Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembl...Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled intoliposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl^- channel activities by patch-clamp technique.Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100//pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 ± 8.3) pS,(117.0±5.7) pS and (144.7±4.5) pS, respectively, were detected. Their activities were voltage-dependent and allwere blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependentmanner. By constructing the open and close dwell time distribution histograms and then fitting them with exponentialfunction, two time constants were obtained in both the open and the close states. The burst activity and conductancesubstate of the channels were observed. Conclusion; There exist three kinds of Cl^- channels with different conduc-tance in human sperm membrane at least. (Asian J Androl 2001 Sep; 3: 185 - 191)展开更多
The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the...The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.展开更多
AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into ...AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine ceils with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels （KATP）, voltage-dependent potassium channels （Kv）, and voltage-dependent calcium channels （KcA） in β-cells were identified by patch clamp technique. RESULTS： After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly （MTT value from 0.024 ±0.003 to 3.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L）. Then MTT value and insulin secretion increased quickly from d 5 to d 10 （MTT value from 0.028 ±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L）, then reached high plateau （MTT value 〉0.052±0.008, insulin secretion 〉18.3±2.6 mIU/L）. In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d （MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L）, but afterwards they reduced gradually （MTT value 〈0.031 ±0.011, insulin secretion 〈8.2±1.5 mIU/L）, and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, Kv, and KCA. CONCLUSION： Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.展开更多
We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. T...We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. The minimum activation potentials of voltage-dependent SV channels tied in 25+/-5 mV. The increase in cytoplasmic Ca2+ led to enhancement of SV-type currents. It was found that the threshold potential of activation shifted towards more depolarized values whenever cytoplasmic Ca2+ was increased. When 10(-10) mol/L free La3+ was added to the bath, SV-type current was suppressed by 60 similar to 75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.展开更多
To attempt to the feasibility of whole-cell patch-clamp recordings on the cardiac ventricular slices of newborn (P3-P7) Sprague-Dawley (S-D) rats and find a good substitute of the single cardiac myocytes with enzymati...To attempt to the feasibility of whole-cell patch-clamp recordings on the cardiac ventricular slices of newborn (P3-P7) Sprague-Dawley (S-D) rats and find a good substitute of the single cardiac myocytes with enzymatic treatment. High resistance seals (】1 GΩ) could be obtained in the cardiac ventricle tissue on this preparation without enzymatic treatment. Then, the cell-attached and whole-cell patch-clamp techniques can be achieved in the thin (200μm) cardiac slices. Averaged sodium current (n=ll cells) was recorded in cell-attached mode, displayed similar features to that previously reported from isolated rat ventricular myocytes. The outward potassium current, Hyperpolarization-activated cation channel or If channel (HCN channel) and action potential (AP) were recorded in whole-cell configuration (n=2 cells) and the similar properties can be seen from the cardiac slices. Cell-attached mode is a stable and reliable mode to record the ion treatment. Resting potential for cardiac slice, measured from Current-clamp recording in whole-cell mode, is about -50 - - 70 mV. The resting potential value from the cardiac slice has similar property except that it is positive to the isolated cardiomyocytes by enzymes. Application of patch-clamp techniques to cardiac slices allows single channel recordings without complicated procedures of cell isolation. Moreover, possible alteration of channel properties caused by proteolytic enzymes can be avoided. In this paper, whole-cell patch-clamp recordings could be achieved on the cardiac slice and we affirmed the feasibility and values of the both recording modes on it. At the same time, there is difficulty and limitation of the application of whole-cell patch-clamp on the cardiac slice for the existence of large mount of connective tissue even in the newborn rats.展开更多
The single ion channel signal is an ionic current that can be recorded by the patch clamp technique. Hidden Markov model (HMM) algorithm has been used to convert the low signal noise ratio (SNR) noisy recording into a...The single ion channel signal is an ionic current that can be recorded by the patch clamp technique. Hidden Markov model (HMM) algorithm has been used to convert the low signal noise ratio (SNR) noisy recording into an idealized quantal one in the case of white background noise. The traditional HMM algorithm is extended and adapted to the colored background noise. A new algorithm called EHMM (Extended HMM) algorithm is proposed, and mainly validated by simulation. Results show that it’s effective.展开更多
One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship whe...One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship when BLMs was formed and it could be confirmed by Gramicidin method.Ion current was increased by dihexyl (C_ (12)) modified ssDNA fixed on the BLMs and also indicated linear relationship to ssDNA's concentration due to the interaction of (C_ 12)-ssDNA and BLMs.Further more,the regression equations were different from BLMs fixed with ssDNA probe and a blank control BLM in the same experimental conditions.The ssDNA probe was successfully fixed on patch-clamp pipette supported-BLMs.Based on our studies,a biosensor with reactive element of patch-clamp pipette-supported BLMs has been established.展开更多
背景:膜片钳技术作为研究离子通道的“金标准”,已有40多年的发展历史。然而,科研机构的研究内容相对独立,没有对现有研究成果进行系统总结,导致现有研究存在重复性高、创新性弱的现象。因此,急需对膜片钳技术做一个全面的回顾,以明晰...背景:膜片钳技术作为研究离子通道的“金标准”,已有40多年的发展历史。然而,科研机构的研究内容相对独立,没有对现有研究成果进行系统总结,导致现有研究存在重复性高、创新性弱的现象。因此,急需对膜片钳技术做一个全面的回顾,以明晰现今的研究现状、热点和未来发展方向。目的:总结近10年膜片钳技术领域的研究现状和发展趋势。方法:使用Web of Science核心合集数据库收集了2013-2023年关于膜片钳技术的出版物。采用CiteSpace和VOSviewer软件对出版物数量进行量化分析,并分析文献条目网络,包括国家、机构、期刊、作者、关键词、高被引文献和共被引参考文献。结果与结论:①近10年间,膜片钳技术领域研究已逐步进入稳定发展阶段。②中国和美国是这方面的领先国家,中国科学院是具有核心影响力的机构,《Journal of Neuroscience》是主要出版刊物,PARK,WON SUN团队(韩国全北国立大学)和CHU,LI团队(中国河北省心脑血管病中医药防治研究重点实验室)在该领域作出了杰出的贡献,但团队之间的协作与交流较少,尚未形成网络合作模式。③膜片钳技术主要应用在神经系统的电生理特性及其疾病的病理机制方面,是研究人员持续关注的焦点。④在心血管系统电生理特性及其疾病病理机制的研究方面,对原代心肌细胞、诱导多能干细胞衍生的心肌细胞的电生理特性和心房颤动、心脏毒性、心源性猝死和高血压等心血管疾病的病理机制方面的研究,是近几年来研究的热点。⑤在膜片钳技术与其他生物技术的结合应用方面,关注的是与光遗传学、双光子钙成像等技术的交叉融合,将是一个重要的研究方向。⑥在药物筛选及治疗靶点的识别研究方面,尤其对于膜片钳技术和中药复方的研究,将成为未来组分中药研究中的一大助力。展开更多
The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “o...The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “onozuka” R\|10, 0\^01% pectolyase Y\|23,0\^15% macerozyme R\|10 and 0\^1% hemicellulase at 25℃. Outward and inward single channels in plasma membrane were observed using cell\|attached recording of patch\|clamp technique. In this study, single channel records showed that more than one species of channel were obtained. These attempts in protoplast isolation and ion channel recording offers the opportunity to characterize cellular mechanisms of salt tolerance in tree species.展开更多
文摘Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl^- channels inhuman sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled intoliposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydration-rehydration procedure. The giant liposomes were used to study the Cl^- channel activities by patch-clamp technique.Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100//pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.1 ± 8.3) pS,(117.0±5.7) pS and (144.7±4.5) pS, respectively, were detected. Their activities were voltage-dependent and allwere blocked by SITS (4-acetamido-4'-isothiocyanato-stilbene-2', 2'-disulfonic acid) in a concentration-dependentmanner. By constructing the open and close dwell time distribution histograms and then fitting them with exponentialfunction, two time constants were obtained in both the open and the close states. The burst activity and conductancesubstate of the channels were observed. Conclusion; There exist three kinds of Cl^- channels with different conduc-tance in human sperm membrane at least. (Asian J Androl 2001 Sep; 3: 185 - 191)
文摘The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.
基金Supported by the National Natural Science Foundation of China,No. 30472254
文摘AIM： To induce the pancreatic duct cells into endocrine cells with a new natural protocol for electrophysiological study. METHODS： The pancreatic duct cells of neonatal rats were isolated, cultured and induced into endocrine ceils with 15% fetal bovine serum for a period of 20 d. During this period, insulin secretion, MTT value, and morphological change of neonatal and adult pancreatic islet cells were comparatively investigated. Pancreatic β-cells were identified by morphological and electrophysiological characteristics, while ATP sensitive potassium channels （KATP）, voltage-dependent potassium channels （Kv）, and voltage-dependent calcium channels （KcA） in β-cells were identified by patch clamp technique. RESULTS： After incubation with fetal bovine serum, the neonatal duct cells budded out, changed from duct-like cells into islet clusters. In the first 4 d, MTT value and insulin secretion increased slowly （MTT value from 0.024 ±0.003 to 3.028±0.003, insulin secretion from 2.6±0.6 to 3.1±0.8 mIU/L）. Then MTT value and insulin secretion increased quickly from d 5 to d 10 （MTT value from 0.028 ±0.003 to 0.052±0.008, insulin secretion from 3.1±0.8 to 18.3±2.6 mIU/L）, then reached high plateau （MTT value 〉0.052±0.008, insulin secretion 〉18.3±2.6 mIU/L）. In contrast, for the isolated adult pancreatic islet cells, both insulin release and MTT value were stable in the first 4 d （MTT value from 0.029±0.01 to 0.031±0.011, insulin secretion from 13.9±3.1 to 14.3±3.3 mIU/L）, but afterwards they reduced gradually （MTT value 〈0.031 ±0.011, insulin secretion 〈8.2±1.5 mIU/L）, and the pancreatic islet cells became dispersed, broken or atrophied correspondingly. The differentiated neonatal cells were identified as pancreatic islet cells by dithizone staining method, and pancreatic β-cells were further identified by both morphological features and electrophysiological characteristics, i.e. the existence of recording currents from KATP, Kv, and KCA. CONCLUSION： Islet cells differentiated from neonatal pancreatic duct cells with the new natural protocol are more advantageous in performing patch clamp study over the isolated adult pancreatic islet cells.
基金The authors acknowledge the support of the National Natural Science Foundation of ChinaProvincial Natural Science Foundation of Shanxi.
文摘We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. The minimum activation potentials of voltage-dependent SV channels tied in 25+/-5 mV. The increase in cytoplasmic Ca2+ led to enhancement of SV-type currents. It was found that the threshold potential of activation shifted towards more depolarized values whenever cytoplasmic Ca2+ was increased. When 10(-10) mol/L free La3+ was added to the bath, SV-type current was suppressed by 60 similar to 75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.
文摘To attempt to the feasibility of whole-cell patch-clamp recordings on the cardiac ventricular slices of newborn (P3-P7) Sprague-Dawley (S-D) rats and find a good substitute of the single cardiac myocytes with enzymatic treatment. High resistance seals (】1 GΩ) could be obtained in the cardiac ventricle tissue on this preparation without enzymatic treatment. Then, the cell-attached and whole-cell patch-clamp techniques can be achieved in the thin (200μm) cardiac slices. Averaged sodium current (n=ll cells) was recorded in cell-attached mode, displayed similar features to that previously reported from isolated rat ventricular myocytes. The outward potassium current, Hyperpolarization-activated cation channel or If channel (HCN channel) and action potential (AP) were recorded in whole-cell configuration (n=2 cells) and the similar properties can be seen from the cardiac slices. Cell-attached mode is a stable and reliable mode to record the ion treatment. Resting potential for cardiac slice, measured from Current-clamp recording in whole-cell mode, is about -50 - - 70 mV. The resting potential value from the cardiac slice has similar property except that it is positive to the isolated cardiomyocytes by enzymes. Application of patch-clamp techniques to cardiac slices allows single channel recordings without complicated procedures of cell isolation. Moreover, possible alteration of channel properties caused by proteolytic enzymes can be avoided. In this paper, whole-cell patch-clamp recordings could be achieved on the cardiac slice and we affirmed the feasibility and values of the both recording modes on it. At the same time, there is difficulty and limitation of the application of whole-cell patch-clamp on the cardiac slice for the existence of large mount of connective tissue even in the newborn rats.
文摘The single ion channel signal is an ionic current that can be recorded by the patch clamp technique. Hidden Markov model (HMM) algorithm has been used to convert the low signal noise ratio (SNR) noisy recording into an idealized quantal one in the case of white background noise. The traditional HMM algorithm is extended and adapted to the colored background noise. A new algorithm called EHMM (Extended HMM) algorithm is proposed, and mainly validated by simulation. Results show that it’s effective.
文摘One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship when BLMs was formed and it could be confirmed by Gramicidin method.Ion current was increased by dihexyl (C_ (12)) modified ssDNA fixed on the BLMs and also indicated linear relationship to ssDNA's concentration due to the interaction of (C_ 12)-ssDNA and BLMs.Further more,the regression equations were different from BLMs fixed with ssDNA probe and a blank control BLM in the same experimental conditions.The ssDNA probe was successfully fixed on patch-clamp pipette supported-BLMs.Based on our studies,a biosensor with reactive element of patch-clamp pipette-supported BLMs has been established.
文摘背景:膜片钳技术作为研究离子通道的“金标准”,已有40多年的发展历史。然而,科研机构的研究内容相对独立,没有对现有研究成果进行系统总结,导致现有研究存在重复性高、创新性弱的现象。因此,急需对膜片钳技术做一个全面的回顾,以明晰现今的研究现状、热点和未来发展方向。目的:总结近10年膜片钳技术领域的研究现状和发展趋势。方法:使用Web of Science核心合集数据库收集了2013-2023年关于膜片钳技术的出版物。采用CiteSpace和VOSviewer软件对出版物数量进行量化分析,并分析文献条目网络,包括国家、机构、期刊、作者、关键词、高被引文献和共被引参考文献。结果与结论:①近10年间,膜片钳技术领域研究已逐步进入稳定发展阶段。②中国和美国是这方面的领先国家,中国科学院是具有核心影响力的机构,《Journal of Neuroscience》是主要出版刊物,PARK,WON SUN团队(韩国全北国立大学)和CHU,LI团队(中国河北省心脑血管病中医药防治研究重点实验室)在该领域作出了杰出的贡献,但团队之间的协作与交流较少,尚未形成网络合作模式。③膜片钳技术主要应用在神经系统的电生理特性及其疾病的病理机制方面,是研究人员持续关注的焦点。④在心血管系统电生理特性及其疾病病理机制的研究方面,对原代心肌细胞、诱导多能干细胞衍生的心肌细胞的电生理特性和心房颤动、心脏毒性、心源性猝死和高血压等心血管疾病的病理机制方面的研究,是近几年来研究的热点。⑤在膜片钳技术与其他生物技术的结合应用方面,关注的是与光遗传学、双光子钙成像等技术的交叉融合,将是一个重要的研究方向。⑥在药物筛选及治疗靶点的识别研究方面,尤其对于膜片钳技术和中药复方的研究,将成为未来组分中药研究中的一大助力。
文摘The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “onozuka” R\|10, 0\^01% pectolyase Y\|23,0\^15% macerozyme R\|10 and 0\^1% hemicellulase at 25℃. Outward and inward single channels in plasma membrane were observed using cell\|attached recording of patch\|clamp technique. In this study, single channel records showed that more than one species of channel were obtained. These attempts in protoplast isolation and ion channel recording offers the opportunity to characterize cellular mechanisms of salt tolerance in tree species.
