大鼠背根节神经元后超极化中Ca~(2 )激活K~ 电流的蜂毒明肽敏感成分I_(AHP)

为了明确大鼠背根节（DRG）神经元中存在慢的Ca2 激活K 电流成分,本实验在新鲜分散的DRG神经元胞体上,采用全细胞电压箝技术,给予DRG神经元一定强度的去极化刺激,记录刺激结束后30ms时的尾电流幅度。结果发现：（1）随着去极化时间从1ms延长至180ms时,尾电流幅度由9．3±2．8pA逐渐增大至64.1±3．4pA（P＜0．001）;（2）当去极化结束后的复极化电位降低时,尾电流幅度先逐渐下降到零,然后改变方向,逆转电位约为-63mV;（3）细胞外施加500μmol／LCd2 或细胞内液中施加11mmol／LEGTA时尾电流明显减小甚至完全消失;（4）尾电流中慢成分的幅度在细胞外给与200nmol／L蜂毒明肽后,减小了约26.32±3。9％（P＜0。01）;（5）细胞外施加10mmol／LTEA,可明显降低尾电流中的快成分。结果提示,在DRG神经元启超极化中存在Ca2 激活K 电流的蜂毒明肽敏感成分──IAHP。     

大鼠背根节神经元后超极化中Ca2+激活 K+电流的蜂毒明肽敏感成分ⅠAHP

为了明确大鼠背根节(DRG)神经元中存在慢的Ca2+激活K+电流成分,本实验在新鲜分散的DRG神经元胞体上,采用全细胞电压箝技术,给予DRG神经元一定强度的去极化刺激,记录刺激结束后30 ms时的尾电流幅度.结果发现:(1)随着去极化时间从1 ms延长至180 ms时,尾电流幅度由9.3±2.8 pA逐渐增大至64.1±3.4 pA(P＜0.001);(2)当去极化结束后的复极化电位降低时,尾电流幅度先逐渐下降到零,然后改变方向,逆转电位约为-63 mV;(3)细胞外施加500μmol/L Cd2+或细胞内液中施加11 mmol/L EGYA时尾电流明显减小甚至完全消失;(4)尾电流中慢成分的幅度在细胞外给与200 nmol/L蜂毒明肽后,减小了约26.32±3.9%(P＜0.01);(5)细胞外施加10 mmol/L TEA,可明显降低尾电流中的快成分.结果提示,在DRG神经元后超极化中存在Ca2+激活K+电流的蜂毒明肽敏感成分--ⅠAiHP.     

神经元钙激活钾电流的特征和功能

钙激活钾电流是由动作电位激活的一类外向钾电流,它是由不同类型的钙激活钾通道所介导。钙激活钾电流参与动作电位复极化和后超极化的电位的形成,并通过调节神经元的放电频率和影响神经元的放电类型参与神经元的多种上生理功能。     

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.     

二氧化硫衍生物对大鼠背根神经元瞬间外向钾电流和延迟整流钾电流的影响

运用全细胞膜片钳技术研究二氧化硫衍生物对大鼠背根神经元瞬间外向钾电流(IA和ID)和延迟整流钾电流(IK)的影响。结果发现二氧化硫衍生物剂量依赖性地增大钾通道的电导,电压依赖性地增大钾电流的幅度,且这种增大作用部分可逆。二氧化硫非常显著地使延迟整流钾电流的激活过程向超极化方向移动,使瞬间外向钾电流的失活过程向去极化方向移动。10μmol/L二氧化硫衍生物作用前后,延迟整流钾电流的半数激活电压分别是(20.3±2.1)mV和(15.0±1.5)mV;IA和ID的半数失活电压分别朝去极化方向移动了6mV和7.4mV。这些结果表明二氧化硫改变了钾通道的特性,改变了神经元的兴奋性。     

缓激肽对大鼠背根神经节分离神经元ATP激活电流的调制作用

在新鲜分离大鼠背根神经节（ＤＲＧ）的５６个细胞标本上,应用全细胞膜片箝技术进行记录。胞外加缓激肽（ＢＫ,１０＾－６￣１０＾－４ｍｏｌ／Ｌ）引坊的ＤＲＧ细胞膜反应结果如下：（１）７１．４％的细胞为内向电流,其电流反应的幅值具有明显的浓度信赖性;（２）１２．５％的细胞为外向电流;（３）１６．１％的细胞未引起可检测的膜反应,单独给予ＡＴＰ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）在大多数受栓细胞（５４／５６）     

新生大鼠脊髓薄片交感节前神经元的长时程后超极化电位

在新生大鼠胸腰段脊髓薄片,对经腹根逆行刺激鉴定的交感节前神经元（ＳＰＮｓ）进行细胞内记录．发现部分ＳＰＮｓ有长时程后超极化电位（１１－ＳＨＰ）,其达峰时间为０．２－０．７ｓ．时程１－１０ｓ,幅度７－２０ｍＶ。１１－ＡＨＰ前部常为６—３０ｍｓ达峰、短于４００ｍｓ时程的快ＡＨＰ成分。１１－ＡＨＰ除伴有膜输入电阻降低外,还呈膜电位依赖性,翻转电位为－９０至－１００ｍＶ。结果证明１１－ＡＨＰ能起着控制ＳＰＮ的放电频率的重要作用。     

咖啡因对大鼠背根神经节急性分离神经元GABA-激活电流的抑制作用

应用全细胞膜片钳记录技术,在大鼠新鲜分离背根神经节(dorsal root ganglion,DRG)神经元上,观察预加咖啡因对GABA-激活电流(IGABA)的调制作用。实验中,大部分受检细胞(97．4％,l13／116)对外加GABA敏感。1-1000μmol／L GABA引起一剂量依赖性、有明显上敏感作用的内向电流。在受检的108个DRG细胞中,约有半数(53．7％,58／108)对胞外加咖啡因(0．1-100μmol／L)敏感．产生一幅值很小的内向电流。倾加咖啡因(0．1～100μmol／L)30s后再加GABA能明显抑制GABA(100μmol／L)激活电流的幅值。预加咖啡因后GABA量效曲线明显下移;GABA-激活电流的最人值较之对照下降约57％;而Kd值(30μmol／L)几乎不变,表示此种抑制为非竞争性的。预加安定(diazepam,1μmol／L)对GABA(100μmol／L)激活电流有增强作用,而预加咖啡因(10μmol／L)有拈抗安定增强IGABA的作用。胞内透析H-8后,几乎可以完全消除咖啡因对,IGABA的抑制作用。已知GABA作用于初级感觉神经元能引起初级传入去极化,因而实验结果提示,咖啡因有可能在初级传入末梢产生对抗突触前抑制的效应。     

大鼠三叉神经节不同直径神经元ATP-激活电流的特征

目的:探讨大鼠三叉神经节不同直径神经元ATP-激活电流的特征。方法:应用全细胞膜片钳技术进行实验。结果:①92.3%(60/65)的细胞对ATP敏感,有反应的细胞可记录到三种型式的ATP-激活电流:快速激活快速失活型(Fast type,F型)、快速激活缓慢失活型(Intermediate type,I型)和缓慢激活缓慢失活型(Slowtype,S型)。三种电流均具有浓度依赖性。②小直径的细胞多表现为F型特征,大直径的细胞多表现为S型特征,而中等大小的细胞多表现为I型特征。③动力学特征:三种类型的ATP激活电流上升相从10%到90%的时间:F型:(33.6±4.5)ms;Ⅰ型:(62.2±9.9)ms;S型:(302.1±62)ms。去敏感相从10%到90%的时间:F型:(399.4±58.2)ms;S型:>500ms。④I-V曲线:三种电流均表现为内向整流的特性,而且翻转电位均为0~5mV。⑤量-效关系:Ⅰ型的量-效曲线居中间,F型的下移,S型的上移,三种类型电流量-效曲线的EC50非常接近。结论:三种型式的ATP-激活电流可能是由不同亚单位组合的P2X受体各亚型所介导,这些亚型分布于不同大小的三叉神经节神经元,从而传导不同的信息。     

奥卡西平对偏头痛大鼠三叉神经节神经元钙电流的影响*

目的:研究奥卡西平(Oxcarbazepine,OXC)对大鼠三叉神经节神经元钙电流的调控作用。方法:SD大鼠随机分为3组(n=8):生理盐水组(NS组),致炎剂组(IS组),OXC预防组。应用膜片钳技术,采用全细胞记录方式,观察OXC对偏头痛大鼠急性分离的三叉神经节的高电压激活钙电流(HVA-ICa)的调控作用。结果:OXC能够抑制钙电流,使钙电流的激活曲线向去极化方向移动,使钙电流的失活曲线向超级化方向移动。结论:OXC可能通过抑制钙离子进入细胞膜,来预防偏头痛的发作。同时OXC可能对外周神经系统及伤害感受的传入的兴奋性起到调控作用。     

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons

Auxiliary beta-subunits associated with pore-forming Slo1 alpha-subunits play an essential role in regulating functional properties of large-conductance, voltage- and Ca(2+)-activated K(+) channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by beta-subunits (beta1, beta2, beta3, or beta4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rbeta2 (but not rbeta3-subunit) was identified as a molecular component in rat lumbar L4-6 dorsal root ganglia (DRG) by RT-PCR responsible for inactivating large-conductance Ca(2+)-dependent K(+) currents (BK(i) currents) in small sensory neurons. The properties of native BK(i) currents obtained from both whole-cell and inside-out patches are very similar to inactivating BK channels produced by co-expressing mSlo1 alpha- and hbeta2-subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BK(i) channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BK(i) currents. Single BK(i) channel currents derived from inside-out patches revealed that one BK(i) channel contained three rbeta2-subunits (on average), with a single-channel conductance about 217 pS under 160 K(+) symmetrical recording conditions. Blockade of BK(i) channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after-hyperpolarization. We propose that the BK(i) channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS).     

Ca(2+)-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca(2+)-activated Ca2+ release.

We examined the possibility that Ca2+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (GkCa,1) and a slower component that had a distinct rising phase (GkCa,2). Both components required influx of extracellular Ca2+ for their activation, and neither was blocked by extracellular TEA (10 mM). GkCa,1 was selectively blocked by apamin, whereas GkCa,2 was selectively reduced by noradrenaline. The time course of GkCa,2 was markedly temperature sensitive. GkCa,2 was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with ruthenium red. These results suggest that influx of Ca2+ directly gates one class of K+ channels and leads to release of Ca2+ from intracellular stores, which activates a different class of K+ channel.     

CaMKII inactivation by extracellular Ca(2+) depletion in dorsal root ganglion neurons

A mechanism by which Ca(2+)/CaM-dependent protein kinase (CaMKII) is autophosphorylated by changes in extracellular calcium in the absence of detectable changes in cytoplasmic [Ca(2+)] has been identified. We find that when the external Ca(2+) concentration ([Ca(2+)](O)) is lowered, Ca(2+) is released from intracellular stores to maintain a constant cytoplasmic Ca(2+) level, gradually depleting the endoplasmic Ca(2+) stores. Accompanying the store-depletion is a rapid decrease in CaMKII activity. Approximately 25% of the measured CaMKII autophosphorylation in DRG neurons in culture can be regulated by Ca(2+) flux from intracellular stores caused by manipulating [Ca(2+)](O), as shown by blocking refilling of store-operated Ca(2+)-channels with SK&F 96365, Ruthenium Red, and a partial block with Ni(2+). Blocking voltage-gated Ca(2+)-channels with either isradipine or SR 33805, had no effect on CaMKII autophosphorylation induced by restoring Ca(2+)(O) to normal after depleting the intracellular Ca(2+) stores. These results show that removal of Ca(2+)(O) has profound effects on intracellular Ca(2+) signaling and CaMKII autophosphorylation, in the absence of measurable changes in intracellular Ca(2+). These findings have wide-ranging significance, because [Ca(2+)](O) is manipulated in many experimental studies. Moreover, this explanation for the paradoxical changes in CaMKII phosphorylation in response to manipulating [Ca(2+)](O) provides a possible mechanism linking activity-dependent depletion of Ca(2+) from the synaptic cleft to a protein kinase regulating many neuronal properties.     

Glutathione modulates Ca(2+) influx and oxidative toxicity through TRPM2 channel in rat dorsal root ganglion neurons

Glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells and maintains thiol redox in the cells. GSH depletion has been implicated in the neurobiology of sensory neurons. Because the mechanisms that lead to melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition in response to glutathione depletion and 2-aminoethyldiphenyl borinate (2-APB) administration are not understood, we tested the effects of 2-APB and GSH on oxidative stress and buthionine sulfoximine (BSO)-induced TRPM2 cation channel currents in dorsal root ganglion (DRG) neurons of rats. DRG neurons were freshly isolated from rats and the neurons were incubated for 24 h with BSO. In whole-cell patch clamp experiments, TRPM2 currents in the rat were consistently induced by H₂O₂ or BSO. TRPM2 channels current densities and cytosolic free Ca²⁺ content of the neurons were higher in BSO and H₂O₂ groups than in control. However, the current densities and cytosolic Ca²⁺ release were also higher in the BSO + H₂O₂ group than in the H₂O₂ alone. When intracellular GSH is introduced by pipette TRPM2 channel currents were not activated by BSO, H₂O₂ or rotenone. BSO and H₂O₂-induced Ca²⁺ gates were blocked by the 2-APB. Glutathione peroxidase activity, lipid peroxidation and GSH levels in the DRG neurons were also modulated by GSH and 2-APB inhibition. In conclusion, we observed the protective role of 2-APB and GSH on Ca²⁺ influx through a TRPM2 channel in intracellular GSH depleted DRG neurons. Since cytosolic glutathione depletion is a common feature of neuropathic pain and diseases of sensory neuron, our findings are relevant to the etiology of neuropathology in DRG neurons.     

Versatile regulation of cytosolic Ca2+ by vanilloid receptor I in rat dorsal root ganglion neurons

Analysis of small dorsal root ganglion (DRG) neurons revealed novel functions for vanilloid receptor 1 (VR1) in the regulation of cytosolic Ca(2+). The VR1 agonist capsaicin induced Ca(2+) mobilization from intracellular stores in the absence of extracellular Ca(2+), and this release was inhibited by the VR1 antagonist capsazepine but was unaffected by the phospholipase C inhibitor xestospongins, indicating that Ca(2+) mobilization was dependent on capsaicin receptor binding and was not due to intracellular inositol-1,4,5-trisphosphate generation. Confocal microscopy revealed extensive expression of VR1 on endoplasmic reticulum, consistent with VR1 operating as a Ca(2+) release receptor. The main part of the capsaicin-releasable Ca(2+) store was insensitive to thapsigargin, a selective endoplasmic reticulum Ca(2+)-ATPase inhibitor, suggesting that VR1 might be predominantly localized to a thapsigargin-insensitive endoplasmic reticulum Ca(2+) store. In addition, VR1 was observed to behave as a store-operated Ca(2+) influx channel. In DRG neurons, capsazepine attenuated Ca(2+) influx following thapsigargin-induced Ca(2+) store depletion and inhibited thapsigargin-induced inward currents. Conversely, transfected HEK-293 cells expressing VR1 showed enhanced Ca(2+) influx and inward currents following Ca(2+) store depletion. Combined data support topographical and functional diversity for VR1 in the regulation of cytosolic Ca(2+) with the plasma membrane-associated form behaving as a store-operated Ca(2+) influx channel and endoplasmic reticulum-associated VR1 possibly functioning as a Ca(2+) release receptor in sensory neurons.     

Ca(2+) mobilization through dorsal root ganglion Ca(2+)-sensing receptor stably expressed in HEK293 cells

The rat dorsal root ganglion (DRG) Ca²⁺-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca²⁺] ([Ca²⁺]_e) from 0.5 to 1 mM resulted in increases in [Ca²⁺]_i levels, which were blocked by 30 µM 2-aminoethyldiphenyl borate. [Ca²⁺]_e-response studies indicate a Ca²⁺ sensitivity with an EC₅₀ of 1.75 ± 0.10 mM. NPS R-467 and Gd³⁺ activated the CaR. When [Ca²⁺]_e was successively raised from 0.25 to 4 mM, peak [Ca²⁺]_i, attained with 0.5 mM, was reduced by 50%. Similar reductions were observed with repeated applications of 10 mM Ca²⁺, 1 and 10 µM NPS R-467, or 50 and 100 µM Gd³⁺, indicating desensitization of the response. Furthermore, Ca²⁺ mobilization increased phosphorylated protein kinase C (PKC) levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca²⁺ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Ca_e²⁺. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca²⁺]_i transients by 49.9 ± 5.2% (at 1 mM Ca²⁺) and 40.5 ± 6.5% (at 2 mM Ca²⁺), compared with controls. The findings suggest involvement of PKC in the pathway for Ca²⁺ mobilization following CaR activation. desensitization; protein kinase C     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.     

Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels

In this study, single-channel recordings of high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)-blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed-blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening.