乙酰胆碱对豚鼠外毛细胞的电压依赖性外向整流钾电流的影响

目的 观察乙酰胆碱（ＡＣｈ）对不同长度豚鼠耳蜗外毛细胞（ＯＨＣ）电压依赖性外向整流钾电流的影响，分析 ＡＣｈ对钾电流激活动力学的影响。方法 全细胞膜片钳技术。结果１００μｍｏｌ／Ｌ的ＡＣｈ对短ＯＨＣ电压依赖性外向整流钾电流的影响较大，刺激电压为５０ｍＶ时，最大外向电流的幅度增加了３４．８％。ＡＣｈ对峰电流的影响大于稳态电流，改变了外向整流钾电流的动力学特征。ＡＣｈ将ＯＨＣ的零电流电位向超极化方向移位约５ｍＶ。1００μｍｏｌ／Ｌ的ＡＣｈ使ＯＨＣ电压依赖性外向整流钾电流的激活动力学发生改变，Ｖ（１／２）＝（－５２．３８±３．９８）ｍＶ，较作用前明显超极化，激活的电压敏感性也提高，Ｓ＝（４０±４．１４）ｍＶ（ｎ＝５）。结论ＡＣｈ增加了ＯＨＣ电压依赖性外向整流钾通道的电导，使通道的激活电压向超极化方向移位。ＡＣｈ的作用是使ＯＨＣ超极化。     

豚鼠耳蜗单离Deiters细胞的钾电流

目的 研究豚鼠耳蜗单离Ｄｅｉｔｅｒｓ细胞的钾电流及其特性。方法 运用膜片钳技术,在全细胞模式下记录正常细胞外液中钾电流,不同Ｋ＾＋浓度的细胞外液对细胞反转电位和外向钾电流的影响,四氨基吡啶（４－ａｍｉｎｏｐｙｒｉｄｉｎｅ,４－ＡＰ）和四乙工胺（ｔｅｔｒａｅｔｈｙｌａｍｍｏｎｉｕｍ,ＴＥＡ）对钾电流成分的阻滞作用,探讨外向钾流通道的激活和失活动力学。结果 单离Ｄｅｉｔｅｒｓ细胞具有电压依赖的外向整流     

水杨酸钠对豚鼠单离外毛细胞外向钾电流和静息电位及膜电容 …

目的 探讨水杨酸钠对豚鼠单离外毛细胞（ＯＨＣ）钾电流、静息电位（ＲＰ）和膜电容（ＭＥＣ）的影响，分析水杨酸钠影响毛细胞功能的细胞电生理机制。方法 应用膜片钳全细胞记录技术，测试０．１和１．０ｍｍｏｌ／Ｌ水杨酸钠用药前后ＯＨＣ的Ｉｋ、ＲＰ和ＭＥＣ变化。结果 水杨酸钠对Ｉｋ的影响具有时效和量效关系，即用药后有Ｉｋ先增加后降低的双相作用，而高浓度水杨酸钠无论是对Ｉｋ的增加作用还是降低作用都比低浓度时明显     

豚鼠耳蜗血管纹边缘细胞钾离子通道特性的研究

目的 研究单个豚鼠耳蜗血管纹边缘细胞的基本电生理特性。方法 应用耳蜗血管纹组织块培养技术和全细胞膜片钳技术，观察单个培养的豚鼠血管纹边缘细胞在电压钳模式下的电流特性。结果 边缘细胞上记录到钾离子电流，该钾通道有以下特性：①具有电压依赖性；②通道的活动可被4-氨基吡啶（4-aminopyridine，4-AP）和四乙铵（tetraethylammonium，TEA）在相对高浓度下阻滞；③细胞外钙离子浓度的减少可导致该钾通道电流的降低。结论 豚鼠耳蜗血管纹边缘细胞钾离子通道电流包括钙离子依赖性钾电流、外向延迟整流钾电流和A型钾电流。     

豚鼠Ⅱ型前庭毛细胞乙酰胆碱敏感性钾电流的钙离子依赖性

目的研究豚鼠Ⅱ型前庭毛细胞乙酰胆碱(acetylcholine,ACh)敏感性钾电流对钙离子的依赖性。方法应用全细胞膜片钳技术研究新鲜单离的豚鼠Ⅱ型前庭毛细胞ACh-敏感性钾电流在细胞内外钙离子浓度改变时其电流幅值的变化。结果①细胞外ACh激活一缓慢持久的外向性钾电流,其反转电位为(-70±10)mV;②ACh-敏感性钾电流电流对细胞外四乙胺(10mmol/L)敏感,而对细胞外4-氨基吡啶(100μmol/L)不敏感;③ACh-敏感性钾电流的幅值大小依赖于细胞外的钙离子浓度,无钙外液中ACh激活一很小的电流,4mmol/L外钙溶液中ACh-敏感性钾电流的幅值达到最大值,而0·5mmol/L外钙溶液中ACh-敏感性钾电流的幅值抑制至(36·5±6·5)%;④细胞内三磷酸肌醇-钙离子释放过程不参与ACh-敏感性钾电流的激活,细胞内透析肝素8mg/ml后30min,ACh-敏感性钾电流的幅值没有明显改变;⑤ACh敏感性钾电流对钙通道阻断剂Cd2+敏感。结论豚鼠Ⅱ型前庭毛细胞ACh-敏感性钾电流的激活依赖于细胞外的钙离子浓度,ACh与豚鼠Ⅱ型前庭毛细胞胆碱能受体结合后,首先激活膜上钙通道引起细胞外钙离子内流,毛细胞内游离钙离子浓度的升高进一步激活钙依赖性钾电流。     

顺铂对大鼠耳蜗螺旋神经节细胞外向延迟整流钾电流的影响

目的:观察记录顺铂作用下急性分离新生大鼠耳蜗螺旋神经节细胞(SGNs)延迟整流钾通道的电流曲线,分析顺铂对SGNs钾电流激活动力学的影响,并初步探讨其耳毒性机制。方法:采用全细胞膜片钳技术记录SGNs外向延迟整流钾电流及顺铂对此电流的影响。结果:钳制电压为-60mV,刺激电压从-60mV到 80mV逐渐去极化,阶跃电压为10mV,持续时间为500ms,可在SGNs上记录到外向钾电流,该电流对TEA-Cl(4-乙基胺)敏感,具有延迟整流特性;在细胞外液中加入10μmol/L顺铂,能明显抑制SGNs延迟整流钾电流;顺铂对此电流的抑制作用与细胞外液中顺铂的浓度呈剂量依赖性;外液洗脱后SGNs电流可基本恢复正常。结论:钾通道与SGNs动作电位的产生密切相关,顺铂可抑制SGNs钾通道电流,导致听觉功能障碍。     

牛蛙球囊毛细胞钙离子通道

用细胞贴附式膜片钳技术在牛蛙内耳球囊的单离毛细胞侧膜记录到两种单通道钙电流。Ｌ型钙通道的电导为２６ｐＳ，在去极化电压下仍可激活，活化范围为－１０～＋４０ｍＶ，硝苯吡啶类药物对其有特异性激活或抑制作用。另一种钙通道的电导为２０ｐＳ，在－８０ｍＶ或－４０ｍＶ均可激活，活化范围为－５０～＋１０ｍＶ，硝苯吡啶类药物无作用。此通道的性质待定，疑系Ｎ型钙通道。     

牛蛙球囊毛细胞钙离子通道

用细胞贴附式膜片钳技术在牛蛙在耳球囊的单离毛细胞侧膜记录到两种单通道钙电流。Ｌ型钙通道的电导为２６ｐＳ，在去极化电压下仍可激活，活化范围为－１０～＋４０ｍＶ，硝苯吡啶类药物对其有特异性激活或抑制作用，另一种钙通道的电导为２０ｐＳ，在－８０ｍＶ或－４０ｍＶ均可激活，活化范围为－５０～＋１０ｍＶ，硝苯吡啶类药物作用，此能宾性质待定，疑系Ｎ型钙通道。     

小鼠耳蜗螺旋神经节细胞电生理学特性的研究

目的：应用膜片钳技术记录小鼠耳蜗螺旋神经节细胞的全细胞电流,了解电压依赖性离子通道的基本电生理学特性,并比较耳蜗顶、底转螺旋神经节细胞电生理学特性的差异。方法：应用全细胞构型电压钳制技术,采用不同的电极内液及阻断剂,在不同的刺激参数下记录耳蜗顶、底转螺旋神经节细胞的电压依赖性离子通道电流,并进行分析比较。结果：实验记录到了内向的钠电流、延迟整流钾电流、超极化激活内向阳离子通道电流及瞬时外向钾电流,并发现耳蜗顶、底转螺旋神经节细胞的延迟整流钾电流及瞬时外向钾电流的电生理学特性具有显著性差异（P〈0．05）。结论：实验记录到的各种离子电流数据表明耳蜗螺旋神经节细胞具有完成动作电位的形成、传导并对其功能进行调节的离子通道基础;耳蜗顶、底转螺旋神经节细胞电生理学特性的差异有助于听觉的形成过程。     

水杨酸钠对大鼠下丘神经元钾通道的抑制作用

目的了解瞬时外向钾通道和延迟整流钾通道在水杨酸钠导致耳鸣的机制中所起的作用。方法利用全细胞膜片钳技术研究水杨酸钠对急性分离的大鼠下丘神经元瞬时外向钾通道和延迟整流钾通道的影响。结果水杨酸钠能够抑制瞬时外向钾通道电流（IK（A））和延迟整流钾通道电流（IK（DR））的幅度，而且此抑制作用具有浓度依赖性（0．1～10mmol／L）。水杨酸钠抑制IK（A）和，IK（DR）的50％抑制浓度（IC50）值分别为2．27mmol／L和0．80mmol／L。1mmol／L水杨酸钠不改变，IK（A）的稳态激活曲线和稳态失活曲线的动力学特征，却将IK（DR）的稳态激活曲线和稳态失活曲线分别向超极化方向显著移动11mV和24mV。结论水杨酸钠以浓度依赖的方式抑制IK（A）和IK（DR），但是只影响IK（DR）的稳态激活和失活动力学特征。水杨酸钠对IK（A）和IK（DR）的影响，尤其对IK（DR）的影响可能与水杨酸钠导致耳鸣的机制有关。     

豚鼠耳蜗单离外毛细胞的外向整流钾电流

目的 :观察豚鼠耳蜗单离外毛细胞 (OHC)的电生理特性 ,记录不同长度OHC的外向整流钾电流 ,分析区分外向整流钾电流所包含的通道电流成分 ,研究外向整流钾电流的动力学特征。方法 :采用酶消化法及机械分离OHC。运用全细胞膜片钳技术 ,在电压钳下记录K+ 通道电流。结果 :OHC的全细胞膜电容为 (30 .96±2 .79) pF(n =2 9) ,零电流电位 (30± 2 .1)mV(n =16 ) ,反转电位为 (- 5 1.6 7± 1.84 )mV(n =9)。不同长度OHC的外向整流钾电流存在系统差异 ,短OHC表现出大的钾电导 ,长OHC则相反。 10 0 μmol/L的氯化镉 (Cd Cl2 )抑制了OHC外向整流钾电流的最大电流幅度的 6 0 % ,且改变了电流的动力学特征 ,对峰电流的影响明显大于稳态电流 (P<0 .0 1,n =5 ) ;1mmol/L的四氨基吡啶 (4 AP)抑制了最大电流幅度的 4 3% ,没有改变电流的动力学特征。外向整流钾电流的激活符合Boltzmann方程 ,V1/ 2 =(- 11.0 7± 0 .2 6 )mV ,S =(6 .6 2± 1.74 )mV(n=13)。结论 :外向整流钾电流包含有钙离子激活的钾离子电流、外向延迟整流钾电流和A型电流     

Subpixel tracking for the analysis of outer hair cell movements

CONCLUSION: Videomicroscopy with subpixel analysis is an excellent system for quantification of outer hair cell (OHC) movements. The resolution of a few nanometers is accurate enough to show induced differences of electromotility. OBJECTIVE: Electromotility of OHCs is a voltage-dependent process resulting from a membrane protein named prestin. Voltage sensitivity is conferred to prestin by intracellular anions. Reduction of these anions reduces electromotility. Videomicroscopy and subpixel tracking combine video-based analysis with a resolution of few nanometers. The aim of this study was to show the feasibility of a system for quantification of OHC movements. MATERIALS AND METHODS: Electromotility was investigated under normal and reduced intracellular chloride conditions. Cells were stimulated by the patch-clamp technique. Voltage steps were 500 ms long, ranging from -170 to +30 mV in 10 mV steps. RESULTS: As in previous studies our results show the following. The direction of OHC movement depends on the polarity of voltage steps, length changes are not equal for symmetrical voltage steps of opposite polarity, average shortening for a depolarizing step (-70 mV to +30 mV) is about 13 nm/mV. Hyperpolarization (-70 mV to -170 mV) on average evokes elongations of about 3 nm/mV. Half maximal chloride concentration reduces motility by 14%; half maximal electromotility is reached by a 94% reduction of chloride.     

Suppression and recovery of voltage-gated currents after cocaine treatments of olfactory receptor cells

Objective

Cocaine (1–5% concentrations) is commonly used as a local anesthetic for the otorhinolaryngeal surgery of the nasal cavity. Recent reports indicate that some patients complain of olfactory deficits after surgery, and decreased olfaction is found in cocaine abusers. In spite of these reports, the effects of cocaine on the olfactory receptor cells are unknown.

Methods

Effect of cocaine was examined in olfactory receptor cells isolated from the newt. Under the voltage clamp with the whole-cell recording configuration, the voltage-gated currents were recorded when the membrane potential was depolarized from a holding potential of −100 mV in a step wise between −90 mV and +40 mV.

Results

When cocaine was applied by a puff pressure (5%) and the extracellular solution, the voltage-gated currents, including inward and outward components, were significantly reduced. The dose–suppression curves of cocaine for sodium and potassium currents could be fitted by the Hill equation. Half-blocking concentration of sodium and potassium currents were 43 μM and 557 μM; Hill coefficient was 1.1 and 0.9, respectively.

Conclusion

This rapid and complete recovery from the suppression was confirmed even after the treatments with the high concentration cocaine. This fact implies that cocaine does not affect olfactory ability after locally high dose treatments of nasal cavity in surgical operation.     

水杨酸钠对豚鼠单离外毛细胞外向钾电流和静息电位及膜电容影响

目的　探讨水杨酸钠对豚鼠单离外毛细胞（ｏｕｔｅｒｈａｉｒｃｅｌｌ,ＯＨＣ） 钾电流（ｐｏｔａｓｓｉｕｍ ｃｕｒｒｅｎｔ,ＩＫ）、静息电位（ｒｅｓｔｉｎｇ ｐｏｔｅｎｔｉａｌ,ＲＰ） 和膜电容（ｍｅｍｂｒａｎｅ ｅｌｅｃｔｒｉｃ ｃａｐａｃｉｔａｎｃｅ,ＭＥＣ） 的影响,分析水杨酸钠影响毛细胞功能的细胞电生理机制。方法　应用膜片钳全细胞记录技术,测试０．１ 和１ ．０ ｍｍｏｌ／Ｌ水杨酸钠用药前后ＯＨＣ的ＩＫ、ＲＰ和ＭＥＣ 变化。结果　水杨酸钠对ＩＫ 的影响具有时效和量效关系,即用药后有ＩＫ 先增加后降低的双相作用,而高浓度水杨酸钠无论是对ＩＫ 的增加作用还是降低作用都比低浓度时明显。ＲＰ平均约为－６０ ｍＶ,ＭＥＣ 平均约为３９ ｐＦ。水杨酸钠有降低ＲＰ和ＭＥＣ的作用,且均存在着量效关系,即高浓度时的作用大于低浓度时。水杨酸钠对ＲＰ和ＭＥＣ影响的时效关系不明显。结论　水杨酸钠具有影响ＯＨＣ两侧膜的Ｋ＋ 电导和细胞内外Ｋ＋ 分布的作用,并通过对ＩＫ、ＲＰ和ＭＥＣ的影响改变ＯＨＣ的兴奋性和机械活动特性,可能是水杨酸影响毛细胞功能的耳蜗机制之一。     

Stretch-activated ion channels in guinea pig outer hair cells.

Two types of stretch-activated (SA) ion channels have been found in the lateral wall of isolated outer hair cells (OHC) from the guinea pig cochlea. One type had a reversal potential of -12 mV and was non-selective to cations, passing Ca2+ as well as monovalent ions. The channel had a conductance of 38-50 pS and the amplitude of the current through the open SA channel was independent of suction. The probability of the channel being open increased with applied suction and was voltage dependent with the maximum probability occurring at pipette potentials of -40 to -60 mV. The second type of SA channel had a conductance of approximately 150 pS and a reversal potential of approximately -50 mV. The ionic selectivity of this channel has not yet been determined, but it is probably K+ selective. OHCs have been shown to undergo a slow change in length in response to acoustic stimulation directed at the lateral wall of the OHC. The SA channels reported here could affect the motile response by altering the membrane potential or by allowing the entry of free Ca2+ which could lead to a change in OHC length through the interaction of actin and myosin. SA channels could also play an important role in regulating the osmotic pressure of OHC thereby influencing its electro-mechanical response.     

人喉癌Hep-2细胞膜钾离子通道与RNA编辑酶1的相关性

目的研究人喉癌细胞系Hep-2细胞膜钾离子通道特性及其与RNA编辑酶1(RNA-dependent adenosinedeaminase1,ADAR1)的相关性.方法以Hep-2细胞为研究对象,采用穿孔膜片钳全细胞记录法研究钾离子通道特性,用逆转录聚合酶链反应检测四乙胺(tetraethylammonium,TEA)阻断钾离子通道前后Hep-2细胞ADAR1 mRNA的表达.结果 Hep-2细胞膜的静息膜电位为(-29.8±1.9)mV,在钳制电压-40 mV,阶跃电压在-80～ 80 mV,记录到一种跨膜电流,该跨膜电流具有电压依赖、外向整流特性,该电流在延迟25ms后最大激活,800 ms内不失活,可被阻断剂TEA阻断.Hep-2细胞钾离子通道被TEA阻断前后ADAR1mRNA相对表达量存在显著性差异.结论 Hep-2细胞膜上存在延迟整流钾离子通道,此钾离子通道可能和ADAR1 mRNA的表达密切相关.     

Inactivating and non-activating delayed rectifier K+ currents in hair cells of frog crista ampullaris.

The possible presence of different types of delayed rectifier K+ current (I(K)) was studied in vestibular hair cells of frog semicircular canals. Experiments were performed in thin slice preparations of the whole crista ampullaris and recordings were made using the whole-cell patch-clamp technique. We found that an apparent homogeneous I(K), isolated from the other K+ currents, could be pharmacologically separated into two complementary components: a capsaicin-sensitive current (I(Kc)) and a barium-sensitive current (I(K,b)). I(K,c) was recruited at potentials more positive than -60 mV and showed a slow activation having a time constant (tau(a)) ranging on average from 12 ms at 40 mV to 32 ms at -20 mV. This current inactivated slowly with two voltage-independent time constants (ta(d1) and tau(d2) were 300 ms and 4 s respectively) and more than 80% of the channels were in an inactivated state at the cell resting potential. I(K,b) was also recruited at potentials more positive than -60 mV, but in contrast to I(K,c), it activated more rapidly (tau(a) ranged on average from 1 ms at 40 mV to 4.5 ms at -20 mV) and it did not exhibit any inactivation process. Current clamp experiments revealed that I(K,b), at variance with I(K,c), contributes to the cell resting potential and represents the main repolarizing current when sensory cells are depolarized from rest. I(K,c) could have a role in hair cells when they are depolarized after hyperpolarizing stimuli, a condition that removes channel inactivation.     

Potassium current properties in apical and basal inner hair cells from guinea-pig cochlea

Inner hair cells (IHCs) of guinea-pigs were separately isolated from the apical and basal turn and the potassium currents were measured by the whole-cell voltage-clamp technique. The potassium current flows through two types of membrane conductance: a fast (I_k,f), tetraethylammonium (TEA)-sensitive conductance and a slow (I_k,s), TEA-resistant conductance. Membrane conductance demonstrated no significant differences between apical IHCs and basal IHCs. Reversal potentials were −65±2 mV and −68±5 mV in apical and basal IHCs, respectively. The rate of outward current activation was voltage dependent and faster in basal IHCs than in apical IHCs. TEA effect was stronger on basal IHCs than on apical IHCs, suggesting that I_k,f is dominant in basal IHCs.