三磷酸腺苷敏感性钾通道Kir6.2 E23K多态性对大鼠心脏结构及功能的影响

目的初步探讨在扩张型心肌病中E23K多态性对心脏结构及功能的影响。方法构建携带人心肌特异性启动子的α-MHC-Kir6.2KK DNA表达载体,运用显微注射法获得Kir6.2E23K多态性的大鼠模型(F0代),F0代大鼠与SD大鼠杂交,产出F1代大鼠并进行基因鉴定。将F1代雄性大鼠分为WT(Wild Type:不含有Kir6.2E23K多态性)组,E23K(含有Kir6.2E23K多态性)组,WT-DCM(Wild Type-Dilated Cardiomyopathy)组和E23KDCM(E23K-Dilated Cardiomyopathy)组,每组10只。于8~12周时腹腔注射阿霉素构建扩张型心肌病(DCM)模型,对照组腹腔注射等量生理盐水。于模型构建完成后2周分别记录并分析4组大鼠心脏超声数据,并用天狼星红染色法检测其心肌胶原容积。结果成功构建携带人Kir6.2E23K多态性的载体α-MHC-KK并获得转基因大鼠。超声结果显示,经阿霉素诱导的大鼠(WT-DCM组和E23K-DCM组)与对照组(WT组和E23K组)相比心腔明显扩大,心功能显著降低,且E23K-DCM组大鼠较WT-DCM组大鼠心脏损伤更为明显(P0.05);与WT-DCM组相比,E23K-DCM组大鼠心肌纤维化程度更为严重(P0.05)。结论在DCM中Kir6.2E23K多态性对大鼠心脏结构和功能均有显著影响。     

2型糖尿病血管并发症患者细胞间黏附分子-1基因多态性研究

目的研究细胞间黏附分子-1（ICAM-1）469K〉E基因多态性位点等位基因分布频率及其与2型糖尿病（T2DM）血管病变的关系。方法采用聚合酶链式反应（PCR）方法检测62例T2DM血管并发症患者ICAM-1469KK、KE及EE基因型出现的频率。并检测其空腹血糖（BS）、甘油三酯（TG）、糖化血红蛋白（HbAIc）、血浆游离ICAM-1（sICAM-1）和总胆固醇（TC）水平，并与70例无血管并发症的T2DM患者及121例正常对照组比较。结果KK、KE、EE3种基因型在3组中的分布频率有明显差异（，=6．313，P=0．043）．DM血管病变组等位基因E的频率明显高于对照组和无血管病变组（P〈0．01），T2DM血管病变组ICAM-1469E等位基因携带者sICAM-1水平明显高于K等位基因携带者（P〈0．01）。结论ICAM—1469E等位基因与DM血管病变及ICAM-1水平升高有关。     

磺脲类受体1基因16-3c/t多态性与格列齐特降糖疗效的关系

目的 研究磺脲类受体1（SUR1）基因16—3C／T多态性对格列齐特降糖疗效的影响。方法 153名2型糖尿病患者服用格列齐特8周，用水解探针技术检测病人SUR1 16—3C／T多态性，比较不同基因型患者治疗前后血糖、HbA1c、HOMA-β、HOMA—IR的变化。结果 基线水平时3种基因型患者的临床表型相似，治疗结束后T／T纯合子患者HbA1c下降和HOMA-β上升程度明显高于其他基因型患者（P均〈0．050）。结论 16—3C／T多态性对格列齐特的降糖疗效有修饰作用，T／T型患者服用格列齐特后胰岛β细胞功能明显提高，取得较好的HbA1c控制。     

钾离子内向整流通道蛋白基因多态性对格列美脲治疗高血压合并糖尿病降糖调脂的作用

目的 探讨钾离子内向整流通道蛋白J亚单位11号成员(KCNJ11)E23K基因多态性在高血压合并糖尿病的表达意义及其对格列美脲降糖调脂作用的影响.方法 选择2型糖尿病患者300例,根据是否合并高血压分为糖尿病组125例和糖尿病合并高血压组(合并组)175例,同期纳入我院体检中心体检的100例健康者为对照组.检测KCNJ...     

ICAM-1基因多态性与HBV感染不同临床结局之间的关系

目的探讨细胞间黏附分子l（ICAM-1）基因多态性与HBV感染不同临床结局之间的相关性。方法应用病例．对照研究和聚合酶链反应-序列特异性引物法（PCR-SSP）检测118例慢性持续性HBV感染患者（包括无症状HBV携带者、慢性乙型肝炎、乙肝后肝硬化患者）和60例HBV急性自限性感染者的ICAM-1基因G241R（G／A）、K469E（A／G）两个位点的多态性，比较各组间基因型和等位基因频率，并对数据进行统计分析。结果①ICAM-l G241R（G／A）位点总GG基因型频率在HBV慢性持续性感染组高于急性自限性感染组，但差异无统计学意义（X^2=1．38，P〉0．05）。②ICAM-1 K469E（A／G）位点，进展性肝病组（慢性乙型肝炎和肝硬化）总KK基因型和总K等位基因的频率与无症状携带者组和自限性感染组相比显著增高（X^2=8．60，P〈0．05；X^2=5．07，P〈0、05），而在自限性感染和无症状携带者之间却无显著差异。结论携带ICAM-1 K469E KK基因型和K等位基因的患者容易进展成慢性乙型肝炎甚至肝硬化，可致慢性HBV感染患者病情进展。     

载脂蛋白E基因多态性与脑梗死的关系

目的探讨载脂蛋白E（apoE）基因多态性与脑梗死的关系。方法应用聚合酶链式反应-限制性片断长度多态性（PCR—RFLP）技术检测78例脑梗死患者和90名健康对照者apoE基因多态性分布特征。结果发现5种apoE基因型，脑梗死组E3／4基因型频率（23．1％）及ε4频率（14.7%）显著高于对照组（7．8％，5．0％），P〈0．05。而E3／3基因型频率（56．4％）及ε3频率（75.6%）显著低于对照组（78．9％，88．3％），P〈0．05。不同apoE基因型间胆固醇和低密度脂蛋白胆固醇水平差异有统计学意义（P〈0．05）。结论apoE基因多态性与脑梗死有关，并影响血脂水平。     

阿尔茨海默病CYP2D6*10基因多态性与多奈哌齐疗效

目的研究阿尔茨海默病（AD）患者CYP2D6＊10基因多态性对多奈哌齐疗效的影响。方法筛选AD患者110例（AD组）和健康体检者124例（对照组）,应用限制性片段长度多态性一聚合酶链反应（RFLP—PCR）方法对两组患者进行CYP2D6＊10基因多态性测定。AD组给予多奈哌齐治疗6个月,于服药前后,应用简易精神状态量表（MMSE）和阿尔茨海默病评定量表认知分量表（ADAS-Cog）进行认知功能测定;应用高效液相色谱一质谱法测定患者血浆中多奈哌齐的稳态血药浓度。结果AD组与对照组CYP2D6＊10的基因型和等位基因频率分布差异无统计学意义（P〉0．05）;AD组CYP2D6＊10基因突变型（T/T型与C／T型）的血药浓度、MMSE和ADAS．Cog评分与野生型（C／C型）相比,差异均有统计学意义（P〈0．05）。结论多奈哌齐对阿尔茨海默病CYP2D6＊10基因突变型患者的疗效优于野生型患者。     

载脂蛋白E基因多态性对血脂及高血压病的影响

为探讨载脂蛋白E基因多态性对血脂及高血压病的影响，采用聚合酶链式反应－限制性片段多态性方法测定112例原发性高血压病人及118例非高血压病对照的载脂蛋白E基因型。结果发现高血压病患者ε4等位基因频率及载脂蛋白E ε4携带者显著高于对照组（P＜0．05）。载脂蛋白E ε4携带者的总胆固醇和低密度脂蛋白胆固醇水平显著高于载脂蛋白E ε2携带者（P＜0．05），载脂蛋白E ε3／ε3携带者的低密度脂蛋白胆固醇水平也显著高于载脂蛋白E ε2携带者（P＜0．05）。载脂蛋白E ε4携带是高血压病的独立危险因素（P＜0．05）。研究表明载脂蛋白E基因多态性影响个体的血脂和脂蛋白水平，载脂蛋白E ε4为高血压病的一种重要遗传标志。     

广东粤北地区瑶族载脂蛋白E基因多态性与原发性高血压的关系

目的探讨广东粤北地区瑶族人群载脂蛋白E（apolipoprote E,apoE）基因多态性与原发性高血压的关系、方法采刚聚合酶链反应-限制性片段长度多态性方法（PCR-RFLP）对200名60岁～80岁广东粤北地区瑶族人群进行apoE基因多态性检测,分析瑶族人群apoE基因频率分布特点,并比较分析不同apoE基凶型与原发性高血压的关系。结果共检测瑶族人群6种apoE基因型,以E3／3型最常见,占64．0％．其次为E3／4型．占17．0%,E2／3、E2／4、E4／4、E2／2基因型分别占15．5％、1．5％、1．0％、1．0％.apoE有3种等位基因：以ε3频率分布最高．为80．3％,其次是ε4,分布的频率为10.3％。ε2频率分布最低,为9．5%。男性和女性apoE基因型分布差异无统计学意义（P〉0．05）。ε4基凶携带者在原发性高血压患者中的分布高于对照组（P〈0．05）。结论广东粤北地区瑶族人群的血压水平受apoE基因多态性的影响,ε4等位基因可能是瑶族人群患原发性高血压的危险因素之一。     

载脂蛋白E基因多态性与老年人血脂、血压的相关性研究

目的探讨载脂蛋白E（ApoE）基因多态性与老年人血脂、血压的关系。方法采用聚合酶链式反应-限制性片段长度多态性分析方法（PCR—RFLP）测定252例老年人的ApoE基因型。结果在不同基因型组间，收缩压水平按E3／4＋E4／4〉E3／3〉E2／2＋E2／3顺序递减（P〈0．05）。而舒张压水平在不同基因型组间无差异（P〉0．05）；总胆固醇（TC）和低密度脂蛋白胆固醇（LDL-C）水甲均按E3／4＋E4／4〉E3／3〉E2／2＋E2／3顺序递减（P〈0．05），甘油三酯（TG）、高密度脂蛋白胆固醇（HDL—C）水平在各组问比较无差异（P〉0．05）。结论ApoE基因多态性影响老年人血脂血压水平，甜等位基因携带者除具有较高的TC、LDL．C水平外，还有较高的收缩压水平。     

Single mutations convert an outward K+ channel into an inward K+ channel

Shaker-type K(+) channels in plants display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, an Arabidopsis K(+) channel (SKOR) and a tomato K(+) channel (LKT1) share high amino acid sequence similarity and identical domain structures; however, SKOR conducts outward K(+) current and is activated by positive membrane potentials (depolarization), whereas LKT1 conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the "opposite" voltage-sensing properties of SKOR and LKT1 remains unknown. Using a screening procedure combined with random mutagenesis, we identified in the SKOR channel single amino acid mutations that converted an outward-conducting channel into an inward-conducting channel. Further domain-swapping and random mutagenesis produced similar results, suggesting functional interactions between several regions of SKOR protein that lead to specific voltage-sensing properties. Dramatic changes in rectifying properties can be caused by single amino acid mutations, providing evidence that the inward and outward channels in the Shaker family from plants may derive from the same ancestor.     

Vitamin K1 supplementation to improve the stability of anticoagulation therapy with vitamin K antagonists: a dose-finding study

Background

Poor anticoagulant stability in patients using vitamin K antagonists is a risk factor for both bleeding and thrombosis. In previous studies supplementation with low dose vitamin K₁ was shown to improve the stability of anticoagulant control. We set up a study to confirm earlier reports and to determine the optimal daily dose of vitamin K₁ in preparation of a large study with clinical endpoints.

Design and Methods

Four hundred patients from two anticoagulation clinics starting with vitamin K antagonists, independently of a possible history of instable anticoagulation, were randomized to receive either placebo or 100, 150 or 200 μg of vitamin K₁ together with their treatment with vitamin K antagonists. The treatment was administered for 6 to 12 months. Anticoagulation stability, expressed as the percentage of time that the International Normalized Ratio was within the therapeutic range, was compared between the groups.

Results

After adjustment for age, sex, vitamin K antagonist used, anticoagulation clinic and interacting drugs as confounding factors the difference in percentage of time with the International Normalized Ratio within the therapeutic range between the placebo group and the vitamin K₁ groups was 2.1% (95% CI: −3.2% – 7.4%) for the group taking 100 μg, 2.7% (95% CI: −2.3% –7.6%) for the group taking 150 μg and 0.9% (95% CI: −4.5% – 6.3%) for the group taking 200 μg vitamin K₁ group, in favor of the vitamin K₁ groups. The patients from both the 100 μg group and the 150 μg group had a 2-fold higher chance of reaching at least 85% of time with the International Normalized Ratio within the therapeutic range. There were no differences in thromboembolic or hemorrhagic complications between the groups.

Conclusions

In patients starting vitamin K antagonists, supplementation with low dose vitamin K₁ resulted in an improvement of time that anticoagulation was within the therapeutic range. Differences between doses were, however, small and the improvement is unlikely to be of clinical relevance. For future studies we recommend selecting only patients with instable anticoagulant control. (This study was registered at www.isrctn.org as ISRCTN37109430)     

Net Clinical Benefit of Non-Vitamin K Antagonist vs Vitamin K Antagonist Anticoagulants in Elderly Patients with Atrial Fibrillation

BackgroundThe risks of thromboembolic and hemorrhagic events in patients with atrial fibrillation both increase with age; therefore, net clinical benefit analyses of anticoagulant treatments in the elderly population are crucial to guide treatment. We evaluated the 1-year clinical outcomes with non-vitamin-K antagonist and vitamin K antagonist oral anticoagulants (NOACs vs VKAs) in elderly (≥75 years) patients with atrial fibrillation in a prospective registry setting.MethodsData on 3825 elderly patients were pooled from the PREFER in AF and PREFER in AF PROLONGATION registries. The primary outcome was the incidence of the net composite endpoint, including major bleeding and ischemic cardiovascular events on NOACs (n = 1556) compared with VKAs (n = 2269).ResultsThe rates of the net composite endpoint were 6.6%/year with NOACs vs 9.1%/year with VKAs (odds ratio [OR] 0.71; 95% confidence interval [CI], 0.51-0.99; P = .042). NOAC therapy was associated with a lower rate of major bleeding compared with VKA use (OR 0.58; 95% CI, 0.38-0.90; P = .013). Ischemic events were nominally reduced too (OR 0.71; 95% CI, 0.51-1.00; P = .050). Major bleeding with NOACs was numerically lower in higher-risk patients with low body mass index (BMI; OR 0.50; 95% CI, 0.22-1.12; P = .07) or with age ≥85 years (OR 0.44; 95% CI, 0.13-1.49; P = .17).ConclusionsOur real-world data indicate that, compared with VKAs, NOAC use is associated with a better net clinical benefit in elderly patients with atrial fibrillation, primarily due to lower rates of major bleeding. Major bleeding with NOACs was numerically lower also in higher-risk patients with low BMI or age ≥85 years.     

The anomalous behavior of the density of water in the range 30 K < T < 373 K

The temperature dependence of the density of water, rho(T), is obtained by means of optical scattering data, Raman and Fourier transform infrared, in a very wide temperature range, 30 < T < 373 K. This interval covers three regions: the thermodynamically stable liquid phase, the metastable supercooled phase, and the low-density amorphous solid phase, at very low T. From analyses of the profile of the OH stretching spectra, we determine the fractional weight of the two main spectral components characterized by two different local hydrogen bond structures. They are, as predicted by the liquid-liquid phase transition hypothesis of liquid water, the low- and the high-density liquid phases. We evaluate contributions to the density of these two phases and thus are able to calculate the absolute density of water as a function of T. We observe in rho(T) a complex thermal behavior characterized not only by the well known maximum in the stable liquid phase at T = 277 K, but also by a well defined minimum in the deeply supercooled region at 203 +/- 5 K, in agreement with suggestions from molecular dynamics simulations.     

Nucleation of Helium in Liquid Lithium at 843 K and High Pressures

Fusion energy stands out as a promising alternative for a future decarbonised energy system. In order to be sustainable, future fusion nuclear reactors will have to produce their own tritium. In the so-called breeding blanket of a reactor, the neutron bombardment of lithium will produce the desired tritium, but also helium, which can trigger nucleation mechanisms owing to the very low solubility of helium in liquid metals. An understanding of the underlying microscopic processes is important for improving the efficiency, sustainability and reliability of the fusion energy conversion process. The spontaneous creation of helium droplets or bubbles in the liquid metal used as breeding material in some designs may be a serious issue for the performance of the breeding blankets. This phenomenon has yet to be fully studied and understood. This work aims to provide some insight on the behaviour of lithium and helium mixtures at experimentally corresponding operating conditions (843 K and pressures between 108 and 1010 Pa). We report a microscopic study of the thermodynamic, structural and dynamical properties of lithium–helium mixtures, as a first step to the simulation of the environment in a nuclear fusion power plant. We introduce a new microscopic model devised to describe the formation of helium droplets in the thermodynamic range considered. Our model predicts the formation of helium droplets at pressures around 109 Pa, with radii between 1 and 2 Å. The diffusion coefficient of lithium (2 Å2/ps) is in excellent agreement with reference experimental data, whereas the diffusion coefficient of helium is in the range of 1 Å2/ps and tends to decrease as pressure increases.     

Properties of Slo1 K+ channels with and without the gating ring

High-conductance Ca²⁺- and voltage-activated K⁺ (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca²⁺ and Mg²⁺ sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca²⁺- and Mg²⁺-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca²⁺ and Mg²⁺ activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.Slo1 channels are expressed in most human tissues and play key roles in many important physiological processes, including smooth muscle contraction, neurotransmitter release, neuronal excitability, hair cell tuning, and action potential termination (1–6). Slo1 channels also are named BK (Big K⁺) or MaxiK channels because of their high single-channel conductance (∼300 pS in 150-mM symmetrical K⁺). Slo1 channels are activated synergistically by both depolarization and intracellular calcium (7–9), linking these two activators in a negative feed-back system to restore negative membrane potential which, in turn, closes voltage-activated Ca²⁺ channels. The dual regulation by voltage and calcium led Hille (10) to predict that BK channels function like the classical Hodgkin–Huxley delayed rectifier channel, except that the range of voltage activation was set by the intracellular Ca²⁺ concentration. The cloning (11) and analysis of the Slo1 channel structure seemed to validate this prediction, in that Slo1 appeared to be modular in its construction, having a core domain containing a voltage sensor controlling a K⁺-selective pore and a long C-terminal tail forming a gating ring structure comprised of four pairs of regulators of the conductance of K⁺ (RCK) domains for sensing and transducing the effect of Ca²⁺ binding to the core.One of the four identical α subunits that assemble to form the Slo1 WT channel (Slo1-WT) is shown in Fig. 1 Top. For the mbr5 cDNA (12) used in this study, the “core” consists of 342 residues including seven transmembrane segments (S0–S6) and the S6–RCK1 linker sequence, which is attached to a long tail of 827 residues. The tail sequence of Slo1-WT is distinct from the cytoplasmic domains of other members of the K⁺ channel extended family. Structure–function studies of the tail have shown the existence of two high-affinity Ca²⁺ binding sites (13, 14) and one low-affinity Mg²⁺ site (14, 15). Modulation of the channel also occurs by additional biological factors, including protons, heme, carbon monoxide, phosphorylation, and oxidation (16–20), all of which may function via their interaction with the tail. Thus, the large tail accommodates a variety of regulatory domains which sense different intracellular factors, leading to pushing or tugging against the core to facilitate or inhibit channel gating. These complicated allosteric interactions between core and tail almost certainly involve several transduction pathways (21–23), all of which alter the properties of the core. Thus, a logical starting point to begin investigating the allosteric interactions would be to understand the baseline properties of the isolated core. However, this approach has been hampered by the inability to express functional cores in the absence of the tail. Previous analysis of truncated expression constructs of Slo1 channels found that their processing stalls in the endoplasmic reticulum (ER), they are not assembled into tetramers, they fail to be exported to the plasma membrane, or they are nonfunctional (24). We now show that core constructs without gating rings can be expressed by leaving a short region required for subunit tetramerization and by appending a small tail domain which facilitates processing and efficient export to the plasma membrane. Thus, we now are able to investigate gating in the absence of a gating ring.Open in a separate windowFig. 1.Slo1 channel constructs used in this study. The Slo1 channel constructs used in this study are based on the mouse mbr5 cDNA (12) and the mouse Shaker family Kv1.4 channel (25). The “Slo1 core and tail” refers to the first 342 and the last 827 amino acid residues. The “Kv1.4 tail” refers to the last 74 amino acid residues of Kv1.4. The different channel constructs are designated as follows: Slo1-WT is Slo1 full-length WT; Slo1C-KvT is a Slo1 core with a 74-residue Kv1.4 tail; Slo1C-Kv-minT is a Slo1 core with a Kv1.4 11-residue mini tail; Slo1C-KvT_NAFQ is a Slo1 core with a 74-residue Kv1.4 tail with NAFQ substituted for KKFR in the tail; Slo1C-KvT R207E is Slo1C-KvT with R207E in S4 in the core.     

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.     

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K⁺ channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K⁺ channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K⁺ channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.Voltage-gated K⁺ channels are highly conserved among bilaterian metazoans and play a central role in the regulation of excitation in neurons and muscle. Understanding the functional evolution of these channels may therefore provide important insights into how neuromuscular excitation evolved within the Metazoa. Three major gene families, Shaker, KCNQ, and Ether-a-go-go (EAG) encode all voltage-gated K⁺ channels in bilaterians (1, 2). In this study, we examine the functional evolution and origins of the Shaker and KCNQ gene families. Shaker family channels can be definitively identified by a unique subunit structure that includes both a voltage-gated K⁺ channel core and a family-specific cytoplasmic domain within the N terminus known as the T1 domain. T1 mediates assembly of Shaker family subunits into functional tetrameric channels (3, 4). KCNQ channels are also tetrameric but lack a T1 domain and use a distinct coiled-coil assembly domain in the C terminus (5, 6). KCNQ channels can be identified by the presence of this family-specific assembly motif and high amino acid conservation within the K⁺ channel core. Both channel families are found in cnidarians (1, 7) and thus predate the divergence of cnidarians and bilaterians, but their ultimate evolutionary origins have not yet been defined.Shaker family K⁺ channels serve diverse roles in the regulation of neuronal firing and can be divided into four gene subfamilies based on function and sequence homology: Shaker, Shab, Shal, and Shaw (8, 9). The T1 assembly domain is only compatible between subunits from the same gene subfamily (4, 10) and thus serves to keep the subfamilies functionally segregated. Shaker subfamily channels activate rapidly near action potential threshold and range from rapidly inactivating to noninactivating. Multiple roles for Shaker channels in neurons and muscles have been described, but their most unique and fundamental role may be that of axonal action potential repolarization. Shaker channels are clustered to the axon initial segment and nodes of Ranvier in vertebrate neurons (11–13) and underlie the delayed rectifier in squid giant axons (14). The Shaker subfamily is diverse in cnidarians (15, 16), and the starlet sea anemone Nematostella vectensis has functional orthologs of most identified Shaker current types observed in bilaterians (16).The Shab and Shal gene subfamilies encode somatodendritic delayed rectifiers and A currents, respectively (17–20). Shab channels are important for maintaining sustained firing (21, 22), whereas the Kv4-based A current modulates spike threshold and frequency (17). Shab and Shal channels are present in cnidarians, but cnidarian Shab channels have not been functionally characterized, and the only cnidarian Shal channels expressed to date display atypical voltage dependence and kinetics compared with bilaterian channels (23). Shaw channels are rapid, high-threshold channels specialized for sustaining fast firing in vertebrates (24, 25) but have a low activation threshold and may contribute to resting potential in Drosophila (19, 26, 27). A Caenorhabditis elegans Shaw has slow kinetics but a high activation threshold (28), and a single expressed cnidarian Shaw channel has the opposite: a low activation threshold but relatively fast kinetics (29). Thus, the ancestral properties and function of Shaw channels is not yet understood. Further functional characterization of cnidarian Shab, Shal, and Shaw channels would provide a better understanding of the evolutionary status of the Shaker family in early parahoxozoans.KCNQ family channels underlie the M current in vertebrate neurons (30) that regulates subthreshold excitability (31). The M current provides a fundamental mechanism for regulation of firing threshold through the Gq G-protein pathway because KCNQ channels require phosphatidylinositol 4,5-bisphosphate (PIP₂) for activation (32, 33). PIP₂ hydrolysis and subsequent KCNQ channel closure initiated by Gq-coupled receptors produces slow excitatory postsynaptic potentials, during which the probability of firing is greatly increased (32, 33). The key functional adaptations of KCNQ channels for this physiological role that can be observed in vitro are (i) a requirement for PIP₂ to couple voltage-sensor activation to pore opening (34, 35), and (ii) a hyperpolarized voltage–activation curve that allows channels to open below typical action potential thresholds. Both key features are found in vertebrate (30, 34, 36–38), Drosophila (39), and C. elegans (40) KCNQ channels, suggesting they may have been present in KCNQ channels in a bilaterian ancestor. Evolution of the M current likely represented a major advance in the ability to modulate the activity of neuronal circuits, but it is not yet clear when PIP₂-dependent KCNQ channels first evolved.Here, we examine the origins and functional evolution of the Shaker and KCNQ gene families. If we assume the evolution of neuronal signaling provided a major selective pressure for the functional diversification of voltage-gated K⁺ channels, then we can hypothesize that the appearance of these gene families might accompany the emergence of the first nervous systems or a major event in nervous system evolution. Recent phylogenies that place the divergence of ctenophores near the root of the metazoan tree suggest that the first nervous systems, or at least the capacity to make neurons, may have been present in a basal metazoan ancestor (41–43) (Fig. S1). One hypothesis then is that much of the diversity of metazoan voltage-gated channels should be shared between ctenophores and parahoxozoans [cnidarians, bilaterians, and placozoans (44)]. However, genome analysis indicates that many “typical” neuronal genes are missing in ctenophores and the sponges lack a nervous system, leading to the suggestion that extant nervous systems may have evolved independently in ctenophores and parahoxozoans (42, 45). Thus, a second hypothesis is that important steps in voltage-gated K⁺ channel evolution might have occurred separately in ctenophores and parahoxozoans. We tested these hypotheses by carefully examining the phylogenetic distribution and functional evolution of Shaker and KCNQ family K⁺ channels. Our results support a model in which major innovations in neuromuscular excitability occurred specifically within the parahoxozoan lineage.     

PI3K polymorphism in patients with sporadic Parkinson’s disease

Parkinson’s disease (PD) is a common irreversible neurodegenerative disease associated with cognitive impairment. To investigate the serum level of phosphatidylinositol-3-kinase (PI3K) and the distribution of the genotypes and alleles of 3 PI3K single-nucleotide polymorphisms (RS37,30,087, RS37,30,088, and RS37,30,089) in PD patients with different clinical characteristics. A total of 54 PD patients and 50 healthy individuals were recruited. The serum PI3K level was measured using the enzyme-linked immunosorbent assay. The severity of PD was assessed using the modified Hoehn-Yahr scale. The cognitive function of PD patients was evaluated using the Mini-Mental State Examination scale and the Montreal Cognitive Assessment. The distribution of the alleles and genotypes of PI3K single-nucleotide polymorphisms (SNPs) was calculated using the Hardy-Weinberg equilibrium. PD patients showed a significantly higher serum level of PI3K compared to healthy individuals. Increased serum PI3K level was observed in PD patients with more severe disease, longer disease duration, and impaired cognitive function. Additionally, no significant differences were observed in the distributions of the genotypes and alleles of 3 PI3K SNPs between PD patients with normal cognitive function and those with cognitive impairment. PD patients with different levels of disease severity, disease duration, and cognitive function had significantly different serum levels of PI3K. However, the PI3K SNPs in patients with normal cognitive function were not significantly different from those in patients with cognitive impairment. These findings contribute to a better understanding of the roles of PI3K and SNPs of the PI3K gene in PD.