-dependent manner. The inhibition of current amplitude by AgCl was voltage-dependent and increased with increasing amplitude of the depolarizing pulse. Furthermore, AgCl but not AgNO3 accelerated the kinetics of current activation. No effect on current inactivation or steady-state inactivation of the channel was observed for AgCl or AgNO3.