β—淀粉样蛋白31—35片段对海马神经元分离膜片Ca^2＋激活大电导钾通道的抑制

为了确定β－淀粉样蛋白（AβP）在影响神经元电生理特性并导致神经毒作用时的最短活性序列,实验采用片钳技术,在急性分离的大鼠海马CA1区锥体细胞的“内面向外”式膜片上,观察了AβP的31－35和25－35片段对C^2 a激活大电导钾（BK）通道活动的影响,结果显示,浴液中给预5umol/L的AβP31－35后,BK通道的平均开放概率（P0）和开放频率在1－3min内分别减少了85．8％（P＜0．01）和72．1％（P＜0．01）,平均开放时间减少了41．1％（P＜0．01）,平均电流幅度则无明显改变（P＞0．05）,给予同样摩尔浓度的AβP25－35后,BK通道平均P0减少了85．5％（P＜0．01）,平均开放时间减少51．4％,（P＜0．05）,结果提示：两种AβP片段对海马神经元BK通道具有抑制作用,。这可能与AβP的神经性作用有关,AβP－31－35片段可能是AβP分子中影响细胞电生理特性的最小活性序列。     

大鼠背根神经节神经元上失活 BK 通道特性的研究

大电导的电压和 Ca²⁺ 激活的 K⁺ 通道 (BK 通道 ) 在哺乳动物的组织中广泛表达,起着多种多样的作用 . 目前只有少数组织中 BK 通道的性质被深入地研究,而且鲜见有失活的 BK 通道 (BK_i) 的报道,尤其是在神经元中 . 发现在大鼠小直径的背根神经节 (DRG) 神经元中,普遍存在失活的 BK 通道 . 失活的 BK 电流成分是 Ca²⁺敏感的,可以被大电导的 BK 通道特异阻断剂 ChTX 所阻断,而且木瓜蛋白酶可以从胞外改变通道失活的特性 .     

p-淀粉样蛋白31-35片段对海马神经元分离膜片Ca2+激活大电导钾通道的抑制

为了确定β-淀粉样蛋白(AβP)在影响神经元电生理特性并导致神经毒作用时的最短活性序列,实验采用膜片钳技术,在急性分离的大鼠海马CA1区锥体细胞的"内面向外”式膜片上,观察了AβP的31-35和25-35片段对Ca2+激活大电导钾(BK)通道活动的影响.结果显示,浴液中给予5μmol/L的AβP31-35后,BK通道的平均开放概率(Po)和开放频率在1～3 min内分别减少了85.8%(P＜0.01)和72.1%(P＜0.01);平均开放时间减少了41.1%(P＜0.01);平均电流幅度则无明显改变(P＞0.05);给予同样摩尔浓度的AβP25-35后,BK通道平均Po减少了85.5%(P＜0.01),平均开放时间减少了51.4%,(P＜0.05).结果提示,两种AβP片段对海马神经元BK通道具有抑制作用,这可能与AβP的神经毒性作用有关;AβP 31-35片段可能是AβP分子中影响细胞电生理特性的最小活性序列.     

新生大鼠下丘脑神经元L—Ca^2＋通道电流活动特征

目的研究新生大鼠下丘脑神经元L-Ca2+通道单通道特性;Ca2+通道激动剂BayK8644对Ca2+通道单通道特性的影响.方法采用神经元急性分离技术;用膜片钳细胞贴附式记录方式进行研究.结果大鼠下丘脑神经元L-Ca2+通道是一种电导相对较大的Ca2+通道,其电导为(29.5±3.1)pS,平均开放时间(τ0)为0.28ms,平均关闭时间的短关闭时间常数(τc1)为2.91ms,长关闭时间常数(τc2)为53.22ms.此通道几乎不存在时间依赖性失活.BayK8644显著增加通道的开放概率,通道平均开放时间增加为1.61ms.结论下丘脑神经元存在L-Ca2+通道,该通道具有明显电压依赖性,而无显著的时间依赖性.通道特征与文献报道的其它神经元上L-Ca2+通道相似,也有明显不同,显示下丘脑神经元L-Ca2+钙通道的独特性.     

血管紧张素Ⅱ对血管平滑肌细胞中大电导钙激活钾通道的多重调节作用（英文）

肾素-血管紧张素系统(renin-angiotensin system, RAS)是影响血管平滑肌细胞张力的重要因素。RAS主要活性物质血管紧张素Ⅱ (angiotensin Ⅱ, Ang Ⅱ)可通过激活血管紧张素Ⅱ-1型受体(angiotensin Ⅱ type 1 receptor, AT1R)升高胞内Ca~(2+)浓度,收缩平滑肌细胞。大电导钙激活钾(large-conductance Ca~(2+)-and voltage-activated potassium, BK)通道是血管平滑肌细胞中分布最广、表达最多的钾离子通道,在维持细胞膜电位和胞内钾钙平衡中发挥重要作用。血管平滑肌细胞上的BK通道主要包含α与β1两种亚基。其中功能亚基BKα上分布有膜电位及Ca~(2+)感受器。因此当膜电位或细胞内Ca~(2+)浓度升高时会反馈性引起BK通道开放。然而,越来越多的研究显示,尽管Ang Ⅱ可升高胞内Ca~(2+)浓度,但却通过激活PKC通路、促进AT1R与BKα通道形成的异源二聚体内吞、加快α与β1亚基解离等途径抑制BK通道的表达和功能。在一些情况下,Ang Ⅱ对BK通道也可表现出激活作用,但机制尚不完全明确。该综述总结了Ang Ⅱ对BK通道抑制或激活两方面效应的可能原因,为改善细胞内离子失衡提供理论依据。     

新生大鼠下丘脑神经元L-Ca~(2 )通道电流活动特征

目的 :研究新生大鼠下丘脑神经元L Ca2 通道单通道特性 ;Ca2 通道激动剂BayK 86 44对Ca2 通道单通道特性的影响。方法 :采用神经元急性分离技术 ;用膜片钳细胞贴附式记录方式进行研究。结果 :大鼠下丘脑神经元L Ca2 通道是一种电导相对较大的Ca2 通道 ,其电导为 (2 9.5± 3.1)pS ,平均开放时间 (τ0 )为 0 .2 8ms,平均关闭时间的短关闭时间常数 (τc1)为 2 .91ms,长关闭时间常数 (τc2 )为 5 3.2 2ms。此通道几乎不存在时间依赖性失活。BayK86 44显著增加通道的开放概率 ,通道平均开放时间增加为 1.6 1ms。结论 :下丘脑神经元存在L Ca2 通道 ,该通道具有明显电压依赖性 ,而无显著的时间依赖性。通道特征与文献报道的其它神经元上L Ca2 通道相似 ,也有明显不同 ,显示下丘脑神经元L Ca2 钙通道的独特性     

模拟失重增强大鼠脑动脉血管平滑肌细胞大电导钙激活钾通道功能

本工作旨在探讨短期模拟失重大鼠脑动脉血管平滑肌细胞（vascular smooth muscle cells,VSMCs）大电导钙激活钾通道（large conductance calcium-activated potassium channels,BKCa channels）功能的改变。以尾部悬吊大鼠模型模拟失重对脑血管的影响。应用激光扫描共聚焦显微镜测定VSMCs胞内游离钙浓度（[Ca^2＋]i）;采用细胞贴附模式,记录BKCa通道的单通道活动。结果表明,模拟失重1周后,大鼠脑动脉VSMCs的[Ca^2＋]i比对照组显著升高（P〈0．05）：BKCa通道的开放概率（Po）与平均开放时间（To）显著增加（P〈0．05）,而单通道电导与平均关闭时间（Tc）则无显著变化。总之,1周模拟失重可引起脑动脉VSMCs的BKCa通道功能显著增强,且与细胞[Ca^2＋]i的升高同步出现。结果提示,脑动脉VSMCs的离子通道机制可能参与介导模拟失重引起的脑血管适应性变化。     

游泳训练后大鼠心肌细胞cAMP、cGMP含量的变化

目的:探讨游泳训练后大鼠心肌环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)含量的变化。方法:用心脏重量(mg)/体重(g)计算心脏系数,放射免疫法测定心肌cAMP、cGMP含量。结果:8周游泳训练导致心脏系数显著增高(P<0.05),心肌cAMP水平升高不显著(P>0.05),cGMP水平显著升高(P<0.05)。结论:8周游泳训练导致心脏代偿性肥大,cAMP/cGMP比值减小。     

过氧化氢诱发的小脑皮质凋亡神经元膜K+通道电流的变化

用新生ＳＤ大鼠小脑皮质细胞进行培养,用Ａｒａ－Ｃ抑制非神经元生长,以Ｈ２Ｏ２诱发神经元凋亡。用膜片细胞贴附式观察了凋亡神经元膜钾离子通道电流的变化,结果表明,凋亡小脑皮质神经元膜Ｋ通道在不同箍位电压下,通道电流（ＩＫ）幅度小于正常神经元的,单位电导小于正常神经元的,通道的平均开放时间,开放概率、短开放及长开放时间常数亦均小于正常神经元的。说明小脑皮质凋亡神经元Ｋ通道活动减弱     

下丘脑神经元电压依赖性钾电流活动在大鼠出生后发育过程中的变化

采用膜片钳细胞贴附式技术,比较研究SD大鼠下丘脑神经元电压依赖性钾通道(voltage-dependent potassium channel,Kv)单通道电流活动的动力学特性,在出生后发育过程中的变化。出生不同天数的大鼠,其下丘脑神经元上Kv通道的电流强度和电导无显著差别(P>0.05),通道电导接近120 pS;单位时间内封接膜片上N个通道的开放概率的总和升高,由第1天的0.19±0.08(n=10)上升到第9天的0.30±0.09(n=10,P<0.05),单通道活动密度增加,由0.14 channel/μm2升高至0.26 channel/μm2。上述结果提示大鼠下丘脑神经元在出生发育过程中,Kv单通道活动的动力学发生显著变化。     

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK_Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK_Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK_Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca²⁺ and voltage without altering the unitary conductance and the K⁺ selectivity. Our results suggest a novel mechanism by which exercise training increases the K⁺ currents by changing the BK_Ca channel activities and kinetics.     

Effects of mechanical stretch on the functions of BK and L-type Ca2+ channels in vascular smooth muscle cells

It is well recognized that pathologically increased mechanical stretch plays a critical role in vascular remodeling during hypertension. However, how the stretch modulates the functions of ion channels of vascular smooth muscle cells (VSMCs) remains to be elucidated. Here, we demonstrated the effects of mechanical stretch on the activity of large conductance calcium, voltage-activated potassium (BK) and L-type Ca²⁺ channels. In comparison with 5% stretch (physiological), 15% stretch (pathological) upregulated the current density of L-type Ca²⁺ and BK channels as well as the frequency and amplitude of calcium oscillation in VSMCs. 15% stretch also increased the open probability and mean open time of the BK channel compared with 5% stretch. BK and L-type Ca²⁺ channels participated in the mechanical stretch-modulated calcium oscillation. Our results suggested that during hypertension, pathological stretch altered the activity of BK and L-type Ca²⁺ channels and manipulated the calcium oscillation of VSMCs.     

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.     

Mechanosensitive Properties of BK Channels from Embryonic Rat Neuroepithelium

The mechanosensitive properties of large-conductance Ca²⁺-activated K⁺ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed in on-cell patches. This mechanosensitivity was not mediated by Ca²⁺ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment. Received: 1 February 1996/Revised: 25 March 1996     

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca²⁺ concentration, [Ca²⁺]_i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca²⁺-activated K⁺ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca²⁺]_i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca²⁺-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca²⁺]_i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca²⁺]_i and of cell migration. Thus, BK channels function to maintain elevations in [Ca²⁺]_i needed to sustain increases in DBTRG cell migration.     

Activation of galanin receptor 2 stimulates large conductance Ca-dependent K (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca²⁺-activated K⁺ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca²⁺ flux and Ca²⁺-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8 ± 16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca²⁺-free solution, suggesting that extracellular Ca²⁺ is not essential. Chelating intracellular Ca²⁺ by either the slow Ca²⁺ buffer EGTA or the fast Ca²⁺ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca²⁺. The role of Ca²⁺ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca²⁺ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca²⁺ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca²⁺ is due to Ca²⁺ efflux from ER through IP3R sequentially opening BK channels.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Ca2+-activated K channels in parotid acinar cells: The functional basis for the hyperpolarized activation of BK channels

Fluid secretion relies on a close interplay between Ca²⁺-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca²⁺ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca²⁺ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca²⁺ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca²⁺ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca²⁺. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes.Key words: ion channels, Ca²⁺-activated K channels, maxi-K channels, IK1 channels     

Involvement of TRP channels in the signal transduction of bradykinin in human osteoblasts

Bradykinin (BK), a mediator of pain and inflammation, is involved in bone metabolism. We have previously reported that BK increased the synthesis of interleukin-6 and prostaglandin E₂ via phosphorylation of ERK1/2 in human osteoblasts, SaM-1. In the present study, we investigated the signal transduction pathway of BK focusing on intracellular Ca²⁺ kinetics in SaM-1 cells. Bath-applied BK increased intracellular Ca²⁺ concentration through the activation of B₂ receptors. Removal of extracellular Ca²⁺ attenuated the effects of BK. Additionally, thapsigargin, endoplasmic reticulum Ca²⁺ pump inhibitor, completely inhibited BK-induced increase of intracellular Ca²⁺. These results suggested that bath-applied BK activated store-operated Ca²⁺ channels (SOCCs) following Ca²⁺ store depletion via B₂ receptor. Although the molecular components of SOCCs have yet to be conclusively identified in all cell types, recent studies demonstrated that transient receptor potential canonical (TRPC) channels are candidates for them. TRPC1, TRPC3, TRPC4 and TRPC6 were expressed in SaM-1 cells and inhibitors of TRP channel, 2-aminoethoxydiphenyl borate, GdCl₃, LaCl₃ and flufenamic acid, inhibited the effects of BK. These findings suggested that BK activated SOCCs and induced Ca²⁺ influx via B₂ receptor in human osteoblasts. Molecular components of the SOCCs are suggested to be TRPC channels.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.