单离豚鼠内耳Deiters细胞的形态及电生理特性

目的：分离豚鼠内耳活性良好的单离Ｄｅｉｔｅｒｓ细胞,观察其形态,探讨该细胞的基本电生理特性。方法：采用酶孵育加机械分离法分离Ｄｅｉｔｅｒｓ细胞;利用膜电钳技术在全细胞模式下检举同细胞电容,记录在正常外液中的钾电流,零电流电位,反转电位,并根据相应公式计算离子通道Ｋ与Ｎａ＾＋的相对通透性比率及Ｋ平衡电位（ＥＫ）《结果：单离Ｄｅｉｔｅｒｓ细胞多呈逗点状,分为胞体,细柄和指突三部分,核靠近胞体中央,平均     

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)和四乙基胺 (tetraethylammonium ,TEA)对钾电流成分的阻滞作用 ,探讨外向钾电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离子选择性通道 ;钾通道阻滞剂 4 AP和TEA可使峰电流和迟电流幅度下降 ,表明存在两种类型的钾通道 ,或者这种通道有两种不同的状态 ;通道的激活和失活符合Boltzmann方程。未记录到Deiters细胞的内向钙电流。结论　Deiters细胞外向整流钾电流的作用可能是缓冲细胞周围间隙钾离子浓度     

利诺吡啶对豚鼠耳蜗外毛细胞和支持细胞钾电流的影响

目的　观察KCNQ家族钾通道特异性阻滞剂利诺吡啶 (linopirdine)对豚鼠耳蜗单离外毛细胞和Deiters细胞 (支持细胞 )总钾电流的影响 ,初步探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的分布。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 8个外毛细胞和5个Deiters细胞的总钾电流 ,并观察 10 0 μmol/L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果　在正常细胞外液中 ,单离外毛细胞可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流 (theK+ currentactivatedatnegativepotential,IKn)两种钾电流 ,而单离Deiters细胞中只记录到外向整流性钾电流。加入 10 0 μmol/L利诺吡啶后 ,外毛细胞中的四乙基二乙胺敏感的钾电流减小 ,IKn被完全抑制 ;而Deiters细胞中的外向整流性钾电流大小无变化。结论KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞 ,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分 ,并构成全部的IKn;但KCNQ家族钾通道不存在于豚鼠耳蜗Deiters细胞。     

利诺吡啶对豚鼠耳蜗外毛细胞和支持细胞钾电流的影响

目的 观察KCNQ家族钾通道特异性阻滞剂利诺吡啶(linopirdine)对豚鼠耳蜗单离外毛细胞和Deiters细胞(支持细胞)总钾电流的影响，初步探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的分布。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8个外毛细胞和5个Deiters细胞的总钾电流，并观察100μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果 在正常细胞外液中，单离外毛细胞可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential，IKn)两种钾电流，而单离Deiters细胞中只记录到外向整流性钾电流。加入100μmol／L利诺吡啶后，外毛细胞中的四乙基二乙胺敏感的钾电流减小，IKn被完全抑制；而Deiters细胞中的外向整流性钾电流大小无变化。结论 KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞，其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分，并构成全部的IKn；但KCNQ家族钾通道不存在于豚鼠耳蜗Deiters细胞。     

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。     

豚鼠耳蜗单离外毛细胞外向钾电流的特性

目的：探讨豚鼠耳蜗单离外毛细胞（ＯＨＣ）钾电流（Ｉｋ）的正常值及其特性。方法：应用膜片钳全细胞记录技术及多种辅助方法,测试在不同细胞内、外液和电压刺激条件下的Ｉｋ、钾尾电流（Ｉｋｔａｉｌ）和反转电位。结果：Ｉｋ具有明显的电压依赖性和时间依赖性,在２０ｍｓ内达峰值,平均激活电压约为－３２．７ｍＶ,从激活电压至０电压时Ｉｋ增长最快,在４０ｍＶ时接近饱和。正常条件下Ｉｋ无明显“ｒｕｎ－ｄｏｗｎ现象”。细     

豚鼠上橄榄外侧核γ—氨基丁酸阳性神经元向耳蜗和和耳…

为探讨豚鼠上橄榄外侧核内γ－氨基丁酸（ＧＡＢＡ）阳性神经元是否同时支配同侧Ｃｏｒｔｉ器和耳蜗核，采用逆行追踪的方法，将Ｆａｓｔ ｂｌｕｅ（ＦＢ）和Ｄｉａｍｉｄｉｎｅ黄（ＤＹ）两种不同性质的荧光素分别注入耳蜗鼓阶及同侧耳蜗核，观察上橄榄外侧核内ＧＡＢＡ阳性神经元的分支投身。结果发现，同侧上橄榄外侧内ＦＢ单标细胞占记细胞的８０．８％；ＤＹ单标细胞占１２．４％；ＦＢ－ＤＹ双细胞占６％；ＧＡＢＡ、ＦＢ、Ｄ     

硝苯地平对三磷酸腺苷引起的Hensen细胞内向性离子流的抑制作用

目的探讨硝苯地平对高浓度三磷酸腺苷（ATP）引起的豚鼠耳蜗单离Hensen细胞非选择性内向性离子流的影响。方法采用酶消化和机械分离的方法单离豚鼠耳蜗Hensen细胞，选择胞膜清晰、胞质透明的单离细胞通过压力注射仪分别给予0．1mmol／LATP、1mmol／LATP、10mmol／LATP、0．1mmol／LATP＋0．1mmol／L舒拉明、单独细胞外液、140mmol／LCsCl＋1mmol／LATP、40mmol／L四乙基铵（tetraethylammonium，TEA）＋1mmol／LATP、1mmol／LATP＋10μmol／L硝苯地平，并行全细胞膜片钳记录。结果当分别给予Hensen细胞0．1mmol／L（n=10），1mmol／L（n=10），10mmol／L（n=6）的ATP刺激时，均可记录到内向电流，并随ATP浓度的增加而增强，呈现浓度依赖性。该内向电流可被0．1mmol／L舒拉明（n=5），140mmol／LCsCl（n=5）和40mmol／LTEA（n=5）所抑制。单独给予细胞外液刺激后未见内向及外向离子流。当同时给予1mmol／LATP和10μmol／L硝苯地平刺激时，内向性电流消失，转而出现外向性电流。结论高浓度ATP可引起Hensen细胞的内向性电流，此电流与钾通道密切相关，并无机械转化通道参与，硝苯地平可抑制这种内向性电流并出现与正常情况相似的外向性电流。提示硝苯地平可通过Hensen细胞改善钾离子循环，起到部分保护耳蜗功能的作用。     

听力学

20 0 4 0 82 0豚鼠耳蜗单离Hensen细胞钾电流特性及三磷酸腺苷对其影响 /李建雄… / /中华耳鼻咽喉科杂志 2 0 0 3;38(5 ) 343～ 346目的 :研究豚鼠耳蜗单离Hensen细胞的钾离子电流及其特性以及三磷酸腺苷 (ATP)对Hensen细胞电生理特性的影响。方法 :采用传统全细胞膜片钳技术 ,对单离Hensen细胞进行记录。ATP通过压力注射仪对单离Hensen细胞给药。结果 :Hensen细胞的钾电流呈明显的外向整流性 ,只有延迟整流性钾电流 (delayedrectificationpotassiumcurrent ,IK) ,没有瞬间外向性钾电流 (transientoutwordpotassiumcur rent ,IA)…     

听力学

20021291豚鼠耳蜗单离外毛细胞的外向整流钾电流/杨军…//临床耳鼻咽喉科杂志一2002,16(1)一27一30 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。运动全细胞膜片钳技术,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士2.79)pF(n=29),零电流电位(30士2.1)mV(n=16),反转电位为(一51.67士1.84)mV(n一9)。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…     

Dissociation enzyme effects on the potassium currents of inner hair cells isolated from guinea-pig cochlea

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. Some enzymes using for IHCs dissociation affect these inactivation kinetics. IHCs were dissociated from guinea-pig cochlea by 1 mg/ml trypsin or 0.25 mg/ml protease VIII, and the properties of the K+ currents were compared using conventional whole-cell voltage-clamp recordings. TEA-sensitive potassium currents showed fast inactivation kinetics in both trypsin-dissociated cells and protease VIII-dissociated cells. The time constant of the inactivation phase in trypsin-treated cells was similar to that in protease VIII-treated cells. However, the rate of inactivation (compared by the ratio between the steady-state current and initial peak current) in protease VIII-treated cells was larger than that in trypsin-treated cells. In protease VIII-dissociated cells, the time constant of recovery from inactivation elucidated by paired-pulse protocol was 3.5 ms. Papain is another enzyme that is sometimes used for dissociating IHCs, so effects of papain were observed. Extracellular papain application (8 unit/ml) demonstrated a slight increase of the outward potassium currents.     

Temperature Enhances Activation and Inactivation Kinetics of Potassium Currents in Inner Hair Cells Isolated from Guinea-Pig Cochlea

Objectives

Until recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs.

Methods

IHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25℃) and physiological temperatures (35℃-37℃).

Results

IHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36℃ than at room temperature. Half-time for activation was shorter at 36℃ than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q₁₀ for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36℃ than at room temperature between the membrane potentials of -20 and +60 mV. Q₁₀ for the inactivation time constant was 3.19.

Conclusion

The results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.     

利用膜片钳技术对急性分离小鼠螺旋神经节神经元的电生理研究

目的 探讨小鼠螺旋神经节神经元(spiral ganglion neurons，SGN)的电生理特性。方法 应用全细胞电压钳技术对急性酶分离的小鼠耳蜗SGN细胞膜上的离子通道电流进行记录和分析。结果 在SGN细胞膜上记录到对氯化四乙胺、4-氨基吡啶敏感的外向延迟整流钾电流和瞬时钾电流，以及对河豚毒素敏感的内向钠电流。并且离子通道活动具有明显的电压依赖性，在没有记录到钠电流的细胞记录到延迟整流钾电流。结论 急性酶分离的小鼠耳蜗SGN细胞的电生理特性可为听觉传导机制的研究提供了重要的参考，并指导下一步通道药理学的研究。     

Inactivating and non-inactivating potassium currents in isolated inner hair cells from guinea pig cochlea

Using conventional whole-cell voltage-clamp recordings, we examined the 4-aminopyridine (4-AP)- and tetraethylammonium (TEA)-sensitive K(+) currents in the cochlear inner hair cells (IHCs) of guinea pigs. 4-AP-sensitive currents were activated slowly and sustained the same current level, whereas TEA-sensitive currents were activated rapidly, followed by inactivation. The inactivation time course of TEA-sensitive currents was voltage-dependent, becoming faster at more depolarized levels. The inactivation of TEA-sensitive currents almost recovered within 5 ms. 4-AP- and TEA-sensitive K(+) currents coexisted in the same IHC.     

乙酰胆碱和三磷酸腺苷介导的豚鼠外毛细胞和Deiters细胞的离子电流

OBJECTIVE: To determine ACh- and ATP-induced currents in isolated outer hair cells of guinea pig cochlea, respectively. METHOD: The whole-cell patch-clamp technique was used to investigate potential and concentration dependence of ACh- and ATP-induced ion currents. RESULTS: ACh(100 mumol/L) induced an early inward current followed by a late outward current when the cells were voltage-clamped at -70 mV. Only outward current occurred when potential was over -60 mV and its reversal potential was (-80 +/- 5.61) mV(n = 6). When depolarization protocols were applied, amplitude of inward currents activated by ATP(100 mumol/L) decreased and reversed at (3.2 +/- 0.23) mV(n = 8). Ion currents activated by ATP (100 mumol/L) were also recorded from isolated Deiters' cells. CONCLUSION: ACh-induced outward current was carried by K+ and ATP-induced inward current was through non-selective cations channel. Effects of ACh and ATP on OHCs were voltage dependent.     

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     

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。