The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaesthesia (or awake) animals. This technique can be used to study the syn...The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaesthesia (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 end...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 oells 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 to0.028±0.003, insulin secretion from 2.6±0.6to 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±0.8to 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 pancrearic 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
Background The myocardial ATP sensitive potassium channel (KATP channel) has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protectio...Background The myocardial ATP sensitive potassium channel (KATP channel) has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using KATP agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that KATP channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial KATP channel by nicorandil. Methods Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4?C. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IKATP occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 μmol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4?C, 10?C, 20?C, 25?C and 35?C respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 (Graphpad, San Diego, CA, USA). Nonlinear curve fitting was done in Clampfit (Axon) or Sigmaplot (SPSS). Results At 4?C, 10?C, 20?C, 25?C and 35?C, the time needed to open the myocardial KATP channel was (81.0±0) minutes, (50.5±11.7) minutes, (28.8±2.3) minutes, (9.4±10.2) minutes and (2.3±1.0) minutes respectively (P=0.003). The linear relationship between temperature and time needed to open the channel was y (min) = (4348.790-124.277x)/60, where y (min) is time needed to open KATP channel, x is temperature, correlation coefficient r =-0.942 (P=0.00), regression coefficient b =-124.277 (P=0.00). The current densities among different temperatures were statistically different (P=0.022), the current density was greater after the activation of KATP channel at higher temperatures. The lower the temperature, the fewer cells in which KATP channels could be opened. At 4?C, only one cell in which the KATP channel could be opened, took a quite long time (81 minutes)and the I-V curve was quite untypical. Conclusions KATP channel activated bynicorandil is temperature dependent and thetemperature linearly related to time needed toopen KATP channel; the lower the temperature, thelonger the time needed to open channel and thesmaller the current density. At profoundhypothermia, it is difficult to activate KATPchannels.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.) vacuola...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 lied in 25±5 mV. The increase in cytoplasmic Ca2+ led toenhancement 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/Lfree La3+ was added to the bath, SV-type current was suppressed by 60~75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.展开更多
Aim: To record the single-channel currents and characterize the electrophysiological properties of the C1^- channels in human sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassemb...Aim: To record the single-channel currents and characterize the electrophysiological properties of the C1^- channels in human sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled into liposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydrationrehydration procedure. The giant liposomes were used to study the C1^- channel activities by patch-clamp technique. Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-C1 (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 all were blocked by SITS (4-acetamido-4′-isothiocyanato-stilbene-2′, 2′-disulfonic acid) in a concentration-dependent manner. By constructing the open and close dwell time distribution histograms and then fitting them with exponential function, two time constants were obtained in both the open and the close states. The burst activity and conductance substate of the channels were observed. Conclusion: There exist three kinds of C1^- channels with different conductance in human sperm membrane at least.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
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
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 anaesthesia (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 oells 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 to0.028±0.003, insulin secretion from 2.6±0.6to 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±0.8to 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 pancrearic 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This work was supported by a grant from the Natural Science Foundation of China (No. 30371374).
文摘Background The myocardial ATP sensitive potassium channel (KATP channel) has been known for more than two decades, the properties of this channel have been intensively investigated, especially the myocardial protection effect by opening this channel. Numerous studies, including hypothermic, using KATP agonists to achieve a hyperpolarizing cardioplegic arrest, have shown a better myocardial protection than potassium arrest. However, there is no evidence showing that KATP channel could be opened by its agonists under profound hypothermia. We investigated the effect of temperature on activation of myocardial KATP channel by nicorandil. Methods Isolated ventricular myocytes were obtained by collagenase digestion of the hearts of guinea pigs and stored in KB solution at 4?C. With a steady ground current, the myocytes were perfused with 1 mmol/L nicorandil until a steady IKATP occurred. Then the cells were perfused with 1 mmol/L nicorandil plus 1 μmol/L glybenclamide. Currents signals were recorded on whole cells using patch clamp technique at several temperatures. The temperature of the bath solution around myocytes was monitored and was controlled at 4?C, 10?C, 20?C, 25?C and 35?C respectively. About 10 cells were tested at each temperature, the cells were considered useful only when the outward current could be induced by nicorandil and blocked by glybenclamide. All data were analyzed using Graphpad PRISM 3.0 (Graphpad, San Diego, CA, USA). Nonlinear curve fitting was done in Clampfit (Axon) or Sigmaplot (SPSS). Results At 4?C, 10?C, 20?C, 25?C and 35?C, the time needed to open the myocardial KATP channel was (81.0±0) minutes, (50.5±11.7) minutes, (28.8±2.3) minutes, (9.4±10.2) minutes and (2.3±1.0) minutes respectively (P=0.003). The linear relationship between temperature and time needed to open the channel was y (min) = (4348.790-124.277x)/60, where y (min) is time needed to open KATP channel, x is temperature, correlation coefficient r =-0.942 (P=0.00), regression coefficient b =-124.277 (P=0.00). The current densities among different temperatures were statistically different (P=0.022), the current density was greater after the activation of KATP channel at higher temperatures. The lower the temperature, the fewer cells in which KATP channels could be opened. At 4?C, only one cell in which the KATP channel could be opened, took a quite long time (81 minutes)and the I-V curve was quite untypical. Conclusions KATP channel activated bynicorandil is temperature dependent and thetemperature linearly related to time needed toopen KATP channel; the lower the temperature, thelonger the time needed to open channel and thesmaller the current density. At profoundhypothermia, it is difficult to activate KATPchannels.
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 lied in 25±5 mV. The increase in cytoplasmic Ca2+ led toenhancement 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/Lfree La3+ was added to the bath, SV-type current was suppressed by 60~75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.
文摘Aim: To record the single-channel currents and characterize the electrophysiological properties of the C1^- channels in human sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled into liposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10μm by dehydrationrehydration procedure. The giant liposomes were used to study the C1^- channel activities by patch-clamp technique. Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-C1 (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 all were blocked by SITS (4-acetamido-4′-isothiocyanato-stilbene-2′, 2′-disulfonic acid) in a concentration-dependent manner. By constructing the open and close dwell time distribution histograms and then fitting them with exponential function, two time constants were obtained in both the open and the close states. The burst activity and conductance substate of the channels were observed. Conclusion: There exist three kinds of C1^- channels with different conductance in human sperm membrane at least.
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.
