Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes

The potassium channel Kv1.3 has recently been located to the inner mitochondrial membrane of lymphocytes. Here, we show that mouse and human cells that are genetically deficient in either Kv1.3 or transfected with siRNA to suppress Kv1.3-expression resisted apoptosis induced by several stimuli, including Bax over-expression. Retransfection of either Kv1.3 or a mitochondrial-targeted Kv1.3 restored cell death. Bax interacted with and functionally inhibited mitochondrial Kv1.3. Incubation of isolated Kv1.3-positive mitochondria with recombinant Bax, t-Bid, or toxins that bind to and inhibit Kv1.3 successively triggered hyperpolarization, formation of reactive oxygen species, release of cytochrome c, and marked depolarization. Kv1.3-deficient mitochondria were resistant to Bax, t-Bid, and the toxins. Mutation of Bax at K128, which corresponds to a conserved lysine in Kv1.3-inhibiting toxins, abrogated its effects on both Kv1.3 and mitochondria. These findings suggest that Bax mediates cytochrome c release and mitochondrial depolarization in lymphocytes, at least in part, via its interaction with mitochondrial Kv1.3.     

Extracellular acidosis modulates drug block of Kv4.3 currents by flecainide and quinidine

INTRODUCTION: As a molecular model of the effect of ischemia on drug block of the transient outward potassium current, the effect of acidosis on the blocking properties of flecainide and quinidine on Kv4.3 currents was studied. METHODS AND RESULTS: Kv4.3 channels were stably expressed in Chinese hamster ovary cells. Whole-cell, voltage clamp techniques were used to measure the effect of flecainide and quinidine on Kv4.3 currents in solutions of pH 7.4 and 6.0. Extracellular acidosis attenuated flecainide block of Kv4.3 currents, with the IC50 for flecainide (based on current-time integrals) increasing from 7.8 +/- 1.1 microM at pH 7.4 to 125.1 +/- 1.1 microM at pH 6.0. Similar effects were observed for quinidine (IC50 5.2 +/- 1.1 microM at pH 7.4 and 22.1 +/- 1.3 microM at pH 6.0). Following block by either drug, Kv4.3 channels showed a hyperpolarizing shift in the voltage sensitivity of inactivation and a slowing in the time to recover from inactivation/block that was unaffected by acidosis. In contrast, acidosis attenuated the effects on the time course of inactivation and the degree of tonic- and frequency-dependent block for both drugs. CONCLUSION: Extracellular acidosis significantly decreases the potency of blockade of Kv4.3 by both flecainide and quinidine. This change in potency may be due to allosteric changes in the channel, changes in the proportion of uncharged drug, and/or changes in the kinetics of drug binding or unbinding. These findings are in contrast to the effects of extracellular acidosis on block of the fast sodium channel by these agents and provide a molecular mechanism for divergent modulation of drug block potentially leading to ischemia-associated proarrhythmia.     

Selectivity filter ion binding affinity determines inactivation in a potassium channel

Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca²⁺-activated K⁺ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K⁺ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K⁺ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K⁺, similar to KcsA, but that even a single K⁺ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K⁺ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

Ion permeation gating within the selectivity filter (SF) of potassium (K⁺) channels has been proposed to control channel activity in different ways for different family members. In voltage-dependent K⁺ (K_V) channels, the SF has been proposed to underlie C-type inactivation (1–3), resulting in the progressive loss of current following the activation of a channel gate located near the intracellular side of the pore (4). C-type inactivation in K_V channels has been shown to be strongly dependent on the affinity of a particular binding site for permeant ions in the pore, and the affinity of these pore sites has been proposed to depend not only on the SF chemical composition, but also on regions outside of the SF (5–8). A structure-function model for this mechanism has been provided most specifically by studies of the proton-gated KcsA channel (9–11) where opening of the activation gate is correlated with a conformational constriction and a decrease in ion occupancy within the four K⁺ binding sites of the SF (named S1 to S4) (9, 12–15). Experimental and structural studies of KcsA in low K⁺ showed that the SF constriction consists of an outward flip of the carbonyl groups of the Gly77, in the middle of the signature sequence (TVGYG) of K⁺ channels, associated with a loss of K⁺ binding at site S2 of the SF (9, 14, 16–20). These conformational changes were accompanied by the binding of several water molecules behind the SF, stabilizing this constricted (also called flipped) state by sterically preventing the SF from switching back into its conductive state (16, 18–20). While some reports challenge this view (21, 22), this activation gate-coupled collapse of the SF is now generally accepted as the mechanism underlying C-type inactivation in K⁺ channels.Several types of ligand-dependent K⁺ channels, including those opened by binding Ca²⁺, such as the BK and MthK channels (23–27), do not exhibit traditional C-type inactivation, despite possessing an identical SF with K_V and KcsA channels. Furthermore, these channels have been proposed to actually gate at the SF (28–32) although a recent cryogenic electron microscopy (cryo-EM) structure of MthK in the absence of calcium (33) revealed a steric closure at the bundle crossing inner gate region, suggesting that there may be two gates involved in calcium gating. Nevertheless, at this time, the structural correlates of SF gating and the difference from inactivation are unknown.In the present study, we set out to first investigate whether we can capture different gating states of MthK by obtaining X-ray structures of its pore (Fig. 1A) in wide-ranging concentrations of K⁺. MthK channels have been previously shown to display a decrease in activity with depolarization, which is further augmented when external K⁺ concentration is lowered, a signature of SF gating and a hallmark of C-type inactivation (34). We reasoned that K⁺ titration of MthK pore structures may provide insights into the molecular causes for K⁺-dependent SF gating and will indicate whether it shares features with the C-type inactivation observed in KcsA (such as a collapsed SF). Unlike KcsA, MthK SF did not collapse in similarly low K⁺ concentrations, suggesting that the clue to why MthK does not display traditional C-type inactivation may lie in understanding the molecular underpinnings that contribute to SF conformational change. Thus, we next investigated the dependence of SF conformation on ion occupancy and used molecular dynamics (MD) simulations to reveal a uniquely low affinity central S2 site in MthK, which may play a lead role in the SF-based channel closure. Overall, our results illustrate how the exact same sequence and structure of the SF in a K⁺ channel can lead to slight variations in K⁺ binding site chemistry, which in turn can lead to distinct functional phenotypes.Open in a separate windowFig. 1.Structure of the MthK pore in different K⁺ concentrations. (A) Overall architecture of wild-type MthK pore structure (three subunits of the tetrameric pore shown for clarity) crystallized with 150 mM K⁺. The SF is highlighted by dashed lines. Alignment of this structure with that crystallized in 6 mM K⁺ yields an all-atom root-mean-square deviation (RMSD) value of 0.25 Å. (B) K⁺-omit electron density maps (2F_o – F_c contoured at 2.0 σ) for SF atoms from two opposing subunits. Structures were solved in 150, 50, 11, and 6 mM [K⁺], as indicated. Crystallographic statistics are in SI Appendix, Table S1. (C) MD system with the MthK (ribbons) embedded in a lipid bilayer (gray sticks) bathed in 200 mM KCl (K⁺ as green spheres, Cl⁻ as blue spheres, and water as red and white sticks).     

A double-point mutation in the selectivity filter site of the KCNQ1 potassium channel results in a severe phenotype, LQT1, of long QT syndrome

Introduction: Slowly activating delayed-rectifier potassium currents in the heart are produced by a complex protein with α and β subunits composed of the potassium voltage-gated channel KQT-like subfamily, member 1 (KCNQ1) and the potassium voltage-gated channel Isk-related family, member 1 (KCNE1), respectively. Mutations in KCNQ1 underlie the most common type of hereditary long QT syndrome (LQTS). Like other potassium channels, KCNQ1 has six transmembrane domains and a highly conserved potassium selectivity filter in the pore helix called "the signature sequence." We aimed to investigate the functional consequences of a newly identified mutation within the signature sequence.
Methods and Results: Potassium channel genomic DNA from a family with clinical evidence of LQTS was amplified by polymerase chain reaction (PCR), and the resulting products were then sequenced. Three family members had a double-point mutation in KCNQ1 at nucleotides 938 (T-to-A) and 939 (C-to-A), resulting in an isoleucine-to-lysine change at amino acid position 313. These patients displayed prolonged QTc intervals (629, 508, and 500 ms^1/2, respectively) and repetitive episodes of syncope, but no deafness. Three-dimensional structure modeling of KCNQ1 revealed that this mutation is located at the center of the channel pore. COS-7 cells displayed a lack of current when transfected with a plasmid expressing the mutant. In addition, the mutant displayed a dominant negative effect on current but appeared normal with respect to plasma membrane integration.
Conclusion: An I313K mutation within the selectivity filter of KCNQ1 results in a dominant-negative loss of channel function, leading to a long QT interval and subsequent syncope.     

On the selective ion binding hypothesis for potassium channels

The mechanism by which K(+) channels select for K(+) over Na(+) ions has been debated for the better part of a century. The prevailing view is that K(+) channels contain highly conserved sites that selectively bind K(+) over Na(+) ions through optimal coordination. We demonstrate that a series of alternating sites within the KcsA channel selectivity filter exists, which are thermodynamically selective for either K(+) (cage made from two planes of oxygen atoms) or Na(+) ions (a single plane of four oxygen atoms). By combining Bennett free energy perturbation calculations with umbrella sampling, we show that when K(+) and Na(+) are both permitted to move into their preferred positions, the thermodynamic preference for K(+) over Na(+) is significantly reduced throughout the entire selectivity filter. We offer a rationale for experimental measures of thermodynamic preference for K(+) over Na(+) from Ba(2+) blocking data, by demonstrating that the presence of Ba(2+) ions exaggerates K(+) over Na(+) thermodynamic stability due to the different binding locations of these ions. These studies reveal that K(+) channel selectivity may not be associated with the thermodynamics of ions in crystallographic K(+) binding sites, but requires consideration of the kinetic barriers associated with the different multi-ion permeation mechanisms.     

替米沙坦对电压依赖性的Kv1.3和Kv1.5通道电流阻断作用的实验研究

目的 通过观察替米沙坦对电压依赖性的Kv1.3和Kv1.5的阻断作用,探讨替米沙坦对此类通道的阻断可能具有的临床作用.方法 使用双电极电压钳技术记录表达于非洲爪蟾卵母细胞的Kv1.3和Kv1.5钾通道电流,不同浓度灌流观察其对电流影响.结果 (1)替米沙坦浓度依赖性的阻断Kv1.3通道,其阻断的IC50是2.05 μmol/L.替米沙坦对Kv1.3电流的阻断具有电压依赖性.(2)替米沙坦浓度依赖件的阻断Kv1.5通道,其阻断的IC50是2.37 μmol/L.替米沙坦对Kv1.5电流的阻断具有更显著的电压依赖性.结论 替米沙坦阻断开放状态的Kv1.3可能是其发挥免疫调节和抗动脉粥样硬化作用的机制之一.替米沙坦对开放状态的Kv1.5钾通道的阻断可能是其减少心房颤动发生率的作用机制之一.     

The molecular and ionic specificity of antiarrhythmic drug actions

Virtually all clinical antiarrhythmic agents act by reducing ion channel conductance, with sodium (Na+), potassium (K+), and calcium (Ca++) channels the primary targets. Na+ channel blockers increase the risk of ischemic ventricular fibrillation and are relatively contraindicated in the presence of active coronary heart disease. Ca++ channel blockers suppress AV nodal conduction and are used to terminate reentrant supraventricular arrhythmias and control the ventricular response to atrial fibrillation. K+ channels constitute the most diverse group of cardiac ion channels. They are the primary targets of Class III antiarrhythmic drugs, the category of such agents presently undergoing the most active development. The rapid delayed rectifier, IKr, plays a key role in repolarization of all cardiac tissues and is the most common (and often only) target of action potential-prolonging drugs. Unfortunately, because of the ubiquity of IKr and the reverse use-dependent action potential prolongation that results from blocking it, IKr blockers are likely to cause torsades de pointes ventricular proarrhythmia. K+ channel blockers, such as amiodarone and azimilide, that affect the slow delayed rectifier IKs as well as IKr, appear to produce a more desirable rate-dependent profile of Class III action. Recently, much has been learned about the molecular basis of K+ channels based on their role in the congenital long QT syndrome. The availability of molecular clones that encode many of the channels in the human heart allows for the rapid screening of many potential new drugs, making possible the development of "designer" antiarrhythmic drugs with specific profiles of channel-blocking selectivity.     

普罗帕酮对Kv1.4ΔN钾通道的作用及细胞外钾离子和pH浓度变化时的影响

目的:探讨抗心律失常药物普罗帕酮对Kv1.4△N钾通道的作用,以及细胞外钾离子和pH浓度变化时对该作用的影响,并探讨该作用可能的机制.方法:将Kv1.4ΔN的mRNA注射入非洲爪蟾卵母细胞并使用双电极钳制法观察普罗帕酮对Kv1.4ΔN电生理特性的影响,以及细胞外钾离子和pH变化时的电生理特性改变.结果:pH7 4状态下,普罗帕酮对Kv1.4ΔN通道的峰电流有抑制作用,这种阻滞作用具有电压依赖性、浓度依赖性以及频率依赖性,并且随电位的升高而作用加强,符合单指数和线性关系.普罗帕酮加速电流的失活过程.在不同的钾离子浓度下,这种阻滞作用具有pH依赖性,细胞外高钾pH7 4时,不同浓度普罗帕酮灌流显示IC50为121 μmol/L;细胞外酸性环境下(pH6 0)IC50提高到463 μmol/L,碱性化的环境(pH8 0)降至58 μmol/L.结论:普罗帕酮是Kv1.4ΔN的阻滞剂,可能与作用于细胞内的某些位点有关.     

A repulsion mechanism explains magnesium permeation and selectivity in CorA

Magnesium (Mg²⁺) plays a central role in biology, regulating the activity of many enzymes and stabilizing the structure of key macromolecules. In bacteria, CorA is the primary source of Mg²⁺ uptake and is self-regulated by intracellular Mg²⁺. Using a gating mutant at the divalent ion binding site, we were able to characterize CorA selectivity and permeation properties to both monovalent and divalent cations under perfused two-electrode voltage clamp. The present data demonstrate that under physiological conditions, CorA is a multioccupancy Mg²⁺-selective channel, fully excluding monovalent cations, and Ca²⁺, whereas in absence of Mg²⁺, CorA is essentially nonselective, displaying only mild preference against other divalents (Ca²⁺ > Mn²⁺ > Co²⁺ > Mg²⁺ > Ni²⁺). Selectivity against monovalent cations takes place via Mg²⁺ binding at a high-affinity site, formed by the Gly-Met-Asn signature sequence (Gly312 and Asn314) at the extracellular side of the pore. This mechanism is reminiscent of repulsion models proposed for Ca²⁺ channel selectivity despite differences in sequence and overall structure.Among biological divalent cations, Mg²⁺ is not only the most abundant, but also plays an essential role in a wealth of cellular processes, including enzymatic reactions, and the stability of nucleic acids and biological membranes (1). Although the biological importance of Mg²⁺ is well established, the molecular entities and mechanisms that govern its cellular homeostasis are not well understood. In bacteria, Mg²⁺ influx is primarily catalyzed by members of the CorA family of divalent ion transport systems (2, 3). The X-ray structure of CorA has provided an excellent template toward a molecular understanding of the mechanisms underlying Mg²⁺ influx (4–7). However, although CorA has been crystallized in a wide range of conditions, so far all available CorA structures seem to correspond to nonconductive conformations, which obviously limits the basic mechanistic insights regarding Mg²⁺ selectivity and translocation that can be derived from these high-resolution structures. Computational analyses, together with NMR, X-ray absorption, and Raman spectroscopy studies, have established that Mg²⁺ holds to its first hydration shell much more tightly than any other physiological cation (8–11); this implies that any Mg²⁺-selective transport system must either compensate for the high hydration energy (and accommodate the invariable octahedral geometry of this hexacoordinated ion) or establish a selectivity mechanism able to discriminate a hydrated or partially hydrated Mg²⁺ ion from monovalent and other divalent cations.Several hypotheses have been postulated to explain CorA’s function, including its role as a Mg²⁺ -selective channel (12), a Co²⁺ transporter (13), and even as an exporter of divalent cations (14). However, detailed mechanistic evaluation of CorA’s functional properties has been limited by the resolution of existing functional assays (15). Mg²⁺ transport through CorA depends on the combination of three parameters: (i) number of open gates, (ii) the electrical potential across the membrane, and (iii) the Mg²⁺ driving force, none of which can be properly controlled with sufficient time-resolution in in vivo experiments. Although a prokaryotic membrane protein, we have been able to heterologously express CorA in Xenopus oocytes, which, in combination with standard electrophysiological approaches, allowed us to measure CorA-catalyzed divalent macroscopic currents under a variety of ionic conditions. Crystallographic studies have suggested that intracellular Mg²⁺ act as the main regulator of CorA gating under physiological conditions (6). That Mg²⁺ acts as both a gating ligand and charge carrier ultimately complicates functional studies of CorA permeation and selectivity properties. To circumvent this issue we used a mutation at the divalent cation sensor that abolishes CorA Mg²⁺-dependent gating (Fig. 1A). This construct is ideally suited to evaluate ion permeation because it stabilizes steady-state currents by inhibiting the divalent ion-driven negative-feedback loop that defines CorA gating. Our results demonstrate that CorA is a bona fide multioccupancy ion channel, and that its divalent cation permeation and tight selectivity against monovalent cations can be explained on the basis of a block and repulsion mechanism, where the canonical “signature sequence” Gly-Met-Asn (GMN) plays a central role.Open in a separate windowFig. 1.CorA-driven Mg²⁺ currents recorded from TEVC. (A) The divalent cation sensor is highlighted on a cartoon representation of CorA crystal structure. Residues Asp89 and Asp253 are shown as purple sticks (B). The membrane potential (Vm) is clamped and held at −60 mV. The external solution is exchanged between two isosmotic buffers: one containing no monovalent or divalent cation (colored in gray on the horizontal bar), and one containing 20 mM Mg²⁺ (noted Mg²⁺). A representative trace recorded on a CorA–WT-expressing oocyte is shown in teal, and control oocyte trace is shown in gray and D253K in purple. The horizontal dotted line indicates the 0 A current level. (C) Representative traces of CorA D253K mutant in TEVC. The voltage pulse protocol is shown on top of the current traces. The dotted line represents the 0 current levels. (D) The corresponding I/V relationships recorded at different external Mg²⁺ concentrations are shown. The GHK-flux equation fits are displayed as solid lines, and experimental values are dots. (E) Mg²⁺ current recorded at −60 mV under external solution perfusion. The external solution is changed stepwise and the corresponding solution exchange protocol is superimposed to the trace. (F) Mean values (±SD) of several traces (n ≥ 5) were recorded and normalized to the maximum current. The values were plotted against the external [Mg²⁺] and fitted with a single-site binding curve.     

A polyether biotoxin binding site on the lipid-exposed face of the pore domain of Kv channels revealed by the marine toxin gambierol

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC₅₀ of 1.2 ± 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 μM gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1–Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.     

Endocannabinoids and cannabinoid analogues block human cardiac Kv4.3 channels in a receptor-independent manner

Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K⁺ channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K⁺ current (I_to1). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I_Kv4.3 in a concentration-dependent manner, AEA and 2-AG being the most potent (IC₅₀ ∼ 0.3-0.4 µM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I_to1 recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.     

Pathogenic plasticity of Kv7.2/3 channel activity is essential for the induction of tinnitus

Tinnitus, the perception of phantom sound, is often a debilitating condition that affects many millions of people. Little is known, however, about the molecules that participate in the induction of tinnitus. In brain slices containing the dorsal cochlear nucleus, we reveal a tinnitus-specific increase in the spontaneous firing rate of principal neurons (hyperactivity). This hyperactivity is observed only in noise-exposed mice that develop tinnitus and only in the dorsal cochlear nucleus regions that are sensitive to high frequency sounds. We show that a reduction in Kv7.2/3 channel activity is essential for tinnitus induction and for the tinnitus-specific hyperactivity. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. Our in vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. Together, our studies provide an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Moreover, our findings point to previously unknown biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans.     

替米沙坦对表达在卵母细胞上Kv1.5通道的抑制效应

目的:研究替米沙坦对表达在卵母细胞上的克隆人类Kv1.5通道的作用,探讨其在心脏复极中的潜在效应。方法:在非洲爪蟾卵母细胞上异源表达克隆人类Kv1.5通道基因,使用双电极电压钳技术记录全细胞电流,检测药物对Ikur电流的影响。结果:替米沙坦以电压依赖性和浓度依赖性方式抑制Kv1.5通道电流,且对峰电流及1.5s末端电流的抑制效应不同,在1μmol/L浓度下,抑制效应分别达到(7.75±2.39)和(52.64±3.77),其半抑制浓度(IC50)分别为(2.25±0.97)μmol/L和(0.82±0.39)μmol/L。替米沙坦对通道的稳态失活没有显著改变,在对照条件下,V1/2的值为(14.47±3.71)mV,斜坡因子k为(23.24±3.86)mV;在1μmol/L替米沙坦作用下,V1/2和k的值分别为(14.38±4.62)mV和(26.26±5.04)mV(n=6,P>0.05)。同时,替米沙坦显著加速了Kv1.5通道的失活。在对照条件下,Kv1.5通道的失活慢时间常数是(693.74±23.16)ms,在应用1μmol/L替米沙坦后,其失活的慢时间常数下降为(523.85±10.28)ms(n=5,P<0.05)。结论:替米沙坦在临床有效浓度范围内能显著抑制表达在卵母细胞上的Ikur电流,提示它兼有选择性阻滞Kv1.5通道的作用。     

大鼠动脉粥样硬化斑块中T淋巴细胞亚群及Kv1.3通道蛋白的表达

目的探究Kv1.3通道蛋白与动脉粥样硬化(AS)模型中活化的T淋巴细胞间的关系。方法 Wistar雄性大鼠24只,随机分为对照组(n=10)和AS组(n=14),采用高脂饲料喂养方法建立AS模型。分别于实验开始前,实验第8周,实验第12周观察各组大鼠体重变化。于第12周处死大鼠前取血,检测血清中总胆固醇(TC)、低密度脂蛋白(LDL-L)、高密度脂蛋白(HDL-L)和甘油三酯(TG)的水平。通过病理HE染色及免疫组织化学方法观察AS斑块内T淋巴细胞亚群分布和Kv1.3通道蛋白表达的改变。结果 AS组体重、TC、LDL-C较对照组明显升高(P均<0.05);HDL-C和TG两组无差异。AS组主动脉管壁可见明显斑块形成,对照组血管壁各层的组织结构正常。AS组动脉斑块部位内膜下及中膜层可见CD4+与CD8+T淋巴细胞聚集,以CD4+T淋巴细胞聚集为主,在病变部位Kv1.3通道蛋白表达增加。对照组血管内膜、中膜中未见T淋巴细胞聚集及KV1.3通道蛋白的表达。结论 Kv1.3通道可能在调节AS斑块部T淋巴细胞亚群的激活中起着重要作用。     

Ion-binding properties of a K+ channel selectivity filter in different conformations

K⁺ channels are membrane proteins that selectively conduct K⁺ ions across lipid bilayers. Many voltage-gated K⁺ (K_V) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K⁺ channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to K_V channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na⁺ or low [K⁺]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K⁺ ion binding to channels with an inactivated or conductive selectivity filter is different from K⁺ ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.K⁺ channels are found in all three domains of life, where they selectively conduct K⁺ ions across cell membranes. Specific stimuli trigger the activation of K⁺ channels, which results in a hinged movement of the inner helix bundle (1–7). This opening on the intracellular side of the membrane initiates ion conduction across the membrane by allowing ions to enter into the channel. After a period, many channels spontaneously inactivate to attenuate the response (8–17). The inactivation process is a timer that terminates the flow of ions in the presence of an activator to help shape the response of the system. Two dominant types of inactivation have been characterized in voltage-dependent channels: N-type and C-type (18). N-type inactivation is fast and involves an N-terminal positively charged “ball” physically plugging the pore of the channel when the membrane is depolarized. C-type inactivation, on the other hand, is a slower process involving a conformational change in the selectivity filter that is initiated by a functional link between the intracellular gate and the selectivity filter (10, 19).Several experimental observations indicate a role for the selectivity filter in C-type inactivation. First, mutations in and around the selectivity filter can alter the kinetics of inactivation (20–23). Second, increasing concentrations of extracellular K⁺ ions decrease the rate of inactivation, as if the ions are stabilizing the conductive conformation of the channel to prevent a conformational change in the selectivity filter (14, 16, 17, 22). Finally, a loss of selectivity of K⁺ over Na⁺ has been observed during the inactivation process in Shaker channels, suggesting a role for the selectivity filter (24, 25). Together, these data indicate that channels in their inactivated and conductive conformations interact with K⁺ ions differently, and suggest that C-type inactivation involves a conformational change in the selectivity filter. Although several structures of K⁺ channels in their conductive state have been solved using X-ray crystallography, there is at present no universally accepted model for the C-type inactivated channel (1, 3–5, 9, 19, 26–28) (Fig. 1B).Open in a separate windowFig. 1.Macroscopic recordings and structural models of KcsA K⁺ channel. (A) Macroscopic currents of WT KcsA obtained by a pH jump from pH 8 to pH 4 reveal channel inactivation. Two models representing the conformation of the channel are shown below. (B) Conductive [Left, Protein Data Bank (PDB) ID code 1K4C] and constricted (Right, PDB ID code 1K4D) conformations of the selectivity filter are shown as sticks, and the ion-binding sites are indicated with green spheres. The thermodynamic properties of the conductive, constricted, and inactivated (Middle) conformations are the subject of this study.Inactivation in the K⁺ channel from Streptomyces lividians (KcsA) has many of the same functional properties of C-type inactivation, which has made it a model to understand its structural features (20). KcsA channels transition from their closed to open gate upon changing the intracellular pH from high to low (Fig. 1A). The rapid flux of ions through the channel is then attenuated by channel inactivation, where most open WT channels are not conducting, suggesting that crystal structures of open KcsA channels would reveal the inactivated channel. In some crystal structures of truncated WT KcsA solved with an open gate, the selectivity filter appears in the constricted conformation, similar to the conformation observed in structures of the KcsA channel determined in the presence of only Na⁺ ions or low concentrations of K⁺ ions (3, 10, 29, 30) (Fig. 1B). Solid-state and solution NMR also indicate that the selectivity filter of the KcsA channel is in the constricted conformation when the cytoplasmic gate is open (31–33).However, a recently published study shows that even when the constricted conformation of KcsA’s selectivity filter is prevented by a nonnatural amino acid substitution, the channel inactivates like WT channels, suggesting the constricted filter does not correspond to the functionally observed inactivation in KcsA (28). In this study, we use isothermal titration calorimetry (ITC) to quantify the ion-binding properties of WT and mutant KcsA K⁺ channels with their selectivity filters in different conformations and EPR spectroscopy to determine the conformation of the channels’ intracellular gates. A comparison of these ion-binding properties leads us to conclude that the conductive and inactivated filters are energetically more similar to each other than the constricted and inactivated filters.     

Two mechanisms of ion selectivity in protein binding sites

A theoretical framework is presented to clarify the molecular determinants of ion selectivity in protein binding sites. The relative free energy of a bound ion is expressed in terms of the main coordinating ligands coupled to an effective potential of mean force representing the influence of the rest of the protein. The latter is separated into two main contributions. The first includes all the forces keeping the ion and the coordinating ligands confined to a microscopic subvolume but does not prevent the ligands from adapting to a smaller or larger ion. The second regroups all the remaining forces that control the precise geometry of the coordinating ligands best adapted to a given ion. The theoretical framework makes it possible to delineate two important limiting cases. In the limit where the geometric forces are dominant (rigid binding site), ion selectivity is controlled by the ion-ligand interactions within the matching cavity size according to the familiar "snug-fit" mechanism of host-guest chemistry. In the limit where the geometric forces are negligible, the ion and ligands behave as a "confined microdroplet" that is free to fluctuate and adapt to ions of different sizes. In this case, ion selectivity is set by the interplay between ion-ligand and ligand-ligand interactions and is controlled by the number and the chemical type of ion-coordinating ligands. The framework is illustrated by considering the ion-selective binding sites in the KcsA channel and the LeuT transporter.     

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

Effects of azimilide, a new class III antiarrhythmic drug, on reentrant circuits causing ventricular tachycardia and fibrillation in a canine model of myocardial infarction

INTRODUCTION: Azimilide blocks the slow (I(Ks)) and fast (I(Kr)) components of the delayed rectifier potassium channel. It also has blocking effects on sodium (I(Na)) and calcium currents (I(CaL)). Its effects on reentrant circuits in infarct border zones causing ventricular tachyarrhythmias are unknown. METHODS AND RESULTS: Activation in reentrant circuits causing sustained ventricular tachycardia (SVT) and the initial polymorphic tachycardia that leads to ventricular fibrillation (VF) was mapped in the epicardial border zone (EBZ) of 4-day-old canine infarcts. Azimilide prolonged the effective refractory period (ERP) in both normal myocardium and EBZ, but reverse use-dependence in EBZ was prominent. Azimilide abolished SVT initiation by programmed electrical stimulation by prolonging the ERP at the site of stimulation either in normal or EBZ, preventing the occurrence of early premature impulses and the formation of lines of block in the EBZ necessary for formation of reentrant circuits. Azimilide prevented VF initiation by programmed electrical stimulation by causing conduction block of reentrant impulses in the EBZ during the initial beats of rapid polymorphic ventricular tachycardia, despite the reverse use-dependent effects on ERP. CONCLUSION: Azimilide has antiarrhythmic effects to prevent reentry causing SVT and VF in a canine infarct model.     

Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K(+) channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K(+) selective, whereas those with two or three are nonselective and permeate Na(+) and K(+) equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K(+) channels is essential for highly selective and efficient permeation of K(+) ions.