犬心室肌Iks通道的分布及Iso和chromanol 293B对其影响

目的:探讨LQT1发病的分子生物学基础以及LQT1患者在交感神经兴奋时发生室性心律失常的电生理机制。方法:实验分别采用westernblot方法检测慢激活延迟整流性钾电流(Iks)通道在犬左心室肌中层和外层的分布情况,以及用玻璃微电极和特制的金属电极,同步记录犬楔形心室肌中层和外层心肌细胞的跨膜动作电位(TAP)和跨壁心电图(TECG),并观察chromanol293B(Iks阻断剂)和异丙肾上腺素(Iso)对心室肌细胞动作电位时程(APD90)、跨壁复极离散度(TDR)、QT间期的影响。结果:Iks通道在中层的分布明显少于外层(P<0.01),chromanol293B使中层和外层心室肌细胞APD90均匀延长(P<0.05),TDR增加不明显(P>0.05),TECG示QT间期延长(P<0.05)。加入Iso后,中层心室肌细胞APD90延长(P<0.05),并出现早期后除极(EAD),外层心室肌细胞APD90缩短(P<0.05),TDR明显增加(P<0.05),TECG示QT间期延长(P<0.05),并能自发产生室性早搏和室性心动过速。结论:Iks通道在心室肌中层和外层分布不均一,而chromanol293...     

人胚肾(HEK293)细胞自身的钾通道

目的研究人胚肾(HEK293)细胞自身的电压依赖性的钾通道。方法利用全细胞膜片钳技术,记录HEK293细胞上的外向电流及其通道的电生理特点。结果HEK293细胞上未发现氯电流。59.7%的细胞表达一种类似瞬间外向钾电流(Ito)的钾电流,该电流对4-AP敏感,2mmol/L的4-AP可明显抑制此电流。在40mV时,此电流幅度为(175±112)pA。失活曲线显示其通道的半数失活电压为(-3.5±0.9)mV,斜率为(6.3±0.8)mV。该通道从失活状态中恢复的时间常数为(333.3±33.9)ms,其失活动力学曲线呈钟形。结论HEK293细胞自身存在Ito样电流,且此电压依赖性钾通道是其自身表达的最主要的有功能的离子通道,提示在利用此广泛应用的表达系统来研究外源性离子通道的时候,应该加倍注意。     

Stereoselective metabolism of bufuralol racemate and enantiomers in human liver microsomes

A new HPLC method was developed using a chiral column to efficiently separate four 1"-hydroxybufuralol (1"-OH-BF) diastereomers that are major metabolites of bufuralol (BF). Employing this method, we examined diastereomer selectivity in the formation of 1"-OH-BF from BF racemate or enantiomers in four individual samples of human liver microsomes. Three different human liver microsomes showed a selectivity of 1"R-OH < 1"S-OH for BF enantiomers, which was similar to that of recombinant CYP2D6 expressed in insect cell microsomes, whereas one human liver microsomal fraction yielded a selectivity of 1"R-OH > 1"S-OH for BF enantiomers, which was similar to those of recombinant CYP2C19 expressed in insect cell microsomes. Recombinant CYP1A2 and CYP3A4 showed a selectivity similar to that of CYP2D6, but their BF 1"-hydroxylase activities were much lower than those of CYP2D6. In inhibition studies, quinidine, a known CYP2D6 inhibitor, markedly inhibited BF 1"-hydroxylation in the fractions of human liver microsomes that showed the CYP2D6-type selectivity. Furthermore, omeprazole, a known CYP2C19 inhibitor, efficiently suppressed the formation of 1"-OH-BF diastereomers from BF in the microsomal fraction that showed the CYP2C19-type selectivity. From these results, we concluded that the diastereomer selectivity in the formation of 1"-OH-BF from BF differs between CYP2D6 and CYP2C19, both of which can be determinant enzymes in the diastereoselective 1"-hydroxylation of BF in human liver microsomes.     

Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels

Cytosolic reducing cofactors, such as NADPH and NADH, are thought to regulate vascular smooth muscle ion channel activity and vascular tone. In this study, the effects of pentose phosphate pathway (PPP) inhibitors, 6-aminonicotinamide (6-AN), epiandrosterone (EPI), and dehydroepiandrosterone (DHEA), on vascular tone were studied in isolated perfused lungs and pulmonary artery (PA) and aortic rings from rats. In addition, effects of 6-AN on voltage-gated K(+) (K(v)) current in PA smooth muscle cells (SMCs) were also examined. Pretreatment of lungs with 6-AN and EPI reduced the pressor response to acute hypoxia and decreased tissue NADPH levels. 6-AN, EPI, and DHEA relaxed isolated PA and aortic rings precontracted with 30 mM KCl in a dose-dependent manner. The PPP inhibitor-induced PA relaxations were reduced in PA rings precontracted with 80 mM KCl but not by pretreatment with nitro-L-arginine or endothelial removal. Pretreatment of PA rings with tetraethylammonium chloride or 4-aminopyridine caused rightward shifts of concentration-relaxation curves for 6-AN, EPI, and DHEA. In contrast, glybenclamide, charybdotoxin, or apamin did not inhibit the relaxant effects of 6-AN, EPI, and DHEA. 6-AN caused an increase in K(v) current in PASMC. These results indicate that reduction of NADPH by the PPP inhibitors causes vasodilation at least partly through opening of K(v) channels.     

Large conductance, calcium- and voltage-gated potassium (BK) channels: regulation by cholesterol

Cholesterol (CLR) is an essential component of eukaryotic plasma membranes. CLR regulates the membrane physical state, microdomain formation and the activity of membrane-spanning proteins, including ion channels. Large conductance, voltage- and Ca2?-gated K? (BK) channels link membrane potential to cell Ca2? homeostasis. Thus, they control many physiological processes and participate in pathophysiological mechanisms leading to human disease. Because plasmalemma BK channels cluster in CLR-rich membrane microdomains, a major driving force for studying BK channel-CLR interactions is determining how membrane CLR controls the BK current phenotype, including its pharmacology, channel sorting, distribution, and role in cell physiology. Since both BK channels and CLR tissue levels play a pathophysiological role in human disease, identifying functional and structural aspects of the CLR-BK channel interaction may open new avenues for therapeutic intervention. Here, we review the studies documenting membrane CLR-BK channel interactions, dissecting out the many factors that determine the final BK current response to changes in membrane CLR content. We also summarize work in reductionist systems where recombinant BK protein is studied in artificial lipid bilayers, which documents a direct inhibition of BK channel activity by CLR and builds a strong case for a direct interaction between CLR and the BK channel-forming protein. Bilayer lipid-mediated mechanisms in CLR action are also discussed. Finally, we review studies of BK channel function during hypercholesterolemia, and underscore the many consequences that the CLR-BK channel interaction brings to cell physiology and human disease.     

Stereoselective disposition of ibuprofen enantiomers in synovial fluid

The simultaneous disposition of the enantiomers of ibuprofen in synovial fluid and plasma was studied in eight patients with arthritis. Concentrations of the active S-enantiomer in synovial fluid exceeded those of the R-enantiomer at all times in all patients with the ratio of S to R concentrations being 2.1 +/- 0.3 (mean +/- SE). Synovial fluid concentrations fluctuated much less than in plasma and exceeded plasma concentrations from 5.4 +/- 0.3 hours for R-ibuprofen and 5.5 +/- 0.6 hours for S-ibuprofen. Pharmacokinetic analysis suggested that, although the enantiomers diffuse into synovial fluid primarily in the unbound form, there may be significant diffusion of the enantiomers out of synovial fluid in the protein-bound form in some patients. Interpatient differences in the disposition of the enantiomers of ibuprofen in synovial fluid were evident and may contribute to the interindividual variability in response to treatment with ibuprofen.     

Inherited disorders of voltage-gated sodium channels

A variety of inherited human disorders affecting skeletal muscle contraction, heart rhythm, and nervous system function have been traced to mutations in genes encoding voltage-gated sodium channels. Clinical severity among these conditions ranges from mild or even latent disease to life-threatening or incapacitating conditions. The sodium channelopathies were among the first recognized ion channel diseases and continue to attract widespread clinical and scientific interest. An expanding knowledge base has substantially advanced our understanding of structure-function and genotype-phenotype relationships for voltage-gated sodium channels and provided new insights into the pathophysiological basis for common diseases such as cardiac arrhythmias and epilepsy.     

Direct block of human ether-a-go-go-related gene potassium channels by caffeine

The human ether-a-go-go-related gene (hERG) potassium channel is expressed in a variety of cell types, including neurons, tumor cells, and cardiac myocytes. In the heart, it is important for repolarization of the cardiac action potential. Attenuation of hERG current can cause long QT syndrome and cardiac arrhythmias such as torsades de pointes. Caffeine is frequently used as a pharmacological tool to study calcium-dependent transduction pathways in cellular preparations. It raises cytosolic calcium by opening ryanodine receptors and may also inhibit phosphodiesterases to increase cytosolic cAMP. In this study, we show 5 mM caffeine rapidly and reversibly attenuates hERG currents expressed in human embryonic kidney 293 cells to 61.1 +/- 2.2% of control. Caffeine-dependent inhibition of hERG current is not altered by raising cAMP with forskolin, buffering cytosolic calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or inhibition of protein kinase C. Thus, the effects of caffeine are unlikely to be mediated by cAMP or intracellular calcium-dependent mechanisms. Further experiments showed caffeine directly blocks hERG in an open state-dependent manner. Furthermore, caffeine inhibition is greatly reduced by the pore mutants Y562A and F656A hERG, which disrupt block of most previously tested hERG antagonists. Thus, caffeine attenuates hERG currents by binding to a drug receptor located within the inner cavity of the channel. Dietary intake of caffeine is unlikely to cause long QT syndrome because plasma concentrations do not reach sufficiently high levels to significantly inhibit hERG currents. However, the effects of caffeine have implications for its use in examining calcium-dependent pathways in cellular preparations expressing hERG.     

Voltage-gated potassium channels are required for human T lymphocyte activation

The calcium channel blockers, verapamil and diltiazem, inhibit phytohemagglutinin (PHA)-induced mitogenesis at concentrations that block the T lymphocyte K channel currents. K channel blockers also inhibit the allogeneic mixed lymphocyte response in a dose-dependent manner with the same potency sequence as for block of K currents. K channel blockers inhibit PHA-stimulated mitogenesis only if added during the first 20-30 h after PHA addition, but not later, indicating a requirement for functional K channels during this period. We investigated the effect of K channel blockers on various aspects of protein synthesis for two reasons: first, protein synthesis appears to be necessary for the events leading to DNA synthesis, and second, the increase in the protein synthetic rate commences during the first 24-48 h after PHA addition. PHA-induced total protein synthesis was reduced to the level in unstimulated T lymphocytes by K channel blockers in a dose-dependent manner with the same potency sequence as for the block of K currents and inhibition of [3H]thymidine incorporation. Two-dimensional gel electrophoresis demonstrated that although the synthesis of the majority of proteins was reduced by K channel blockers to the level in unstimulated T cells, some proteins continued to be synthesized at an enhanced rate compared with resting cells. Two proteins, S and T, detected by two-dimensional gel electrophoresis in unstimulated T lymphocytes, appeared to be reduced in intensity in gels of PHA-treated T lymphocytes, in contrast to the increased synthesis of the remaining proteins. 4-Aminopyridine (4-AP), at concentrations that inhibit protein synthesis, prevented the apparent PHA-induced reduction of proteins S and T. These proteins may play a role in maintaining the T lymphocyte in a resting state and may be related to the translation inhibitory factors reported to be present at a higher specific activity in quiescent T lymphocytes than in PHA-activated T cells. The expression of the IL-2 receptor (Tac) during T lymphocyte activation was not altered by K channel blockers, whereas the production of interleukin 2 (IL-2) was reduced to the level in unstimulated T lymphocytes. Exogenous IL-2 partially relieved the inhibition of mitogenesis by low, but not by high, concentrations of 4-AP. These experiments clarify the role of K channels in T lymphocyte activation and suggest that functional K channels are required either for protein synthesis or for events leading to protein synthesis.     

Interactions of the antimalarial drug mefloquine with the human cardiac potassium channels KvLQT1/minK and HERG

Mefloquine is a quinoline antimalarial drug that is structurally related to the antiarrhythmic agent quinidine. Mefloquine is widely used in both the treatment and prophylaxis of Plasmodium falciparum malaria. Mefloquine can prolong cardiac repolarization, especially when coadministered with halofantrine, an antagonist of the human ether-a-go-go-related gene (HERG) cardiac K+ channel. For these reasons we examined the effects of mefloquine on the slow delayed rectifier K+ channel (KvQT1/minK) and HERG, the K+ channels that underlie the slow (I(Ks)) and rapid (I(Kr)) components of repolarization in the human myocardium, respectively. Using patch-clamp electrophysiology we found that mefloquine inhibited KvLQT1/minK channel currents with an IC50 value of approximately 1 microM. Mefloquine slowed the activation rate of KvLQT1/minK and more block was evident at lower membrane potentials compared with higher ones. When channels were held in the closed state during drug application, block was immediate and complete with the first depolarizing step. HERG channel currents were about 6-fold less sensitive to block by mefloquine (IC50 = 5.6 microM). Block of HERG displayed a positive voltage dependence with maximal inhibition obtained at more depolarized potentials. In contrast to structurally related drugs such as quinidine, mefloquine is a more effective antagonist of KvLQT1/minK compared with HERG. Block of KvLQT1/minK by mefloquine may involve an interaction with the closed state of the channel. Inhibition by mefloquine of KvLQT1/minK in the human heart may in part explain the synergistic prolongation of QT interval observed when this drug is coadministered with the HERG antagonist halofantrine.     

Molecular biology of voltage-gated K+ channels in heart

Summary— The recent cloning of numerous voltage-activated K+ channels provides new information concerning the architecture of K+ channel proteins. The combination of molecular genetic and biophysical methods gives us a new insight into the molecular mechanisms of K+ channel pharmacology.     

Effects of inhibition of ATP-sensitive potassium channels on metabolic vasodilation in the human forearm

Experimental data suggest that vascular ATP-sensitive potassium (K(ATP)) channels may be an important determinant of functional hyperaemia, but the contribution of K(ATP) channels to exercise-induced hyperaemia in humans is unknown. Forearm blood flow was assessed in 39 healthy subjects (23 males/16 females; age 22+/-4 years) using the technique of venous occlusion plethysmography. Resting forearm blood flow and functional hyperaemic blood flow (FHBF) were measured before and after brachial artery infusion of the K(ATP) channel inhibitors glibenclamide (at two different doses: 15 and 100 microg/min) and gliclazide (at 300 microg/min). FHBF was induced by 2 min of non-ischaemic wrist flexion-extension exercise at 45 cycles/min. Compared with vehicle (isotonic saline), glibenclamide at either 15 microg/min or 100 microg/min did not significantly alter resting forearm blood flow or peak FHBF. The blood volume repaid at 1 and 5 min after exercise was not diminished by glibenclamide. Serum glucose was unchanged after glibenclamide, but plasma insulin rose by 36% (from 7.2+/-0.8 to 9.8+/-1.3 m-units/l; P =0.02) and 150% (from 9.1+/-1.3 to 22.9+/-3.5 m-units/l; P =0.002) after the 15 and 100 microg/min infusions respectively. Gliclazide also did not affect resting forearm blood flow, peak FHBF, or the blood volume repaid at 1 and 5 min after exercise, compared with vehicle (isotonic glucose). Gliclazide induced a 12% fall in serum glucose (P =0.009) and a 38% increase in plasma insulin (P =0.001). Thus inhibition of vascular K(ATP) channels with glibenclamide or gliclazide does not appear to affect resting forearm blood flow or FHBF in healthy humans. These findings suggest that vascular K(ATP) channels may not play an important role in regulating basal vascular tone or skeletal muscle metabolic vasodilation in the forearm of healthy human subjects.     

High-affinity blockade of human ether-a-go-go-related gene human cardiac potassium channels by the novel antiarrhythmic drug BRL-32872

Human ether-a-go-go-related gene (HERG) potassium channels are one primary target for the pharmacological treatment of cardiac arrhythmias by class III antiarrhythmic drugs. These drugs are characterized by high antiarrhythmic efficacy, but they can also initiate life-threatening "torsade de pointes" tachyarrhythmias. Recently, it has been suggested that combining potassium and calcium channel blocking mechanisms reduces the proarrhythmic potential of selective class III antiarrhythmic agents. BRL-32872 is a novel antiarrhythmic drug that inhibits potassium and calcium currents in isolated cardiomyocytes. In our study, we investigated the effects of BRL-32872 on cloned HERG channels heterologously expressed in Xenopus oocytes. Using the two-microelectrode voltage clamp technique, we found that BRL-32872 caused a high-affinity, state-dependent block of open HERG channels (IC(50) = 241 nM) in a frequency-dependent manner with slow unbinding kinetics. Inactivated channels mainly had to open to be blocked by BRL-32872. The HERG S620T mutant channel, which has a strongly reduced degree of inactivation, was 51-fold less sensitive to BRL-32872 block, indicating that BRL-32872 binding was enhanced by the inactivation process. In an additional approach, we studied HERG channels expressed in a human cell line (HEK 293) using the whole-cell patch-clamp technique. BRL-32872 inhibited HERG currents in HEK 293 cells in a dose-dependent manner, with an IC(50) value of 19.8 nM. We conclude that BRL-32872 is a potent blocker of HERG potassium channels, which accounts for the class III antiarrhythmic action of BRL-32872.     

Effects of irbesartan on cloned potassium channels involved in human cardiac repolarization

We studied the effects of irbesartan, a selective angiotensin II type 1 receptor antagonist, on human ether-a-go-go-related gene (HERG), KvLQT1+minK, hKv1.5, and Kv4.3 channels using the patch-clamp technique. Irbesartan exhibited a low affinity for HERG and KvLQT1+minK channels (IC(50) = 193.0 +/- 49.8 and 314.6 +/- 85.4 microM, respectively). In hKv1.5 channels, irbesartan produced two types of block, depending on the concentration tested. At 0.1 microM, irbesartan inhibited the current in a time-dependent manner (22 +/- 3.9% at +60 mV). The blockade increased steeply with channel activation increasing at more positive potentials. However, at 10 microM, irbesartan induced a time-independent blockade that occurred in the range of potentials of channel opening, reaching its maximum at approximately 0 mV, and remaining unchanged at more positive potentials (24.0 +/- 1.0% at +60 mV). In Kv4.3 currents, irbesartan produced a concentration-dependent block, which resulted in two IC(50) values (1.0 +/- 0.1 nM and 7.2 +/- 0.6 microM). At 1 microM, it inhibited the peak current and accelerated the time course of inactivation, decreasing the total charge crossing the membrane (36.6 +/- 7.8% at +50 mV). Irbesartan shifted the inactivation curve of Kv4.3 channels, the blockade increasing as the amount of inactivated channels increased. Molecular modeling was used to define energy-minimized dockings of irbesartan to hKv1.5 and HERG channels. In conclusion, irbesartan blocks Kv4.3 and hKv1.5 channels at therapeutic concentrations, whereas the blockade of HERG and KvLQT1+minK channels occurred only at supratherapeutic levels. In hKv1.5, a receptor site is apparent on each alpha-subunit of the channel, whereas in HERG channels a common binding site is present at the pore.     

Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins

Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.     

Involvement of voltage-gated sodium channels blockade in the analgesic effects of orphenadrine

Orphenadrine is a drug acting on multiple targets, including muscarinic, histaminic, and NMDA receptors. It is used in the treatment of Parkinson’s disease and in musculoskeletal disorders. It is also used as an analgesic, although its mechanism of action is still unknown. Both physiological and pharmacological results have demonstrated a critical role for voltage-gated sodium channels in many types of chronic pain syndromes. We tested the hypothesis that orphenadrine may block voltage-gated sodium channels. By using patch-clamp experiments, we evaluated the effects of the drug on whole-cell sodium currents in HEK293 cells expressing the skeletal muscle (Nav1.4), cardiac (Nav1.5) and neuronal (Nav1.1 and Nav1.7) subtypes of human sodium channels, as well as on whole-cell tetrodotoxin (TTX)-resistant sodium currents likely conducted by Nav1.8 and Nav1.9 channel subtypes in primary culture of rat DRG sensory neurons. The results indicate that orphenadrine inhibits sodium channels in a concentration-, voltage- and frequency-dependent manner. By using site-directed mutagenesis, we further show that orphenadrine binds to the same receptor as the local anesthetics. Orphenadrine affinities for resting and inactivated sodium channels were higher compared to those of known sodium channels blockers, such as mexiletine and flecainide. Low, clinically relevant orphenadrine concentration produces a significant block of Nav1.7, Nav1.8, and Nav1.9 channels, which are critical for experiencing pain sensations, indicating a role for sodium channel blockade in the clinical efficacy of orphenadrine as analgesic compound. On the other hand, block of Nav1.1 and Nav1.5 may contribute to the proconvulsive and proarrhythmic adverse reactions, especially observed during overdose.