Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

In K⁺ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K⁺ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K⁺ channels.The functional behavior of K⁺ channels is defined by a series of structural rearrangements associated with the processes of activation and inactivation gating (1–6). In response to a prolonged stimulus and in the absence of an N-terminal inactivating particle, most K⁺ channels become nonconductive through a process known as C-type inactivation (7). This C-type inactivation is crucial in controlling the firing patterns in excitable cells and is fundamental in determining the length and frequency of the cardiac action potential (8). C-type inactivation is inhibited by high extracellular K⁺ (9, 10), and the blocker tetraethylammonium (TEA) (11) can also be slowed down in the presence of permeant ions with a long residence time in the selectivity filter (Rb⁺, Cs⁺, and NH⁴⁺) (10).The prokaryotic pH-gated K⁺ channel KcsA shares most of the mechanistic properties of C-type inactivation in voltage-dependent K⁺ channels (5, 6, 12–16). Recent crystal structures of open/inactivated KcsA reveal that there is a remarkable correlation between the degree of opening at the activation gate and the conformation and ion occupancy of the selectivity filter (5). In KcsA, the selectivity filter is stabilized by a hydrogen bond network, with key interactions between residues Glu71, Asp80, and Trp67 and a bound water molecule (17). Disrupting this hydrogen bond network favors the conductive conformation of the selectivity filter (12, 13, 15).Early electrophysiological experiments have suggested that the outer vestibule (around T449 residue in Shaker and Y82 residue in KcsA) undergoes significant conformational rearrangement during C-type inactivation gating (16, 18, 19). However, comparison of the WT KcsA crystal structure, where the filter is in its conductive conformation, with either the structure obtained with low K⁺ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5) does not show major conformational changes in the outer vestibule that would explain these results (Fig. 1A). We have suggested that this apparent discrepancy can be understood if we take into consideration the potential differences in the dynamic behavior of the outer vestibule changes as the K⁺ channel undergoes its gating cycle (16).Open in a separate windowFig. 1.Comparison of outer vestibule conformation in KcsA structures with conductive and collapsed/inactivated filters. (A) High-K⁺ KcsA structure [Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K⁺ KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outer vestibule residues are depicted as red spheres, and relevant residues are labeled. (B) Schematic representation of typical macroscopic currents elicited by pH-jump experiments in WT (inactivating) and E71A (noninactivating) KcsA channels at a depolarizing membrane potential is shown. Conditions that stabilize the closed, open/inactivated, and open/conductive conformations at the steady state are indicated with a black circle. (C) Effect of opening the lower gate on the mobility of spin-labeled outer vestibule residues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidyl glycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and noninactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) and open (pH 4, black) states of KcsA, as determined by continuous wave (CW) EPR. The spectra shown are amplitude-normalized. Details are provided in SI Materials and Methods.We have probed the gating-induced structural dynamics at the outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combination with a recently developed computational method, restrained ensemble (RE) simulations. RE simulation was used to constrain the outer vestibule using experimentally derived distance histograms in different functional states (closed, open/inactivated, and open/conductive) and to monitor the extent of backbone conformational changes during gating. To this end, we took advantage of our ability to stabilize both the open/conductive (E71A mutant) and the open/inactivated (WT) conformations of KcsA upon opening the activation gate under steady-state conditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive conformation is highly dynamic. In addition, the red edge excitation shift (REES) points to a change in hydration dynamics between conductive and nonconductive outer vestibule conformations, suggesting a role of restricted water molecules in C-type inactivation gating. We suggest that, on average, the backbone conformation of the outer vestibule does not change significantly between different functional states but that local dynamics change significantly, underlining the importance of the hydrogen bond network behind the selectivity filter and the microscopic observables (e.g., dynamics of hydration) in K⁺ channel gating and C-type inactivation.     

Transcatheter Aortic Valve Implantation in the Elderly: Who to Refer?

In recent years, experience with transcatheter aortic valve implantation has led to improved outcomes in elderly patients with severe aortic stenosis (AS) who may not have previously been considered for intervention. These patients are often frail with significant comorbid conditions.     

经房间隔穿刺途径消融左室顶部室性心律失常五例

目的分析在三维标测系统指导下,通过经房间隔穿刺途径消融起源于左室顶部的室性早搏、室性心动过速的方法、电生理特点及消融结果。方法对5例体表心电图符合左室流出道附近起源特点的患者,术中在左室流出道未标测到理想靶点,行房间隔穿刺将消融导管跨二尖瓣环倒钩至左室顶部,采用三维电解剖标测法,用冷盐水灌注消融导管以35～40w,43℃,流速17ml／min在该区域进行片状基质消融治疗。结果5例术中在左室顶部均可标测到低电压区,窦性心律时可记录到心室晚电位或室性心动过速发作时可记录到心室舒张中期电位,通过房间隔穿刺途径的即刻消融成功率为100％,随访3个月,有I例复发并出现晕厥。结论对于起源于左室顶部的室性心律失常,经房间隔穿刺途径进行射频消融治疗是有效的、安全的。     

冲洗式射频消融迷宫手术治疗不同病因永久性心房颤动的疗效比较

目的分析和比较我院冲洗式射频消融迷宫手术治疗不同病因永久性心房颤动（房颤）的疗效。方法：收集我院201 1年1月1日-2012年12月31日外科冲洗式射频消融迷宫手术治疗永久性房颤502例病例资料进行分析。根据病因（手术方式）不同分为两组：瓣膜置换并发冲洗式射频消融迷宫手术（瓣膜病组）406例;非瓣膜置换并发冲洗式射频消融迷宫手术（非瓣膜病组）96例。分别于术中心脏复跳、术后1周、3个月和6个月行超声心动图及心电图检查,观察左房、左室大小及窦性心律维持情况。结果：两组术后各时点心脏转复率均无显著差异,两组左房内径均较术前显著缩小。就全部患者而言,术中心脏转为窦性心律者为358例,转复率为71．3％;术后1周为376例,转复率为74．9％;术后3个月为347例,转复率为69．1％。术后6个月为339例,转复率为67．5％。总体手术死亡3例,2例死于低心排,1例死于恶性心律失常（室颤）,手术死亡率为0．6％。结论：冲洗式射频消融迷宫手术治疗不同病因永久性房颤的疗效没有显著差异,疗效均较好。     

湖北省2012年流行株O139霍乱弧菌脉冲场凝胶电泳分型分析

目的分析3起霍乱疫情病原O139霍乱弧菌分子分型特征和遗传相关性,探讨O139霍乱疫情流行特征。方法对2012年湖北省3起霍乱疫情分离鉴定的35株O139霍乱弧菌菌株用水煮法提取DNA,利用聚合酶链反应检测霍乱肠毒素CT基因;取新鲜培养的菌株,制备约4.3个麦氏单位的细菌悬浮液,经裂解、洗涤及限制性内切酶NotI和SfiI酶切后进行脉冲场凝胶电泳(PFGE)分子分型,用凝胶成像仪获取电泳图像,分析DNA片段并用BioNumerics V4.6软件UPGMA方法(复选Dice相关系数)进行聚类分析。结果 35株O139霍乱弧菌ctxA基因PCR扩增产物约为308bp,分离自聚餐食用的凉菜、病人、带菌者及厕所标本(对应病人家)O139霍乱弧菌均为产毒株,经NotI酶切分为9种PFGE带型,SfiI酶切分为6种PFGE带型;A市2株病人分离菌和4株带菌者分离菌PFGE带型为KZGN11O139.CN0077+KZGS12O139.CN0054,B市1株病人分离菌和1株厕所分离菌PFGE带型为KZGN11O139.CN0302+KZGS12O139.CN0058,C市和D市分离菌株优势型为KZGN11O139.CN0276+KZGS12O139.CN0007,包括3株食品分离菌、2株厕所分离菌及16株病人和带菌者分离菌,另有6株PFGE带型呈现多样性。结论 2012年湖北省霍乱疫情特点为散发以及聚餐导致的局限暴发,3起疫情的分离菌株带型各不相同,呈现多样性,其中2起为单一菌型感染,1起为混合菌型感染,传染来源复杂且不明确,提示应加强霍乱的病原学监测。     

脑钠肽与超声心动图评价老年糖尿病并存高血压患者左心室舒张功能的研究

目的 探讨老年糖尿病并存高血压患者左心室功能的变化与脑钠肽(BNP)水平的关系. 方法 128例2型糖尿病患者按是否并存高血压分组,其中不伴高血压组63例,伴高血压组65例,对照组为62例健康查体者.测定各组BNP浓度.常规超声测定左心室结构指标,计算左心室质量指数(LVMI),脉冲多普勒测量二尖瓣口舒张早期血流速度(E)、舒张晚期血流速度(A),计算E/A值.定量组织多普勒技术(QTVI)测量左心室壁二尖瓣环6个位点(侧壁和后间隔、前壁和下壁、前间隔和后壁)处的舒张早期峰值运动速度(Em)和舒张晚期峰值运动速度(Am).计算6个位点平均Em、Am(MEm、MAm)和E/MEm比值. 结果 糖尿病不伴高血压组、伴高血压组LVMI[分别为(91.6±17.3)g/m2、(116.7±20.5)g/m2]、E/MEm(10.3±1.8和12.5±1.4)及BNP浓度[(47.7±29.4)ng/L、(105.7±32.5)ng/L]较对照组[分别为(78.7±19.5)g/m2、8.9±1.6、(20.8±11.63)ng/L]升高,差异有统计学意义(F值分别为11.54、13.83和9.75,P均＜0.05);不伴高血压组、伴高血压组MEm[《6.7±1.0)cm/s、(5.4±0.9)cm/s]较对照组[(5.4±0.9)cm/s]降低,差异有统计学意义(F=11.26,P＜0.05).糖尿病患者BNP浓度与E/A、MEm值呈负相关(r值分别为-0.42、-0.51,均P＜0.01),与LVMI和E/MEm值呈正相关(r值分别为0.48、0.58,均P＜0.01). 结论 糖尿病患者左心室舒张功能降低,并存高血压患者左心室舒张功能障碍更为严重.联合血浆BNP浓度和超声指标有助于准确评估老年糖尿病患者左心室舒张功能.