河蟹眼柄神经分泌细胞离子通道的膜片钳研究

采用全细胞膜片钳技术对培养12－24小时不同形态河蟹眼柄视节端髓X器官（MTXO）神经分泌细胞离子通道进行了研究。结果表明,河蟹眼柄MTXO中分布的A、B、C三种类型神经分泌细胞均可记录到由向电流和外向电流组成的正常全细胞电流。内向电流由高电压激活钙离子通道电流（Lca)和对TTX敏感钠离子通道电流（INa）组成。ICa的激活电压为－30mV,在0－ 20mV电压下达到峰值,在－40mV和－70mV保持电压下记录的ICa激活阈值、初始峰值及I－V曲线无明显差别。外向电流明显,幅值较大,包括对4－AP敏感的快速激活、快速失活钾离子通道电流（IA）和对TEA敏感的缓慢激活、缓慢失活钾离子通道电流（IK）。正常蟹种、二龄成蟹和早熟蟹种MTXO神经分泌细胞均表达电压门控钠、钾、钙离子通道,通道电流和电压特征无明显区别.     

胞外钙对豚鼠耳蜗Deiters细胞钾电流的调制

为观察胞外钙对豚鼠耳蜗单个离体Deiters细胞钾电流的调控作用并探讨其机制,实验记录了Deiters细胞在正常细胞外液和无钙外液中的全细胞钾电流(whole cell K^ currents,IK),并分析了其电生理学特性的改变。结果观察到,Deiters细胞与在正常细胞外液中相比,在祛除细胞外液中的Ca^2 后Ik电流幅值明显增加,弦电导值亦明显增加,但其平衡电位未明显改变。在无钙外液中Ik电流的反转电位向超极化方向明显移位,更接近于按照Ner-nst方程得出的K^ 理论平衡电位;而且其稳态激活曲线亦向超极化方向明显移位,但其激活趋势与正常相比无明显改变。此外,观察了Deiters细胞中钙抑制性钾电流的电流-电压关系和电导-电压关系,发现两者均呈“S”形,提示此钙抑制性钾电流可能存在2种不同的钾电导成分。由此,推测可能有两种机制参与胞外钙对Deiters细胞钾电流的调控：(1)Deiters细胞中的Ik通道可能存在一个Ca^2 敏感结构域,胞外Ca^2 可能通过改变此结构域而对Ik电流产生调制;(2)Deiters细胞中可能存在一种新型的双相门控性钾通道或钾通道耦联型受体或是一种新型的钾通道亚型,祛除胞外Ca^2 可激活此新型钾电导而对L电流产生调制。由此推测,在听觉形成过程中,胞外钙浓度下降可以对Deiters细胞的全细胞钾电流产生调制,从而更有利于Deiters细胞内K^ 外流,进而有效地缓冲外毛细胞周围的K^ 浓度：而且还可以使Deiters细胞产生更快的复极化并有利于维持其静息状态。     

海马脑片锥体神经元钙离子通道的盲法膜片钳研究

目的和方法 :采用大鼠海马脑片盲法膜片钳全细胞记录技术研究CA1区锥体神经元电压门控性Ca2 通道的动力学特征。结果 :大鼠海马脑片CA1区锥体神经元电压门控性Ca2 通道电流具有如下特点 :①激活的阈电位偏低 ,为 (- 4 9.3± 8.6 )mV ,范围为 - 6 5～ - 30mV(n =2 3)。②衰减时间常数τ值较大 ,且变化范围大 (10 0～ 70 0ms) (n =12 ) ,并且衰减具有Ca2 电流幅值的依赖性 ,③稳态失活呈现电压依赖性 ,半失活电压为 (- 5 5 .4± 9.7)mV ,斜率因子为 (5 .3± 0 .9)mV(n =10 )。④当细胞外Ca2 浓度为 2 .5mmol/L时 ,Ca2 通道的反转电位为 (5 5±13)mV(n =10 )。⑤尾电流成分较为单一 ,不表现电压依赖性。另外 ,Ca2 电流对戊脉胺及双氢吡啶类化合物硝苯地平均不敏感。结论 :根据上述Ca2 电流特征 ,海马脑片CA1区锥体神经元上的Ca2 通道主要以N型为主     

白介素1β抑制培养的大鼠皮层神经元钠电流峰值和动作电位幅度

白介素1β(interleukin-1β,IL-1β)是重要的促炎细胞因子,在中枢神经系统的生理学和病理学过程中发挥关键作用。电压门控钠通道是可兴奋细胞电学活动的基础,控制神经元的兴奋性和动作电位。最近的研究又显示了IL-1β与电压门控通道之间的相互作用。为考察中枢神经元中IL-1β与电压门控钠通道之间的相互作用,本研究使用10ng/mL的IL-1β处理培养的大鼠皮层神经元24h,通过电压钳技术测定电压门控钠电流,结果表明IL-1β处理抑制钠电流幅度,但不改变其激活和失活性质。与电压钳记录结果相一致,电流钳记录表明IL-1β降低动作电位幅度但不影响阈值。这些结果显示长时间的IL-1β处理可以抑制电压门控钠电流,这种抑制作用减小了动作电位幅度,这可能改变神经元的电学性质、突触传导等基本功能,并提示了IL-1β在神经系统损伤和疾病中作用的新的思路。     

血竭及其成分龙血素B对背根神经节细胞河豚毒素敏感型钠通道电流的影响

应用全细胞膜片钳技术在急性分离的大鼠背根神经节细胞上观察血竭及其成分龙血素B对河豚毒素敏感型电压门控性钠通道电流的影响. 结果发现, 血竭和龙血素B对河豚毒素敏感型钠通道电流峰值均有浓度依赖的抑制作用, 高浓度的血竭(0.05%)和龙血素B(0.02 mmol/L)使河豚毒素敏感型钠通道电流峰值偏移, 并电压依赖性地影响通道的激活和失活过程. 以上结果表明, 血竭对河豚毒素敏感型钠通道电流的影响主要是其成分龙血素B作用的结果. 血竭的镇痛作用可能部分是通过其成分龙血素B直接干预初级感觉神经元电压门控性钠通道, 阻碍痛觉信息传入而产生的.     

钙激活性氯离子通道的电生理研究

目的:研究大鼠肺动脉平滑肌细胞上钙激活性氯离子通道的电流、电压电流关系等电生理特性.方法:采用急性酶分离法(胶原酶Ⅰ型和木瓜蛋白酶)分离出单个肺动脉平滑肌细胞,应用膜片钳技术,测定各组大鼠肺动脉平滑肌细胞上钙激活性氯离子通道电流和电压电流.结果:急性酶分离法能成功分离出适用于膜片钳记录的单个大鼠肺动脉平滑肌细胞,并测到稳定的钙激活性氯离子通道电流,该电流表现出时间、电压及钙离子依赖性,并呈外向整流特征.结论:大鼠肺动脉平滑肌细胞的钙激活氯离子通道具有时间依赖性、钙离子依赖性和电压依赖性,并具有外向整流特征.     

过氧化氢对老年豚鼠耳蜗外毛细胞大电导钙激活钾通道电流的影响

本文旨在研究氧自由基(oxygen free radical)供体——过氧化氢(H2O2)对老年豚鼠耳蜗外毛细胞大电导钙激活钾通道(large-conductance Ca2+-activated potassium channels,BKCa channels)电流的影响,探讨氧自由基对老年豚鼠耳蜗外毛细胞BKCa通道电流的作用机制。采用急性酶分离方法分离耳蜗外毛细胞,用全细胞膜片钳记录通道电流,鉴别并分析通道特性,观察不同浓度H2O2对BKCa通道电流的影响。结果显示,在膜片钳全细胞模式下,可记录到一串幅值较大、快速激活、几乎不失活的电流,激活电压大于-40~-30 mV,电流随膜电位的增加而增强,电流幅值不断增大,并表现出外向整流的特性,无"rundown"现象;IbTX(100 nmol/L)可完全阻断通道活动,证实该电流为BKCa通道电流。BKCa通道电流表现出明显的H2O2浓度依赖性激活,电流幅值和峰值电流密度随H2O2浓度(1、2、4μmol/L)增加而增大。以上结果提示,外毛细胞可能存在能够调节胞内钙平衡的氧自由基/BKCa途径。     

大鼠海马CA1区锥体神经元I_A和I_K在出生后早期的变化

大脑快速发育期(brain growth spurt,BGS)是神经元生长、突触连接的关键时期;电压门控性K+通道是维持细胞兴奋性和神经元间信息传递的关键通道。本文旨在探究BGS期内大鼠海马CA1区锥体神经元电压门控性K+通道电流及其通道动力学特性的变化,以期找出大鼠海马CA1区锥体神经元电压门控性K+通道发育的关键期。采用全细胞膜片钳技术,研究出生后0~4周大鼠海马CA1区脑片上的锥体神经元全细胞电压门控性K+通道电流及其通道动力学特性。结果显示:在测试电压为+90mV下,以出生后0周为参照,出生后1~4周的瞬时外向K+通道电流(IA)的最大电流密度的增幅分别为(16.14±0.51)%、(81.73±10.71)%、(106.72±5.29)%、(134.58±8.81)%(n=10,P<0.05);延迟整流K+通道电流(IK)的最大电流密度增幅分别为(16.75±3.88)%、(134.01±2.85)%、(180.56±8.49)%、(194.5±8.53)%(n=10,P<0.05),显示K+通道电流密度于1~2周增幅最大;IA的激活曲线向左移,半数激活电压随周龄增加逐渐减小,分别为14.67±0.75、13.46±0.64、8.39±0.87、4.60±0.96、0.54±0.92(mV,n=10,P<0.05);IK的激活曲线向左移,半数激活电压随周龄增加逐渐减小,分别为8.94±0.85、6.65±0.89、0.47±1.15、1.80±0.89、8.56±1.08(mV,n=10,P<0.05)。IA的失活曲线向左移,0周龄与1周龄之间的半数失活电压没有显著性差异,而出生后1~4周随周龄增加半数失活电压逐渐减小(P<0.05),分别为45.68±1.26、46.81±0.78、48.64±0.81、51.96±1.02、58.31±1.35(mV,n=10)。以上结果表明,随着鼠龄的增加,IA和IK电流密度逐渐增加,电压门控性K+通道半数激活、失活电压降低,尤其是出生后1周至2周变化明显,上述变化与海马神经元的逐渐发育成熟及其功能的完善有关。     

熊果酸激活低分化鼻咽癌细胞氯通道和减小细胞容积

本文旨在研究熊果酸对低分化鼻咽癌细胞氯通道的激活作用,以及熊果酸对其细胞容积的影响。采用膜片钳技术记录熊果酸激活的鼻咽癌细胞(CNE-2Z)全细胞氯电流,应用离子置换、改变细胞外渗透压、氯通道阻断剂等观察熊果酸诱导的氯电流的特性,活细胞动态图像分析技术测量细胞容积变化。结果显示,等渗条件下可记录到CNE-2Z细胞微弱且稳定的背景氯电流,细胞外灌流熊果酸可浓度依赖性(1～100nmol/L)诱发氯电流的产生,在±80mV电压钳制下,100nmol/L熊果酸激活的氯电流的平均电流密度为(78.92±6.39)pA/pF和(59.86±4.86)pA/pF,该电流具有较明显的外向优势,不表现明显的时间依赖性和电压依赖性失活。该电流翻转电位为(4.83±0.30)mV,较接近Cl平衡电位(0.9mV)。熊果酸激活的氯通道对不同阴离子的通透性为:Cl-=I->Br->葡萄酸根离子。该电流具有容积敏感性,可被细胞外高渗透压显著抑制;氯通道阻断剂他莫昔芬(tamoxifen)、5-硝基-2-(3-苯丙胺)苯甲酸[(5-nitro-2-(3-phenylpro-pylamino)benzoic acid,NPPB]可抑制该电流。细胞外灌流熊果酸1h后,细胞容积减小,氯通道阻断剂NPPB可抑制该容积变化。以上结果提示,熊果酸可以激活低分化鼻咽癌细胞的氯通道,使Cl外流,进而引起细胞容积减小。     

河蟹眼柄神经内分泌细胞Glu受体研究

作者采用全细胞膜片钳技术测定了中华绒螯蟹 (Eriocheirsinensis)眼柄视神经节端髓X器官 (MTXO)神经内分泌细胞对 0 0 1— 10mmol/L谷氨基酸 (Glu)的反应 ,并结合药理学方法进行了Glu受体研究。结果表明 ,Glu激活A型和B型细胞离子型Cl-通道蛋白受体 ,诱导快速激活、快速失活的配体门控Cl-通道电流 (IGlu)。依据内外液的Cl-浓度比例引发去极化或超极化反应 ,继续施加Glu ,细胞快速出现脱敏反应 ;去除Glu后 ,细胞约需 2 0s恢复对Glu的敏感状态。IGlu幅值呈浓度依赖性 ,量 效关系曲线呈线形 ,激活阈值为 0 0 1mmol/L ,约 5mmol/L达到饱和。河蟹眼柄神经内分泌细胞IGlu明显受到Cl-通道阻断剂picrotoxin(0 5mmol/L)抑制 ;对离子型Glu受体激动剂Quisqualate、Kainate、NMDA、AMPA不敏感。Ibotenicacid(IA)可模拟Glu诱导快速激活、快速脱敏的Cl-电流 ,并与Glu产生交互脱敏作用。Glu和GABA对河蟹眼柄神经内分泌细胞无交叉脱敏和交叉激活作用 ,甘氨酸 (Gly)没有诱导细胞产生任何反应 ,提示中枢神经系统通过Glu和GABA两套系统实现对眼柄神经内分泌系统的精确调控。     

Cysteine modification of a putative pore residue in ClC-0: implication for the pore stoichiometry of ClC chloride channels

The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share approximately 50-60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl(-) less effectively, so the P(o)-V curve was shifted to a more depolarized potential by approximately 45 mV. The K165C-K165 heterodimer showed double-barrel-like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate.     

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl^–, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl^– concentration [Cl^–]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl^–]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents. heat shock; geldanamycin; cellular stress; channel trafficking; transepithelial chloride transport     

Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata

Membranehyperpolarization normally activates the slow gate of theTorpedo voltage-gated chloride channel(ClC-0). To elucidate the structural basis of this process, carboxyterminus truncation mutants and chimeras were constructed, expressed inXenopus oocytes, and evaluated using atwo-microelectrode voltage clamp. Introduction of stop codons atseveral positions between transmembrane domains 12 and 13 (D12 and D13)showed no expression, whereas a truncation just after D13 yieldedwild-type currents. A chimera (022) entailing the substitution of thecarboxy-terminal cytoplasmic tail after Lys-520 with the correspondingregion of ClC-2 lacked slow gating, whereas a more conservativeconstruct (chimera 002), in which D13 was replaced with its ClC-2analog, retained its capacity to slow gate. These findings suggest thatimportant structures reside within the interdomain stretch (IDS)between D12 and D13. Unlike ClC-2, in which transplantation of"ball" structures could restore gating to constitutively openmutants, transplantation of the ClC-0 IDS to the amino terminus ofchimera 022 did not restore gating. Surprisingly, replacement of theIDS by the analogous regions of either ClC-1 or ClC-2 showed slowvoltage-activated gating, although the gating was altered. Our findingslead us to conclude that both the functional expression and the slowvoltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

     

Quantitative analysis of the voltage-dependent gating of mouse parotid ClC-2 chloride channel

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.     

Electrophysiological Characteristics of Six Mutations in hClC-1 of Korean Patients with Myotonia Congenita

ClC-1 is a member of a large family of voltage-gated chloride channels, abundantly expressed in human skeletal muscle. Mutations in ClC-1 are associated with myotonia congenita (MC) and result in loss of regulation of membrane excitability in skeletal muscle. We studied the electrophysiological characteristics of six mutants found among Korean MC patients, using patch clamp methods in HEK293 cells. Here, we found that the autosomal dominant mutants S189C and P480S displayed reduced chloride conductances compared to WT. Autosomal recessive mutant M128I did not show a typical rapid deactivation of Cl⁻ currents. While sporadic mutant G523D displayed sustained activation of Cl⁻ currents in the whole cell traces, the other sporadic mutants, M373L and M609K, demonstrated rapid deactivations. V_1/2 of these mutants was shifted to more depolarizing potentials. In order to identify potential effects on gating processes, slow and fast gating was analyzed for each mutant. We show that slow gating of the mutants tends to be shifted toward more positive potentials in comparison to WT. Collectively, these six mutants found among Korean patients demonstrated modifications of channel gating behaviors and reduced chloride conductances that likely contribute to the physiologic changes of MC.     

Burst properties of a supergated double-barrelled chloride ion channel

The chloride selective channel from Torpedo electroplax, ClC-0, is the prototype of a large gene family of chloride channels that behave as functional dimers, with channel currents exhibiting two non-zero conductance levels. Each pore has the same conductance and is controlled by a subgate, and these have seemingly identical fast gating kinetics. However, in addition to the two subgates there is a single slower 'supergate' which simultaneously affects both channels. In the present paper, we consider a six state Markov model that is compatible with these observations and develop approximations as well as exact results for relevant properties of groupings of openings, known as bursts. Calculations with kinetic parameter values typical of ClC-0 suggest that even simple approximations can be quite accurate. Small deviations from the assumption of independence within the model lead to marked changes in certain predicted burst properties. This suggests that analysis of these properties may be helpful in assessing independence/non-independence of gating in this type of channel. Based on simulations of models of both independent and non-independent gating, tests using binomial distributions can lead to false conclusions in each situation. This is made more problematic by the difficulty of selecting an appropriate critical time in defining a burst empirically.     

The role of a conserved lysine in chloride- and voltage-dependent ClC-0 fast gating

ClC-0 is a chloride channel whose gating is sensitive to voltage, chloride, and pH. In a previous publication, we showed that the K149C mutation causes a +70-mV shift in the voltage dependence of ClC-0 fast gating. In this paper we analyze the effects of a series of mutations at K149 on the voltage and chloride dependence of gating. By fitting our data to the previously proposed four-state model for ClC-0 fast gating, we show which steps in fast-gate opening are likely to be affected by these mutations. Computational analysis of mutant ClC-0 homology models show electrostatic contributions to chloride binding that may partially account for the effects of K149 on gating. The analysis of gating kinetics in combination with the available structural information suggests some of the structural changes likely to underpin fast-gate opening.     

ClC-3 is a candidate of the channel proteins mediating acid-activated chloride currents in nasopharyngeal carcinoma cells

Acid-activated chloride currents have been reported in several cell types and may play important roles in regulation of cell function. However, the molecular identities of the channels that mediate the currents are not defined. In this study, activation of the acid-induced chloride current and the possible candidates of the acid-activated chloride channel were investigated in human nasopharyngeal carcinoma cells (CNE-2Z). A chloride current was activated when extracellular pH was reduced to 6.6 from 7.4. However, a further decrease of extracellular pH to 5.8 inhibited the current. The current was weakly outward-rectified and was suppressed by hypertonicity-induced cell shrinkage and by the chloride channel blockers 5-nitro-2-3-phenylpropylamino benzoic acid (NPPB), tamoxifen, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS). The permeability sequence of the channel to anions was I(-) > Br(-) > Cl(-) > gluconate(-). Among the ClC chloride channels, ClC-3 and ClC-7 were strongly expressed in CNE-2Z cells. Knockdown of ClC-3 expression with ClC-3 small interfering (si)RNA prevented the activation of the acid-induced current, but silence of ClC-7 expression with ClC-7 siRNA did not significantly affect the current. The results suggest that the chloride channel mediating the acid-induced chloride current was volume sensitive. ClC-3 is a candidate of the channel proteins that mediate or regulate the acid-activated chloride current in nasopharyngeal carcinoma cells.     

Involvement of helices at the dimer interface in ClC-1 common gating

ClC-1 is a dimeric, double-pored chloride channel that is present in skeletal muscle. Mutations of this channel can result in the condition myotonia, a muscle disorder involving increased muscle stiffness. It has been shown that the dominant form of myotonia often results from mutations that affect the so-called slow, or common, gating process of the ClC-1 channel. Mutations causing dominant myotonia are seen to cluster at the interface of the ClC-1 channel monomers. This study has investigated the role of the H, I, P, and Q helices, which lie on this interface, as well as the G helix, which is situated immediately behind the H and I helices, on ClC-1 gating. 11 mutant ClC-1 channels (T268M, C277S, C278S, S289A, T310M, S312A, V321S, T539A, S541A, M559T, and S572V) were produced using site-directed mutagenesis, and gating properties of these channels were investigated using electrophysiological techniques. Six of the seven mutations in G, H, and I, and two of the four mutations in P and Q, caused shifts of the ClC-1 open probability. In the majority of cases this was due to alterations in the common gating process, with only three of the mutants displaying any change in fast gating. Many of the mutant channels also showed alterations in the kinetics of the common gating process, particularly at positive potentials. The changes observed in common gating were caused by changes in the opening rate (e.g. T310M), the closing rate (e.g. C277S), or both rates. These results indicate that mutations in the helices forming the dimer interface are able to alter the ClC-1 common gating process by changing the energy of the open and/or closed channel states, and hence altering transition rates between these states.