蝎毒素BmkTXKβ对家兔心房肌细胞瞬间外向钾电流的抑制作用

为研究蝎毒素BmkTXKβ对家兔心房肌细胞瞬间外向钾电流(I_to)的影响,采用全细胞膜片钳技术记录应用BmkTXKβ前后的I_to电流. 结果显示BmkTXKβ（1 μmol/L）使心房肌细胞I_to（刺激电压为+50 mV）从（13.63±0.87）pA/pF减少到（7.98±0.78）pA/pF,抑制率为41.4% (n=16, P＜0.001).冲洗后, I_to部分恢复至(11.18±0.82)pA/pF (n=6, P＜0.01,与给药后比较).在0.01～100 μmol/L范围内BmkTXKβ呈浓度依赖性地抑制I_to,IC₅₀的均值为0.95 μmol/L (n=10, P＜0.01),但无频率依赖性(n=6,P＞0.05). 1 μmol/L的BmkTXKβ可使I_to通道的失活动力学曲线明显左移,V_1/2分别为（－23.6±2.7） mV和（－35.3±3.6） mV（n=8,P＜0.05）,曲线斜率基本不变.同时可使I_to通道的恢复过程明显减慢,恢复曲线右移,τ值从（51.2±8.5） ms延长至（93.5±13.4） ms（n=9,P＜0.01）,但不影响其激活过程.据此推断BmkTXKβ对家兔心房肌细胞I_to具有显著的抑制作用,主要作用于失活过程,延长该通道的恢复时间.     

吗啡后处理对大鼠心肌细胞ATP敏感性钾通道的影响

在氰化钠(NaCN)处理模拟细胞缺血的单个大鼠心肌细胞上,应用膜片钳电压钳制全细胞记录模式,研究吗啡后处理对缺血心肌细胞膜ATP敏感性钾通道的影响,并探讨吗啡后处理可能涉及的阿片受体类型．吗啡后处理可使ATP敏感性钾通道电流( I_KATP )增加(61.4 ± 13.6)%,促进K_ATP通道开放．特异阻断κ-阿片受体不能阻止I_KATP增加,而非特异性阻断阿片受体或特异阻断δ-阿片受体均可阻止I_KATP增加．结果表明, 吗啡后处理促进K_ATP通道开放与δ-阿片受体的激活有关．     

重复膜片钳法研究MPP+对神经元钙电流作用

细胞膜片钳技术是研究膜离子通道的有效方法.在单个细胞上反复形成多次全细胞构型从而在同一细胞上观察某些药物的长时间作用对于通道膜电流的影响.利用全细胞构型下胞浆与电极内液的连通可以方便地向胞内引入药物.以此法研究MPP⁺多巴胺能神经瘤细胞(MN9D)的毒性作用表明MPP⁺导致细胞电压依赖性钙电流(I_Ca)显著下降;MPP⁺作用1 h以内高去极化电压较低去极化电压诱发的钙电流先受MPP⁺影响而下降; MPP⁺对未分化细胞的钙电流无显著作用(n=3).     

乌拉坦对大鼠海马CA1神经元电压门控钠通道和动作电位的作用

乌拉坦对兴奋性和抑制性配体门控通道具有广泛的可检测的作用.作者运用全细胞膜片钳技术研究乌拉坦对wistar大鼠海马CA1神经元电压门控钠通道和动作电位的作用.结果发现乌拉坦可逆并剂量依赖性地抑制钠电流和动作电位,其中,在10mmol/L浓度时可减小钠电流强度达38%,使激活曲线向去极化方向移动,并延长钠通道失活后的恢复时间,降低动作电位的幅值.这些结果表明乌拉坦对电压门控钠通道的抑制作用可能是乌拉坦全身麻醉作用的机制之一.     

小鼠心室肌细胞内向整流钾电流的特性及其与早后去极化的关系

用全细胞膜片钳技术首次研究了成年小鼠单个心室肌细胞内向整流钾电流(I_k1)的整流特性,膜电导和反转电位对胞外钾离子浓度的敏感性及激活、失活动力学等性质。发现小鼠心室肌细胞I_k1最突出的特点就是其电压-电流(I～V)曲线在正于反转电位50mV的范围内无负斜率(negative slope)区,而是保持在一个很低的外向电流水平(59±39)pA。当用3mmol/L K^±和3mmol/L Cs^±灌流时,这一部分的I_k1减小为零,随后产生早后去极化(EAD)。结果提示:小鼠I_k1的这一特点与其EAD易发有关,I_k1受抑是本实验EAD发生的先决条件。     

ATP敏感钾通道的研究进展

ATP敏感钾通道（K_ATP）是一组将细胞膜电活动与细胞代谢联系在一起的重要通道.该通道是由磺酰脲受体（SUR）和内向整流钾通道（K_IR6.x）亚单位组成的异源四聚体（SUR/K_IR6.x）₄.SUR与K_IR6.x基因在染色体上配对存在.K_IR6.x亚单位形成通道的电流孔道,SUR使通道对磺酰脲类药物、钾通道开放剂和Mg²⁺-NDPs等调节因子敏感.不同亚型K_ATP通道特性由SUR与K_IR6.x亚单位组成决定.K_ATP通道门控受［ATP］_i和［ADP］_i调节,膜磷脂(PIPs)抑制通道对ATP的敏感性,细胞磷酸转移系统也参与ATP/ADP对通道的调节机制;磺酰脲类复合物（SUs）抑制K_ATP通道,钾通道开放剂（KCOs）激活K_ATP通道;G蛋白以及PKA、PKC、PKG等信使物质也参与通道的调节.K_ATP通道对胰岛素的分泌、心肌缺血预适应以及血管的张力起重要调节作用.     

皮层SI区伤害感受神经元膜电生理特性研究

用细胞内记录技术, 在16只成年健康猫, 研究了皮层第一躯体感觉区(primary somatosensory cortex area,SI区)伤害感受神经元的电生理特性.SI区伤害感受神经元自发放电频率差异大,放电形式多样.极化电流绝对值≤1.0 nA时,伤害感受神经元I-V极相关(r=0.96),整流作用不明显;极化电流绝对值＞1.0 nA时,在两个方向上发生整流,I-V(电流-电压)曲线表现为“S”型, 其中伤害感受神经元整流作用较非伤害感受神经元明显.伤害感受神经元R_m、τ、C_m明显大于非伤害感受神经元(P＜0.01或P＜0.05).结果提示SI区伤害感受神经元与非伤害感受神经元可能在细胞膜结构、细胞大小等方面存在有意义的差别,从而反映其不同的生理功能.此电学参数特点也可为痛觉的特异性学说提供实验资料.     

中华绒螯蟹眼柄MTXO细胞GABA受体通道研究

采用全细胞膜片钳技术测定了中华绒螯蟹（Eriocheir sinensis）眼柄视神经节端髓X器官（MTXO）三种类型神经内分泌细胞对0.01～5mmol/L γ氨基丁酸（γ-aminobutyric acid,GABA）的反应,并结合选择性拮抗剂和激动剂的使用进行了GABA受体研究.在电流钳模式下,依据不同Nernst Cl^－电位,三种类型细胞均对GABA产生去极化或超级化反应.在电压钳模式下,GABA激活Cl^－通道电流（I_GABA）.I_GABA在灌流GABA后约1 200 ms内激活,800 ms内达到峰值,没有明显的脱敏反应,反转电位接近Nernst Cl^－电位.I_GABA幅值呈浓度依赖性,激活阈值为0.01 mmol/L,约在0.5 mmol/L达到饱和.药理学实验结果表明,中华绒螯蟹眼柄神经内分泌细胞GABA受体是Cl^－通道蛋白,对Cl^－离子通道阻断剂Picrotoxin和Niflumic acid敏感,但是对GABA_A受体拮抗剂Bicuculline和GABA_C受体激动剂cis-4-aminocrotonic acid (CACA)和trans-4-aminocrotonic (TACA)均不敏感.     

低钾胁迫对谷子幼苗叶片光合作用的影响

为了探究低钾胁迫引起谷子幼苗光合作用改变的响应机制,以谷子品种‘晋谷21号’(钾敏感型)和 ‘龙谷25号’(钾非敏感型)为试验材料,设置正常供钾(5.0 mmol·L^-1, K₅)、低钾(1、0.1、0.01 mmol·L^-1, K₁、K_0.1、K_0.01)和无钾(0 mmol·L^-1,K₀)5个处理,通过营养液盆栽实验,分析不同程度低钾胁迫对谷子幼苗生长、光合气体交换参数、叶绿素荧光特性和碳同化关键酶活性的影响。结果表明：(1)低钾胁迫显著抑制了谷子幼苗的生长,株高、茎粗、叶面积及干物质显著降低,同时叶片钾含量减少,K₀处理‘晋谷21号’和‘龙谷25号’叶片钾含量较K₅处理分别显著降低了48.14%和37.85%。(2)K₀处理谷子幼苗光合色素含量较K₅处理显著降低,但K_0.1和K₁处理与K₅处理差异不显著。(3)低钾胁迫导致谷子幼苗叶片净光合速率(P_n)降低,与K₅处理相比,K₀、K_0.01、K_0.1处理P_n、气孔导度(G_s)、蒸腾速率(T_r)、气孔限制值(L_s)显著降低,胞间CO₂浓度(C_i)却显著增加,证明光合速率降低主要由非气孔限制引起。(4)低钾胁迫很易导致光系统Ⅱ(PSⅡ)和光系统Ⅰ(PSⅠ)光化学量子产量降低,K₁处理叶片的PSⅡ实际光化学量子效率(Φ_PSⅡ)、PSⅠ光化学量子产量［Y(Ⅰ)］分别降低了5.32%~9.57%和2.38%~5.63%,K₀处理则分别显著降低了17.15%~20.15%和18.71%~21.28%。(5)K₀处理下‘晋谷21号’的 1,5 二磷酸核酮糖羧化酶(Rubisco)和磷酸烯醇式丙酮酸羧化酶(PEPC)活性较K₅处理分别显著降低了42.86%和42.71%,相应处理的‘龙谷25号’则分别显著降低了26.85%和42.77%。研究发现,低钾胁迫下‘龙谷25号’对钾素吸收利用能力较强,各生长生理参数变化幅度较小,表现出较强的低钾耐性;低钾胁迫导致谷子幼苗净光合速率降低,K₀和K_0.1处理下光合速率降低主要与气孔导度降低、光系统的电子传递与能量转换抑制以及碳同化关键酶Rubisco、PEPC活性下降等有关。     

葛氏鲈塘鳢与七彩神仙鱼心肌细胞钠离子通道的温度响应特征

温度适应对于动物的生存至关重要，而目前对于鱼类这样的外温动物的温度适应相关研究仍然缺乏。葛氏鲈塘鳢(Perccottus glenii)可以在结冰的环境中生存数天，而七彩神仙鱼(Symphysodon aequifasciatus)是被广泛饲养的热带观赏鱼类，本研究将这2种鱼类作为冷水鱼与热带鱼的代表，探究其温度耐受性。为此，测定了它们的临界温度以及不同温度下(0 ~ 38 ℃)的心率，并利用原代细胞急性分离，结合膜片钳电生理技术与控温技术，测定了上述2种鱼类心肌电压门控钠离子通道在不同温度下的电生理特征。结果显示，葛氏鲈塘鳢和七彩神仙鱼的温度耐受范围具有较大差异，分别为﹣2.0 ~ 27.4 ℃以及13.1 ~ 39.3 ℃。葛氏鲈塘鳢的心率在0 ~ 19 ℃之间随着温度的升高而稳定升高，在19 ℃时达到最高，其后逐渐降低，这与葛氏鲈塘鳢心肌细胞上的电压门控钠离子通道在20 ℃时电流峰值最大且通道的开放概率最大这一电生理特征具有一致性。而七彩神仙鱼的心率则在14 ~ 31 ℃之间稳定升高，这与其心肌细胞电压门控钠离子通道电流峰值和开放概率在15 ~ 30 ℃的实验范围内随温度的升高而升高的电生理特性也一致。以上结果说明，这2种鱼类的心率、心肌细胞电压门控钠离子通道复合电流对于温度的响应与其自身的温度耐受范围以及原产栖息地温度紧密相关。因此，鱼类心肌细胞上的电压门控钠离子通道可能存在对温度的适应机制，保证鱼类循环系统在不同温度下正常运行。     

Mechanism of Maxi-K Channel Activation by Dehydrosoyasaponin-I

Dehydrosoyasaponin-I (DHS-I) is a potent activator of high-conductance, calcium-activated potassium (maxi-K) channels. Interaction of DHS-I with maxi-K channels from bovine aortic smooth muscle was studied after incorporating single channels into planar lipid bilayers. Nanomolar amounts of intracellular DHS-I caused the appearance of discrete episodes of high channel open probability interrupted by periods of apparently normal activity. Statistical analysis of these periods revealed two clearly separable gating modes that likely reflect binding and unbinding of DHS-I. Kinetic analysis of durations of DHS-I-modified modes suggested DHS-I activates maxi-K channels through a high-order reaction. Average durations of DHS-I-modified modes increased with DHS-I concentration, and distributions of these mode durations contained two or more exponential components. In addition, dose-dependent increases in channel open probability from low initial values were high order with average Hill slopes of 2.4–2.9 under different conditions, suggesting at least three to four DHS-I molecules bind to maximally activate the channel. Changes in membrane potential over a 60-mV range appeared to have little effect on DHS-I binding. DHS-I modified calcium- and voltage-dependent channel gating. 100 nM DHS-I caused a threefold decrease in concentration of calcium required to half maximally open channels. DHS-I shifted the midpoint voltage for channel opening to more hyperpolarized potentials with a maximum shift of −105 mV. 100 nM DHS-I had a larger effect on voltage-dependent compared with calcium-dependent channel gating, suggesting DHS-I may differentiate these gating mechanisms. A model specifying four identical, noninteracting binding sites, where DHS-I binds to open conformations with 10–20-fold higher affinity than to closed conformations, explained changes in voltage-dependent gating and DHS-I-induced modes. This model of channel activation by DHS-I may provide a framework for understanding protein structures underlying maxi-K channel gating, and may provide a basis for understanding ligand activation of other ion channels.     

The amino terminus of Slob, Slowpoke channel binding protein, critically influences its modulation of the channel

The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.     

The pore structure and gating mechanism of K2P channels

Two-pore domain (K2P) potassium channels are important regulators of cellular electrical excitability. However, the structure of these channels and their gating mechanism, in particular the role of the bundle-crossing gate, are not well understood. Here, we report that quaternary ammonium (QA) ions bind with high-affinity deep within the pore of TREK-1 and have free access to their binding site before channel activation by intracellular pH or pressure. This demonstrates that, unlike most other K(+) channels, the bundle-crossing gate in this K2P channel is constitutively open. Furthermore, we used QA ions to probe the pore structure of TREK-1 by systematic scanning mutagenesis and comparison of these results with different possible structural models. This revealed that the TREK-1 pore most closely resembles the open-state structure of KvAP. We also found that mutations close to the selectivity filter and the nature of the permeant ion profoundly influence TREK-1 channel gating. These results demonstrate that the primary activation mechanisms in TREK-1 reside close to, or within the selectivity filter and do not involve gating at the cytoplasmic bundle crossing.     

Electrophysiological Properties of Ion Channels in Ascaris suum Tissue Incorporated into Planar Lipid Bilayers

Ion channels are important targets of anthelmintic agents. In this study, we identified 3 types of ion channels in Ascaris suum tissue incorporated into planar lipid bilayers using an electrophysiological technique. The most frequent channel was a large-conductance cation channel (209 pS), which accounted for 64.5% of channels incorporated (n=60). Its open-state probability (P_o) was ~0.3 in the voltage range of −60~+60 mV. A substate was observed at 55% of the main-state. The permeability ratio of Cl⁻ to K⁺ (P_Cl/P_K) was ~0.5 and P_Na/P_K was 0.81 in both states. Another type of cation channel was recorded in 7.5% of channels incorporated (n=7) and discriminated from the large-conductance cation channel by its smaller conductance (55.3 pS). Its Po was low at all voltages tested (~0.1). The third type was an anion channel recorded in 27.9% of channels incorporated (n=26). Its conductance was 39.0 pS and P_Cl/P_K was 8.6±0.8. P_o was ~1.0 at all tested potentials. In summary, we identified 2 types of cation and 1 type of anion channels in Ascaris suum. Gating of these channels did not much vary with voltage and their ionic selectivity is rather low. Their molecular nature, functions, and potentials as anthelmintic drug targets remain to be studied further.     

Characterization of a partially degraded Na+ channel from urinary tract epithelium

Summary The mammalian urinary bladder contains in its apical membrane and cytoplasmic vesicles, a cation-selective channel or activating fragment which seems to partition between the apical membrane and the luminal (or vesicular space). To determine whether it is an activating fragment or whole channel, we first demonstrate that solution known to contain this moiety can be concentrated and when added back to the bladder causes a conductance increase, with a percent recovery of 139±25%. Next, we show that using tip-dip bilayer techniques (at 21°C) and a patch-clamp recorder, the addition of concentrated solution resulted in the appearance of discrete current shots, consistent with the incorporation of a channel (as opposed to an activating fragment) into the bilayer. The residency time of the channel in the bilayer was best described by the sum of two exponentials, suggesting that the appearance of the channel involves an association of the channel with the membrane before insertion. The channel is cation selective and more conductive to K⁺ than Na⁺ (by a factor of 1.6). It has a linearI–V relationship, but a singlechannel conductance that saturates as KCl concentration is raised. This saturation is best described by the Michaelis-Menten equation with aK _m of 160mm KCl and aG _max of 20 pS. The kinetics of the channel are complex, showing at least two open and two closed states.Since the characteristics of this channel are similar to a channel produced by the degradation of amiloride-sensitive Na⁺ channels by the proteolytic enzyme kallikrein (which is released by the cortical collecting duct of the kidney), we suggest that this channel then is not synthesized by the cell but is rather a degraded form of the epithelial Na⁺ channel.     

Electrostatic basis of valence selectivity in cationic channels

We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.     

Separation of Gating Properties from Permeation and Block in mslo Large Conductance Ca-activated K+ Channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K⁺ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca²⁺ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca²⁺ block, and a slow voltage-dependent Ba²⁺ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca²⁺ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca²⁺ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca²⁺ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca²⁺ concentration range spanning three orders of magnitude indicating that over this range the internal Ca²⁺ concentration does not limit the ability of the channel to be activated by voltage.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.