Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents,offering critical molecular-level insights essential for drug screening and optimization.By enabling a dee...Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents,offering critical molecular-level insights essential for drug screening and optimization.By enabling a deeper understanding of ion channel behavior,these techniques significantly accelerate the process of drug discovery and development.In recent years,automated patch-clamp technique has undergone substantial advancements,surpassing traditional manual methods with its high throughput,improved data consistency,and automation.However,fully harnessing these advantages requires meticulous optimization of experimental conditions tailored to specific targets.Without such refinement,the high cost of consumables and operational expenses could severely hinder the widespread adoption of this technique.This study focused on the TRPV1 channel,detailing the establishment of an automated patch-clamp detection system for TRPV1 currents,optimization of experimental parameters,and a comparative evaluation of the results against manual patch-clamp techniques.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
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
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
Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission ...Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission are associated with pathophysiological and neurodegenerative disorders,whereas glucagon-like peptide-1 has demonstrated neuroprotective effects.However,it is not yet clear whether diabetes causes alterations in inhibitory input to retinal ganglion cells and whether and how glucagon-like peptide-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to retinal ganglion cells.In the present study,we used the patch-clamp technique to recordγ-aminobutyric acid subtype A receptor-mediated miniature inhibitory postsynaptic currents in retinal ganglion cells from streptozotocin-induced diabetes model rats.We found that early diabetes(4 weeks of hyperglycemia)decreased the frequency of GABAergic miniature inhibitory postsynaptic currents in retinal ganglion cells without altering their amplitude,suggesting a reduction in the spontaneous release ofγ-aminobutyric acid to retinal ganglion cells.Topical administration of glucagon-like peptide-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency,subsequently enhancing the survival of retinal ganglion cells.Concurrently,the protective effects of glucagon-like peptide-1 on retinal ganglion cells in diabetic rats were eliminated by topical administration of exendin-9-39,a specific glucagon-like peptide-1 receptor antagonist,or SR95531,a specific antagonist of theγ-aminobutyric acid subtype A receptor.Furthermore,extracellular perfusion of glucagon-like peptide-1 was found to elevate the frequencies of GABAergic miniature inhibitory postsynaptic currents in both ON-and OFF-type retinal ganglion cells.This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of glucagon-like peptide-1 receptor activation.Moreover,multielectrode array recordings revealed that glucagon-like peptide-1 functionally augmented the photoresponses of ON-type retinal ganglion cells.Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of glucagon-like peptide-1.These results suggest that glucagon-like peptide-1 facilitates the release ofγ-aminobutyric acid onto retinal ganglion cells through the activation of glucagon-like peptide-1 receptor,leading to the de-excitation of retinal ganglion cell circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy.Collectively,our findings indicate that theγ-aminobutyric acid system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy.Furthermore,the topical administration of glucagon-like peptide-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.展开更多
We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-t...We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmo1/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currents increase with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.展开更多
The effects of BaCl_2 on slow vacuolar (SV) currents of radish are studied byusing the whole-vacuolar patch-clamp recording mode. The Ca^(2+)-dependent SV channel can beactivated by cytosolic Ca^(2+). When 1 mmol/L Ba...The effects of BaCl_2 on slow vacuolar (SV) currents of radish are studied byusing the whole-vacuolar patch-clamp recording mode. The Ca^(2+)-dependent SV channel can beactivated by cytosolic Ca^(2+). When 1 mmol/L BaCI_2 is added into pipette solution, SV currents aresuppressed remarkably. Then adding BaCI_2 of different concentrations into the bath solution, SVcurrents reflect different effects. The results show that BaCl_2 with a lower concentration (< 3mmol/L) promotes the channel currents and the currents are saturated when BaCl_2 concentrations arebetween 1 μmol/L and 1 mmol/L, but BaCl_2 with higher concentration (≥ 3 mmol/L) inhibits SVcurrents.展开更多
The planar patch-clamp technique has been applied to high throughput screening in drug discovery. The key feature of this technique is the fabrication of a planar patch-clamp substrate using appropriate materials. In ...The planar patch-clamp technique has been applied to high throughput screening in drug discovery. The key feature of this technique is the fabrication of a planar patch-clamp substrate using appropriate materials. In this study, a planar patch-clamp substrate was designed and fabricated using a silicon-on-insulator （SOI） wafer. The access resistance and capacitance of SOl-based planar patch-clamp substrates are smaller than those of bulk silicon-based planar substrates, which will reduce the distributed RC noise.展开更多
The effect of La3+ on potassium channels in rat ventricular myocytes was investigated using the whole-cell patch-clamp recording mode. The Ca2+-independent voltage- activated outward K+ current was activated by the de...The effect of La3+ on potassium channels in rat ventricular myocytes was investigated using the whole-cell patch-clamp recording mode. The Ca2+-independent voltage- activated outward K+ current was activated by the depolar- izing pulse in enzymatically isolated rat ventricular myocytes. After addition of different concentrations La3+ to the bath solution, the outward K+ current was depressed gradually. The inhibition effect was in a concentration-dependent man- ner. The phenomena of the outward K+ current, being the main repolarizing current suppressed by La3+, suggest that the effect of lanthanides on myocardial function should be exploited further.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
Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion ...Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.展开更多
Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods...Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.展开更多
基金The National Natural Science Foundation of China(Grant No.32000674).
文摘Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents,offering critical molecular-level insights essential for drug screening and optimization.By enabling a deeper understanding of ion channel behavior,these techniques significantly accelerate the process of drug discovery and development.In recent years,automated patch-clamp technique has undergone substantial advancements,surpassing traditional manual methods with its high throughput,improved data consistency,and automation.However,fully harnessing these advantages requires meticulous optimization of experimental conditions tailored to specific targets.Without such refinement,the high cost of consumables and operational expenses could severely hinder the widespread adoption of this technique.This study focused on the TRPV1 channel,detailing the establishment of an automated patch-clamp detection system for TRPV1 currents,optimization of experimental parameters,and a comparative evaluation of the results against manual patch-clamp techniques.
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)
基金supported by the National Natural Science Foundation of China,Nos.32070989(to YMZ),31872766(to YMZ),81790640(to XLY),and 82070993(to SJW)the grant from Sanming Project of Medicine in Shenzhen,No.SZSM202011015(to XLY)。
文摘Diabetic retinopathy is a prominent cause of blindness in adults,with early retinal ganglion cell loss contributing to visual dysfunction or blindness.In the brain,defects inγ-aminobutyric acid synaptic transmission are associated with pathophysiological and neurodegenerative disorders,whereas glucagon-like peptide-1 has demonstrated neuroprotective effects.However,it is not yet clear whether diabetes causes alterations in inhibitory input to retinal ganglion cells and whether and how glucagon-like peptide-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to retinal ganglion cells.In the present study,we used the patch-clamp technique to recordγ-aminobutyric acid subtype A receptor-mediated miniature inhibitory postsynaptic currents in retinal ganglion cells from streptozotocin-induced diabetes model rats.We found that early diabetes(4 weeks of hyperglycemia)decreased the frequency of GABAergic miniature inhibitory postsynaptic currents in retinal ganglion cells without altering their amplitude,suggesting a reduction in the spontaneous release ofγ-aminobutyric acid to retinal ganglion cells.Topical administration of glucagon-like peptide-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency,subsequently enhancing the survival of retinal ganglion cells.Concurrently,the protective effects of glucagon-like peptide-1 on retinal ganglion cells in diabetic rats were eliminated by topical administration of exendin-9-39,a specific glucagon-like peptide-1 receptor antagonist,or SR95531,a specific antagonist of theγ-aminobutyric acid subtype A receptor.Furthermore,extracellular perfusion of glucagon-like peptide-1 was found to elevate the frequencies of GABAergic miniature inhibitory postsynaptic currents in both ON-and OFF-type retinal ganglion cells.This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of glucagon-like peptide-1 receptor activation.Moreover,multielectrode array recordings revealed that glucagon-like peptide-1 functionally augmented the photoresponses of ON-type retinal ganglion cells.Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of glucagon-like peptide-1.These results suggest that glucagon-like peptide-1 facilitates the release ofγ-aminobutyric acid onto retinal ganglion cells through the activation of glucagon-like peptide-1 receptor,leading to the de-excitation of retinal ganglion cell circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy.Collectively,our findings indicate that theγ-aminobutyric acid system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy.Furthermore,the topical administration of glucagon-like peptide-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.
基金This work was supported by the National Natural Science Foundation of China (Grant No. 29890280).
文摘We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmo1/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currents increase with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.
基金We thank Dr. Pei Zhenming for technique advice on the patch clamp. This work was supported by the National Natural Science Foundation of China (Grant No. 29890280).
文摘The effects of BaCl_2 on slow vacuolar (SV) currents of radish are studied byusing the whole-vacuolar patch-clamp recording mode. The Ca^(2+)-dependent SV channel can beactivated by cytosolic Ca^(2+). When 1 mmol/L BaCI_2 is added into pipette solution, SV currents aresuppressed remarkably. Then adding BaCI_2 of different concentrations into the bath solution, SVcurrents reflect different effects. The results show that BaCl_2 with a lower concentration (< 3mmol/L) promotes the channel currents and the currents are saturated when BaCl_2 concentrations arebetween 1 μmol/L and 1 mmol/L, but BaCl_2 with higher concentration (≥ 3 mmol/L) inhibits SVcurrents.
文摘The planar patch-clamp technique has been applied to high throughput screening in drug discovery. The key feature of this technique is the fabrication of a planar patch-clamp substrate using appropriate materials. In this study, a planar patch-clamp substrate was designed and fabricated using a silicon-on-insulator （SOI） wafer. The access resistance and capacitance of SOl-based planar patch-clamp substrates are smaller than those of bulk silicon-based planar substrates, which will reduce the distributed RC noise.
基金This work was supported by the National Natural Science Foundation of China(Grant No.29890280)the Shanxi Provincial and Shanxi University Natural Science Foundation.
文摘The effect of La3+ on potassium channels in rat ventricular myocytes was investigated using the whole-cell patch-clamp recording mode. The Ca2+-independent voltage- activated outward K+ current was activated by the depolar- izing pulse in enzymatically isolated rat ventricular myocytes. After addition of different concentrations La3+ to the bath solution, the outward K+ current was depressed gradually. The inhibition effect was in a concentration-dependent man- ner. The phenomena of the outward K+ current, being the main repolarizing current suppressed by La3+, suggest that the effect of lanthanides on myocardial function should be exploited further.
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Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.
文摘Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.
