一种有效记录成骨细胞L型钙通道电流的穿孔全细胞膜片钳方法

目的:探讨一种有效记录成骨细胞L型钙通道电流的穿孔全细胞膜片钳方法。此方法使玻璃电极与细胞内液保持电化学连续性,使细胞内环境保持稳定,改善常规全细胞膜片钳破膜造成的破膜难和封接不稳,以及常规全细胞膜片钳破膜后造成的封接不稳。方法:以成骨细胞为研究对象,采用β-七叶素穿孔全细胞膜片钳技术,记录骨细胞L型钙通道电流。结果:采用这种穿孔全细胞膜片钳技术,能有效记录到成骨细胞上L型钙通道电流。结论:穿孔膜片钳技术是一种简便而有效的记录全细胞电流的实验方法,该方法的建立为今后更深入的研究疾病中成骨细胞的病理生理机制提供实验基础。     

穿孔膜片钳技术在离子通道电流研究中的应用

目的:传统的全细胞膜片钳技术在离子通道电流的记录中存在机械稳定性差,对细胞的损伤大,以及胞内液的被渗析影响与细胞内信号转导和离子通道调控有关的第二信使物质的正常运行。而穿孔全细胞膜片钳技术应用二性霉素B或制霉菌素在细胞膜上形成特定的孔道,选择性地允许一些离子和大分子物质,从而使细胞内环境保持相对稳定,在一定程度上弥补了上述缺陷,实验成功率也相应提高。本文就穿孔膜片钳技术在全细胞离子通道电流记录中的应用进行探讨。     

兔颈上神经节神经元的急性分离及其通道电流记录

目的 探讨兔颈上神经节神经元分离及电生理研究的方法.方法 为了获得适用于膜片钳实验技术的单个颈上神经节神经细胞,应用酶和机械分离相结合的方法,急性分离20～30 d幼兔的单个颈上神经节神经元.通过倒置显微镜直接观察以及全细胞膜片钳技术的电压钳和电流钳分别对分离得到的颈上神经节神经元的形态学和电生理学特性进行研究.结果 急性分离所得活性较好的颈上神经节神经元胞体具有圆形和顶树突特征,立体感强,光晕明显.在全细胞膜片钳记录模式下可记录到全细胞电流、钠电流、钾电流及动作电位.结论 该方法可以得到形态和生理特性良好的单个颈上神经节神经元;该方法适用于膜片钳技术的研究,对深入探讨药物、物理因子等对神经节神经元离子通道的影响具有重要的应用价值.     

TNFα对单个大鼠心室肌细胞L型钙电流的影响

目的探讨TNFα对心肌细胞L型钙电流的影响。方法分离大鼠单个心室肌细胞,用全细胞膜片钳方法检测TNFα对单个心室肌细胞L型钙电流的影响。结果 TNFα使单个大鼠心室肌细胞L型钙电流持续增强,促进细胞外钙离子持续向细胞内流动。结论 TNFα与感染性休克心脏收缩性改变相关。     

丹参在常氧、急性低氧及氧反常等条件下对心室肌细胞L-Ca电流的影响

目的：用不同浓度的丹参NaCl溶液作用于常氧、低氧、及氧反常的豚鼠心室肌细胞上，观察L-Ca通道电流的相对变化，以期解释丹参减轻及阻止细胞内钙超载的机理。方法： 使用全细胞膜片钳技术研究心室肌细胞L-Ca通道电流的变化。结果： 无论是常氧、缺氧和缺氧后复氧状态下，浓度为32、320、3 200 mg/L的丹参制剂都能有效降低L-Ca通道电流幅值并呈浓度依赖性。此外，低浓度32 mg/L的丹参液对缺氧和氧反常细胞的作用更大于常氧细胞。结论： 丹参溶液能有效降低低氧和氧反常造成的异常增大的L-Ca电流幅值，阻止钙超载发生。     

2.122豚鼠结肠平滑肌细胞L型钙通道特性研究

目的全细胞式膜片钳技术研究豚鼠结肠平滑肌细胞的L型钙通道特性,以加深对胃肠动力调控的认识及有利于胃肠动力药物的开发. 方法豚鼠,200～400g,木瓜蛋白酶酶消化法分离结肠纵肌细胞.全细胞膜片钳法记录单细胞的钙通道电流.     

双分离法记录Wistar大鼠神经元的全细胞电流

背景:细胞培养与通道电流记录是全细胞膜片钳实验的主要难点。目的:介绍一种简单可行的降低全细胞膜片钳实验方法,将细胞急性分离与电流的分离技术结合起来,以提高工作效率,缩短实验的时间,从根本上降低膜片钳实验的难度。方法:SPF级出生4～7d的wistar大鼠40只,雌雄不限。采用改良的急性分离的方法制备Wistar大鼠脑皮质细胞,将大鼠脑皮质切成400～600μm厚度的薄片,在人工脑脊液中通混合气静止1h,并通以氧气。将脑组织块放入含有16u/mL(typeX)和2u/mL(typeXIV)蛋白水解酶的人工脑脊液中,孵育60min,清除消化酶。在全细胞电压钳制模式下,保持电位-80mV,给予-60mV到60mV的去极化脉冲刺激,步阶为+10mV,刺激波宽160ms。记录到跨膜总电流,在全细胞电极液里面加入70mmol/LCsCl,70mmol/LCsF;在外液中先后加入11μmol/L阻断剂河豚毒素、30mmol/L的氯化四乙胺、1mmol/L的4-AP。分别记录内向钠电流,瞬时外向钾电流和延迟整流钾电流,结果用clampfit分析处理。主要观察:①细胞的形态学观察。②全细胞电流的记录。③内向钠电流的记录。④外向钾电流的记录。结果与结论:细胞空间立体结构强,表面光滑,有完整的树突或者轴突,且细胞的活性可以在25℃室温下维持8～10h。在外液中加入1μmol/L的河豚毒素基本上可以阻断钠电流;30mmol/L的氯化四乙胺和1mmol/L的4-AP可以阻断外向钾电流。结果表明,改良的细胞急性分离方法细胞功能完好。通过电流分离技术,不改变细胞外液和电极液,仅需添加特异阻断剂,可记录到内向钠电流,瞬时外向钾电流以及延迟整流钾电流,较之传统方法可显著提高工作效率。     

大鼠肺动脉平滑肌细胞的分离及电压门控性钾电流的记录方法

目的介绍一种大鼠肺动脉平滑肌细胞(PASMCs)的急性分离方法并观察电压门控性钾电流电生理特性。方法应用胶原酶和木瓜蛋白酶联合消化法获得大鼠PASMCs,利用全细胞膜片钳技术记录PASMCs膜上的电压门控性钾通道(Kv)电流。结果在相差显微镜下观察大鼠PASMCs呈舒展梭形,边界清晰,有完整的细胞膜,胞浆均匀,数量多,活性好。结论用酶急性分离的大鼠PASMCs,容易进行全细胞膜片钳记录,方法简单、稳定、可靠。     

冬虫夏草对豚鼠心室肌细胞胞浆钙浓度及L-型钙电流的影响

目的 :冬虫夏草 (CodycepsSinensis ,CS)为麦角科真菌 ,是我国传统的名贵药材。它已被研究证明具有抗多种心律失常的作用 ,尤其适用于难治性缓慢型心律失常、传导阻滞。本实验旨在研究冬虫夏草水提液对豚鼠单个心室肌细胞内钙信号及L型钙电流的影响 ,从而探讨其抗心律失常的机制。方法 :急性分离 (使用胶原酶Ⅱ )豚鼠单个心室肌细胞 ,将细胞以Fluo - 3/AM做荧光标记 ,在激光扫描共聚焦显微镜timeseries程序下对细胞XY平面进行扫描 ,以荧光强度值变化表示 [Ca2 + ]i变化。进一步应用全细胞膜片钳技术测定单个心室肌细胞L -型钙电流 ,实…     

敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 目的: 探讨ClC-3氯通道是否为IK1钾通道的调节靶点,重点研究鼻咽癌细胞IK1钾通道对ClC-3氯通道功能及蛋白表达的影响。方法: 采用siRNA转染技术抑制低分化鼻咽癌上皮细胞(CNE-2Z) IK1 基因的表达;real-time PCR技术检测ClC-3 mRNA的表达;Western blot检测ClC-3的蛋白表达;细胞免疫荧光结合激光共聚焦显微镜技术检测ClC-3和IK1蛋白在细胞内分布;全细胞膜片钳记录细胞氯电流。结果: IK1 siRNA可以成功转染CNE-2Z细胞,有效抑制鼻咽癌细胞IK1钾离子通道的表达;用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后, ClC-3的mRNA表达上调而ClC-3蛋白却表达减少:在低分化鼻咽癌上皮细胞,低渗刺激可激活氯通道,产生一个较大的氯电流,在成功转染IK1 siRNA的细胞,此氯电流明显减弱。结论: 敲低IK1钾离子通道可抑制ClC-3氯离子通道的表达和功能。     

Whole-cell and perforated-patch recordings from O2-sensitive rat carotid body cells grown in short- and long-term culture

We are investigating transduction mechanisms in a major peripheral chemosensory organ, the rat carotid body, using short- and long-term dissociated cell cultures and patch-clamp, whole-cell recording. In this study membrane properties of cultured glomus or type I cells were characterized with conventional whole-cell recording and the new perforated-patch technique during control (160 Torr) and low-PO₂ (20 Torr) conditions. These cells contained voltage-gated channels typical of electrically excitable cells and had large input resistances (approx. 2 G). Under whole-cell voltage clamp the cells produced brief inactivating inward currents, which were largely abolished by 0.2–2.0 M tetrodotoxin, followed by prolonged outward currents, which were reduced by 5 mM tetraethylammonium or abolished by the substitution of Cs⁺ ions for K⁺ ions in the pipette. On exposure to hypoxia the outward K⁺ current was reduced typically by 15%–20% with both conventional whole-cell and perforated-patch recording, which minimizes washout of the cell's cytoplasm. This effect persisted in long-term culture and was specific, since the inward current was unaffected and, moreover, it did not occur in cultured small intensely fluorescent cells, which are closely related to glomus cells. These properties of cultured rat glomus cells are contrasted with those recently reported for freshly isolated rabbit glomus cells.     

Cytoplasmic bridges and gap junctions in an insect cell line (Aedes albopictus).

Cell pairs of an insect cell line (Aedes albopictus, clone C6/36) were used study simultaneously the diffusional and electrical properties of intercellular junctions. Diffusion studies involved injection of fluorescent molecules into one cell of a cell pair and visual inspection of their intercellular redistribution. Electrical measurements involved a dual voltage clamp method and whole-cell recording with patch pipette. The voltage clamp protocol was aimed at examining the dependency of the junctional conductance, gj, on membrane potential, Vm. Cell pairs exhibiting a voltage-dependent gj were found to allow intercellular diffusion of Lucifer Yellow CH (molecular mass, 443 Da), but not of FITC-dextran (molecular mass, 4,400 Da). This response pattern is consistent with the presence of gap junctions in the intercellular junctions. Cell pairs showing no voltage dependence of gj were found to permit intercellular diffusion of both Lucifer Yellow CH and FITC-dextran (dextran labelled with fluorescein isothiocyanate). This behaviour is compatible with the presence of cytoplasmic bridges connecting the two adjacent cells. Hence, in culture the cells investigated express two kinds of intercellular structures, gap junctions and cytoplasmic bridges.     

Giga-seal formation alters properties of sodium channels of human myoballs

The influence of giga-seal formation on the properties of the Na⁺ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters _m and _h were smaller for the patch currents than for the whole cell, and the h curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca²⁺ concentration. The capacitive current generated in the patch by the whole-cell Na⁺ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na⁺ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.     

Photolysis of caged cyclic AMP in the ciliary cytoplasm of the newt olfactory receptor cell

The effects of cyclic nucleotide monophosphate (cNMP) in the ciliary cytoplasm of the olfactory receptor cell were examined by using photolysis of caged cNMP loaded from the whole-cell patch clamp pipette. Illumination of the cilia induced an inward current at −50 mV. The current amplitude was voltage dependent and the polarity was reversed at +10 mV. The amplitude of the light-induced current was dependent on both light intensity and duration. The intensity-response relation was fitted well by the Hill equation with a coefficient ( n _H) of 4.99 ± 2.66 (mean ± s.d. ,   n = 19  ) and the duration-response relation with a coefficient of 4.03 ± 1.43 (   n = 17  ). The activation time course of adenylyl cyclase was estimated by comparing the light-induced response with the odorant-induced response. Adenylyl cyclase was activated approximately 260 ms later from the onset of the odorant-stimulation. The light-induced current developed very sharply. This could be explained by the sequential openings of cAMP-gated and Ca²⁺-activated Cl ⁻ channels. At +100 mV, where Ca²⁺ influx is expected to be very small, the current rising phase became less steep. When the cells were stimulated by long steps of either odour or light, the odorant-induced current showed stronger decay than the light-induced response. This observation suggests that the molecular system regulating desensitization is situated upstream of cAMP production.     

Stretch sensitivity of the lateral wall of the auditory outer hair cell from the guinea pig.

The inner and outer hair cells of the mammalian hearing organ are mechano-transducer cells. Here we report evidence that the lateral wall of outer hair cells (OHCs) is a mechano-receptor. This mechano-sensitivity appears to complement that of the stereocilia. Patch clamping studies showed that stretching of the membrane patches by suction at the pipette activated potassium channels with 130 pS unit conductance specifically localized in the lateral wall. Application of an osmotic tension to the entire cell membrane under whole-cell recording produced a 10 mV hyperpolarization. The reversal potential and the magnitude of the macroscopic current under voltage clamp were consistent with the single-channel properties of stretch-activated potassium channels. The elongated cylindrical cell body of the OHC is optimally positioned in the cochlea to sense axial force due to the vibrations of the basilar membrane during sound stimulation. This sensitivity can explain the production of a predominantly hyperpolarizing response to sound stimuli, unique to the OHC. Coupled with voltage-dependent OHC motility, the stretch-activated channels may play an important role in producing a mechanical feedback, an indispensable element in cochlear tuning.     

牵张应变对小肠Cajal间质细胞起搏电流的作用

目的研究牵张应变刺激对体外培养的小肠Cajal间质细胞(ICC)起搏电流的影响。方法利用II型胶原酶消化并在含有干细胞因子的SmGM培养基中培养ICC,72h后采用免疫荧光细胞化学方法鉴定培养的ICC。利用传统全细胞记录模式膜片钳技术记录正常小肠ICC起搏电流,然后采用对细胞直接施加正压和灌流低渗溶液的两种方法给细胞膜施加张应变刺激,以观察牵张应变对小肠ICC起搏电流的影响。结果培养72h后的ICC在光镜下,胞体呈三角形或梭形,且自胞体发出2￣4条长突起,并与邻近ICC突起相互连接成网络状;荧光显微镜下观察ICC胞体和突起酪氨酸激酶受体c-kit表达呈阳性;在膜电位钳制在-60mV的条件下,可以记录到自发而节律性内向电流,即起搏电流;在传统全细胞记录模式下,两种张应变刺激均可以激活一种内向电流同时明显增加起搏电流的振幅及频率。结论牵张应变对胃肠壁的刺激可能作用于胃肠平滑肌节律性运动的起搏细胞ICC并改变其电生理特性,从而调节胃肠平滑肌运动的基础张力和频率。     

Inositol trisphosphate releases stored calcium to block voltage-dependent calcium channels in single smooth muscle cells

In single cells obtained by enzymic treatment of rabbit small-intestinal smooth muscle, and held under voltage clamp by patch pipette in the whole-cell recording mode, release of inositol trisphosphate (InsP ₃) from its caged precursor by flash photolysis caused complete inhibition of the voltage-dependent calcium current. No inhibition was seen in control experiments where the cage (2-nitrosoacetophenone) was released by flash photolysis from caged ATP. The inhibition by InsP ₃ of the calcium current was prevented if 10 mM EGTA or 2 mg/ml heparin was included in the pipette solution. Heparin is known to block InsP ₃ receptors. These results suggest that release of calcium stores by InsP ₃ raises Ca_i and that calcium ions inhibit the calcium current by acting either directly or otherwise on the internal mouth of the calcium channel.     

A novel approach to in situ characterization of pancreatic beta-cells

The tissue-slice technique has enabled major insights into neural and neuroendocrine physiology. Our aim was to adapt this technique to study the function of the endocrine pancreas. The preparation combines an in situ approach, as in gland perfusion, with a resolution characteristic of electrophysiological studies on single cells. The membrane potential in beta-cells in the slices recorded using the whole-cell patch-clamp was close to the calculated reversal potential for K+. With sufficient ATP in the recording pipette the beta-cells depolarized rapidly on exposure to an increased glucose concentration or stimulation with tolbutamide. The cells preserved bursting and spiking capacity for tens of minutes despite the whole-cell dialysis. In addition, the voltage clamp was used to monitor the changes in the membrane capacitance and to allow correlation of the electrical activity and the cytosolic calcium changes. The pancreatic tissue slice preparation is a novel method for studying the function of the beta- and other pancreatic endocrine and exocrine cells under near-physiological conditions.     

Dual-frequency method for synchronous measurement of cell capacitance,membrane conductance and access resistance on single cells

A dual-frequency method was developed to monitor changes of membrane capacitance, membrane conductance and serial resistance between patch pipette and cytoplasm of the cell in the whole-cell configuration. Measurement of real and imaginary components of cell admittance during excitation with two superimposed sinusoidal voltages of different frequencies provides mathematical solutions for all three variables. The validity of the method was verified with experiments on mast cells and exocrine pancreatic acinar cells. During degranulation of mast cells, induced by GTPS in the pipette solution, a stepwise increase in membrane capacitance could be observed, indicating that the resolution of the method is below 10 fF. Precalibration of the setup allows all calculated data to be expressed as absolute values. The capacitance measurement proved to be rather independent of changes in the access resistance and in the cell membrane resistance over a wide range. The huge changes in membrane conductance of mouse pancreatic acinar cells during hormonal stimulation with acetylcholine produced a relative error of less than 0.3% in the capacitance trace. This allows a clear distinction between changes of membrane conductance and cell capacitance. The method therefore offers great advantages in the study of exocytosis as well as endocytosis in cell types, such as exocrine gland cells, with major changes in membrane conductance during hormonal stimulation.