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…     

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。     

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。     

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Ⅱ型前庭毛细胞乙酰胆碱敏感性大电导钙依赖性钾通道与L型钙通道共存

目的 研究豚鼠Ⅱ型前庭毛细胞乙酰胆碱(acetylcholine,ACh)敏感性大电导钙依赖性钾通道(big conductance,calcium-dependent potassium channel,BK)激活过程中的钙离子内流机制.方法 健康杂色豚鼠52只,断头后取出前庭终器,经胶原酶IA消化后获取Ⅱ型前庭毛细胞.采用全细胞膜片钳技术检测新鲜单离的Ⅱ型前庭毛细胞ACh-敏感性BK电流对细胞外钙通道阻断剂和激动剂的敏感性.结果 ①细胞外ACh激活一缓慢持久的外向性电流,其反转电位为(-70.5±10.6)mV(-x±s,下同,n=10);-50 mV钳制电压下,100 μmol/L ACh激活电流的幅值为(267±106)pA(n=11).②ACh-敏感性钾电流对细胞外IBTX(iberiotoxin,200 nmol/L)敏感,而细胞外蜂毒明肽(apamin,1 μmol/L)对ACh-敏感性钾电流幅值无抑制作用.③ACh-敏感性BK对细胞外钙通道阻断剂NiCl2敏感,可被细胞外钙通道阻断剂CdCl2强力阻断.NiCl2和CdCl2对ACh-敏感性BK的半数抑制浓度分别为(135.5±18.5)μmol/L(n=7)和(23.4±2.6)μmol/L(n=7).④L型钙通道激动剂(-)-Bay-K 8644激活Ⅱ型前庭毛细胞产生一种与ACh-敏感性BK类似的外向性电流,并能被IBTX强力阻断.结论 Ⅱ型前庭毛细胞中ACh-敏感性BK与细胞膜L型钙通道共存,可能具有重要的生理学作用和意义.     

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。     

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

目的研究乙酰胆碱(acetylcholine, ACh)和三磷酸腺苷(adenosine triphosphate, ATP)介导的豚鼠耳蜗单离外毛细胞(outer hair cell, OHC)的离子电流.方法应用全细胞膜片钳技术,记录不同的钳制电压对ACh和ATP介导的离子电流的影响以及不同浓度的ACh和ATP分别引致的离子电流.结果 100 μmol/L ACh在钳制电压为-70 mV时引出一个内向电流和一个外向电流,-60 mV以上只引出了外向电流,其反转电位为(-80±5.61) mV(n=6). 100 μmol/L ATP在给予去极化钳制电压时,所引出的内向电流的幅度越来越小,在(3.2±0.23) mV(n=8)反转为外向电流.在单离的Deiters细胞上也记录出100 μmol/L ATP介导的离子电流.结论 ACh介导的外向电流是一种钾离子电流,ATP介导的内向电流是非选择性的阳离子电流,ACh和ATP对OHC的作用是电压依赖的.     

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     

豚鼠Ⅱ型前庭毛细胞胆碱能受体通道特性

目的 研究豚鼠前庭Ⅱ型前庭毛细胞胆碱能受体介导的离子通道的特性。方法 应用全细胞膜片钳技术研究豚鼠新鲜单离的Ⅱ型前庭毛细胞对细胞外乙酰胆碱(ACh)的反应。结果 Ⅱ型毛细胞对ACh敏感,10~(-6)～10~(-3)MACh可引起一浓度依赖性电流(EC50=54.5μM)。-50mV钳制电压下,标准盐溶液中,10μM ACh激活一缓慢持久的外向电流,电流幅值177±40pA。绝大多数检测的毛细胞再次完全激活时间约为60S。细胞膜电位对此电流的通道电导无影响,1mM ACh增大细胞静息电导0.5～1.0倍。细胞外钠离子不参与此电流的激活。此电流的反转电位依赖于细胞外钾浓度,提示乙酰胆碱激活一钾电流。结论 细胞外ACh激活一缓慢持久的电流,此电流的激活依赖于细胞外钾离子浓度,可能激活一钾电流。     

豚鼠耳蜗单离Hensen细胞的分离技术及其钾通道

目的　探讨适合膜片钳技术的单离Hensen细胞的分离及对其钾通道的研究。方法　取豚鼠耳蜗 ,获得Corti器 ,采用酶消化和机械分离方法对Hensen细胞进行分离 ,并采用传统全细胞膜片钳技术 ,记录单离Hensen细胞的钾电流。结果　每只耳蜗可以获得活性良好的单离Hensen细胞 12± 3个。Hensen细胞的钾电流呈明显的外向整流性 ,只有迟电流 (IK) ,没有峰电流 (IA)。结论　酶消化结合机械分离的方法可获得适用于膜片钳技术研究的单离Hensen细胞 ,并用于讨论了所记录到的Hensen细胞钾电流     

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     

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.     

Slow motility, electromotility and lateral wall stiffness in the isolated outer hair cells

Slow motile length changes of isolated, apical turn outer hair cells (OHCs) (n=36) were induced by perfusion of saline (flow rate: 0.6 microl/min) as a mechanical challenge or by perfusion of 12.5 mM KCl solution for 90 s as a chemical and mechanical challenge with and without ocadaic acid (OA), a serine/threonine protein phosphatase inhibitor. Electromotility was evoked by square pulses from +/-35 mV to +/-240 mV during the slow shortening and recovery period (n=36). Stiffness of the lateral wall was measured by the micropipette aspiration technique (n=20). Saline perfusion caused a reversible shortening of 774+/-87 nm (n=9) as well as K+ of 1465+/-159 nm (n=9). Slow shortening increased lateral wall stiffness (1.25+/-0.02 to 1.52+/-0.03 nN/microm) (n=5-5). Simultaneously, electromotility magnitude decreased (n=9). Ocadaic acid blocked slow shortening, increased lateral wall stiffness, and decreased the magnitude of electromotility. Mechanical or mechanical+chemical stimulation of ocadaic acid treated OHCs do not further change stiffness or electromotility. Isolated OHCs respond with slow shortening and consutive cell stiffness increase to mechanical insult. This phenomenon seems operating with calcium-, and phosphorylation-dependent modifications of the cytoskeletal proteins. The subsequent electromotility gain decrease suggests a slow OHC shortening driven regulation of the cochlear amplifier with simultaneous safety control of the auditory periphery against overstimulation.     

Voltage-dependent K(+) currents in spiral prominence epithelial cells of rat cochlea

It has been suggested that spiral prominence is associated with ion transport, but the characterization of ion channels has not been explored so far. We studied the electrical properties and ion conductances of the spiral prominence epithelial cells (SPECs), which are epithelial cells covering the luminal side of spiral prominence, in the upper turn of neonatal rat cochlea using a whole-cell variant patch clamp technique. The cell capacitance was 16.3+/-2.1 pF (n=33) and the resting membrane potential was -68. 9+/-2.5 mV (n=14) in perilymph-like bath solution. It was found that those SPECs possess a large voltage-activated, outwardly rectifying K(+) current and a small inwardly rectifying K(+) current. The outward K(+) current was activated by depolarizing pulses more positive than -30 mV, and sensitive to tetraethylammonium chloride (20 mM), 4-aminopyridine (10 mM), but not to Ba(2+) (0.5 mM). Tail current analysis revealed that it was primarily K(+)-selective. The time course of activation was well fitted by an exponential function raised to second power. The small inwardly rectifying K(+) current was sensitive to Ba(2+) (0.5 mM), and the Ba(2+)-sensitive current was K(+)-selective. In cell-attached or inside-out patch recordings, no discernible K(+) channel currents were found in the apical membrane of SPECs. Based on these results, we conclude that SPECs have two types of voltage-dependent K(+) currents, which are most likely located in the basolateral membrane.     

豚鼠耳蜗外毛细胞乙酰胆碱敏感性钾电流特性

目的 研究豚鼠耳蜗外毛细胞乙酰胆碱(acetylcholine,ACh)敏感性钾电流离子特性及其受体的药理学特性.方法 健康豚鼠38只,断头后取出基底膜,经胶原酶Ⅳ消化后获取外毛细胞.采用全细胞记录膜片钳技术检测新鲜单离外毛细胞ACh-敏感性钾电流对细胞外钙依赖性钾电流阻断剂和N型胆碱能受体抑制剂的敏感性.结果 ①细胞外ACh激活一快速去敏感化的外向性钾电流,其平均(-x±s,以下同)反转电位为(-67.3±8.2)mV(n=10);-50 mV钳制电压下,100 μmol/LACh激活电流的幅值为(506.6±186.3)pA(n=9).②ACh-敏感性钾电流对细胞外四乙铵(tetraethylammonium,TEA,10 mmol/L)、蜂毒明肽(apamin,1 μmol/L)敏感,而细胞外的IBTX(iberiotoxin,200 nmol/L)对ACh-敏感性钾电流幅值无抑制作用.③ACh-敏感性钾电流的半数激活浓度(EC50)为(33.5±5.7)μmol/L(n=7).④ACh-敏感性钾电流对细胞外γ-氨基丁酸(gammaaminobutyric acid,GABA)-A受体阻断剂荷包牡丹碱(bicuculline)和α9-N型胆碱能受体(α9受体)特异性抑制剂士的宁(strychnine)敏感.士的宁和荷包牡丹碱对ACh-敏感性钾电流的抑制作用具有浓度依赖性,其半数抑制浓度(IC50)分别为(22.3±2.6)nmol/L(n=7)和(1.2±0.4)μmol/L(n=6).结论 细胞外ACh激活豚鼠耳蜗外毛细胞产生小电导钙依赖性钾电流(SK),此电流可能由α9受体介导.     

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。     

Local mechanical properties of guinea pig outer hair cells measured by atomic force microscopy

In this study, mechanical properties of guinea pig outer hair cells (OHCs) were measured by atomic force microscopy (AFM). First, in order to confirm the availability of AFM for measurement of the mechanical properties of the OHC, Young's moduli of the OHCs measured in this study were converted into stiffnesses using a one-dimensional model of the cell and then compared with the values reported in the literature. Next, the difference in local mechanical properties of the OHC along the cell axis was measured. Finally, the relationship between Young's modulus in the middle region of the OHC and the cell length was evaluated. The results were as follows. (1) AFM is an adequate tool for the measurement of mechanical properties of the OHC. (2) Mechanical properties in the apical region of the OHC are a maximum of three times larger than those in the basal and middle regions of the cell. (3) Young's modulus in the middle region of a long OHC obtained from the apical turn of the cochlea and that of a short OHC obtained from the basal turn or the second turn are 2.0+/-0.81 kPa (n=10) and 3.7+/-0.96 kPa (n=10), respectively. In addition, it was found that Young's modulus decreases with an increase in the cell length.     

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     

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.