Patch Clamp Methods And Protocols Pdf

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patch clamp methods and protocols pdf

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Overview DOI: Signaling in the nervous system is mediated by changes in voltage across the membranes of individual neurons. Understanding these bioelectrical signals—their ionic basis, modulation, information content, contributions to intercellular communication,. Understanding these bioelectrical signals—their ionic basis, modulation, information content, contributions to intercellular communication, and ways in which they encode sensory input and behavioral output—is one of the fundamental tasks of contemporary neuroscience research. We provide an overview of the background and principles of whole-cell recording as well as a discussion of the basic complement of equipment including air tables, microscopes, Faraday cages, micromanipulators, recording electrodes, amplifiers and other electronics, solutions, and recording pipettes needed for these experiments.

Whole-Cell Patch-Clamp Recording

The chloride Cl - channel cystic fibrosis transmembrane conductance regulator CFTR is defective in cystic fibrosis CF , and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature.

Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity.

Few works used automated patch clamp APC for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. Using optimized conditions, we recorded both wt- and corrected Fdel-CFTR Cl - currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp.

Also, the results showed good similarities of CFTR currents recording between the two methods. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

The cystic fibrosis transmembrane conductance regulator CFTR is a cAMP-dependent chloride Cl - channel whose encoding gene is mutated in the common autosomal recessive disease cystic fibrosis CF; Riordan et al.

The Fdel mutation induces misfolding of the nascent protein and retention in the endoplasmic reticulum Cheng et al. Cystic fibrosis transmembrane conductance regulator pharmacotherapy is one major therapeutic strategy that involves identifying compounds that correct abnormal CFTR protein.

Most research effort is directed at finding small molecules which interact directly with mutated CFTR to correct its folding and gating, and thus improve protein trafficking and channel function. Two compounds that have yielded promising results are the potentiator Ivacaftor, also called VX Van Goor et al. Lumacaftor was approved for clinical use in combination with Ivacaftor for the treatment of Fdel homozygous patients; however, this combination therapy, called Orkambi TM , provided only modest benefit Boyle et al.

Therefore, the search for innovative small molecules and understanding their mechanisms of action remain a priority for CF therapeutic research. To this end, robust screening assays are essential for rapidly testing large numbers of compounds and performing informative concentration—response curves in vitro.

As multiple CFTR defects need to be corrected, cell-based assays should monitor improvements in the trafficking, membrane stability, and channel function of rescued mutant CFTR. One of the most powerful techniques for characterizing ion channels and studying their pharmacology is patch clamp recording. It allows the recording of channel currents in real time, in a biological environment, and with high resolution Hamill et al.

Unlike other functional assays such as Ussing chamber short-circuit current measurements, flux studies using either radioisotopes or ion-selective electrodes , or fluorescence assays [membrane potential detection Van Goor et al.

Although conventional manual patch clamp MPC techniques have low throughput and are therefore not suitable for screening phase, automated patch clamp APC techniques are available and can be applied to investigate CFTR pharmacology. APC has become the gold standard for early safety assessment when examining cardiac hERG channel to new drug candidates Polonchuk, but has not yet been widely used for CFTR pharmacology studies Liu et al.

Patch-clamp experiments were performed 2—3 days after plating. For conventional MPC, Cl - currents were measured in the whole-cell patch-clamp configuration after compensating electronically for pipette capacitance in the cell-attached mode. Automated whole-cell patch clamp recordings were obtained using two different systems.

Both chip models exhibited a resistance of 3—3. The holding potential was maintained at mV throughout the experiment, and two voltage-clamp protocols were used to measure whole-cell CFTR currents.

The tree of events shows all steps leading to the whole-cell configuration gray rectangle 1 followed by the CFTR recording parameters gray rectangle 2. To monitor the current evolution under drugs application and confirm the absence of significant leak current; a single depolarization from to 0 mV was applied every 5 s for 6—7 min drug time course protocol, green dots.

A Tree of events for whole-cell configuration establishing gray rectangle 1 and personalized CFTR recording gray rectangle 2. After stable sealing of the cell, the seal enhancer solution was replaced by the external solution before performing the whole-cell configuration and recordings.

Stock solutions were prepared in dimethylsulfoxide. All statistical tests were performed using GraphPad Prism, version 6. Automated planar patch clamp is commonly used to detect off-target effects on the potassium channel hERG during the safety testing phase of drug development.

Both systems tested use the general principle of planar patch clamp, which is comparable to MPC with some differences in the experimental conditions Milligan et al. Briefly, planar APC experiments use cartridge chips with a variable number of microfluidic chambers. Cells in suspension are placed in the wells and interact with the glass to form stable giga-seal at the aperture. After giga-seal formation, whole-cell configuration is achieved by physical rupture of the cell-attached patch, then pre-programmed voltage-clamp steps are applied and recording begins.

As APC requires both fresh cell suspension and cell membrane integrity, the first step was to obtain viable and isolated cells in suspension. Success rates varied between cell lines. The rates of achieving seals, entering the whole-cell configuration, and completing the CFTR recording protocol are shown in the table, with the overall success rates in pooled wt- and Fdel-CFTR experiments of Much lower rates were obtained in CFBE epithelial cells 5.

The following discussion focuses mainly on results obtained with BHK cells. TABLE 1. Recordings were collected and analyzed in experiments that met the above criteria.

Several observations suggest that the differences observed between data obtained using APC and MPC were caused by a decrease in the seal resistance while recording.

Dashed lines indicate the zero current level. C Corresponding distribution of current densities recorded at 0 mV.

Error bars, SEM. TABLE 2. Comparison of patch clamp parameters for manual and automated BHK whole-cell recordings. Fdel is by far the most common CF mutation; therefore, the functional screening assay should enable the recording of Fdel-CFTR currents. However, this mutation induces a gating defect in addition to the trafficking defect Dalemans et al. As expected, whole-cell recording detected little for any Cl - current using either method current densities at 0 mV: Figure 3B shows the corresponding distribution of each current density at 0 mV.

Taken together, these results indicate that, aside from a slight contamination by leak current with APC, similar results can be obtained when recording rescued Fdel-CFTR using either method.

B Corresponding distribution of current densities recorded at 0 mV. The recording sequence above is useful when determining the efficiency of candidate corrector molecules or comparing the potencies of several correctors.

However, with MPC, we are potentially able to collect information such as the stability of currents over long time periods and the temperature dependence of channel gating. Alternative application recorded with APC. Black lines are representative linear regressions of the time course curves before and after potentiator addition.

Means of slope values are indicated. Colored symbols are the mean of current densities and gray dashed lines the corresponding error, SEM. To study the stability of whole-cell currents, we performed APC whole-cell recordings on BHK cells over a min period. Such an experiment may be considered when assaying the ability of compounds to increase CFTR stability. In addition, the high quality of the seal and stability of the patch may allow the effects of sequential drug applications to be monitored and determination of EC 50 or IC To evaluate the potentiator effect of the tested compound, linear regression analyses were performed using the points on each time course curve.

For each individual experiment, points during the 2 min before and the 2 min after compound addition were used to calculate the slopes. The example in Figure 4B black lines shows representative linear regression before and after addition of VX Here, the mean slopes computed for eight different cells yielded values that are significantly different 0.

According to the literature Wang et al. Also, it suggests the feasibility of screening for compounds that correct the thermal instability of Fdel-CFTR.

However, as mentioned in Table 1 , we also tested APC with different cell types. Error bars, SEM; ns, not significant. Several cell-based screening assays for CFTR are available that monitor anion flux [e. Although they provide high throughput, they are only semi-quantitative because the net electrochemical across the membrane for the anion changes during the course of the assay. Here, we have shown that planar APC can be used when screening for CFTR modulators and provides medium throughput together with several advantages over other cell-based assays.

Optimizing the success rate is key to using these assays for drug testing. To increase this rate of experiment success and develop the use of more physiological cell lines i. The main difference was that CFTR currents recorded using APC were often contaminated with non-selective leak currents due to a decline in seal quality. Interestingly, APC can use the same protocols as MPC, making it useful for studies of current stability over long periods, the effects of sequential drug applications useful for EC 50 or IC 50 determination, precise measurement of potentiation , and the temperature dependence of channel gating CFTR modulators.

This last point is a real advantage compared to many HTS assay since it enables compounds to be tested at physiological temperature.

As already mentioned, mutant CFTR channels have altered temperature sensitivity, yet, most screening assays are performed at room temperature, which may explain some discrepancies between in vitro results and clinical trials. Once the conditions have been optimized, APC is faster and easier to use than MPC with similar high-resolution results and the possibility to address the same questions.

All steps during APC experiments are finely controlled and executed by a robot, allowing more consistent times and concentrations at which drugs are injected.

The use of microfluidics within the APC chips ensures that volumes are minimized and allows more precise control of the temperature.

A major drawback with APC is the requirement for cells in suspension, which limits the use of epithelial cell lines under physiological conditions, including CFBE, Calu-3, and the human colonic cell line T The normal state of these cells is highly polarized, with tight junctions and apical CFTR.

The effects of CFTR modulators may be influenced by the integrity of the epithelium and the maintenance of cell—cell contacts Fritsch and Edelman, The detachment protocol used in this study or the generation of cell lines adapted to suspension, may also adversely affect interactions between CFTR and its partners, altering channel function and regulation.

We have shown that whole-cell parallel planar APC is suitable for recording CFTR activity and gives robust results similar to those obtained by MPC but with higher throughput and standardized protocols.

Screening with APC is an attractive option for pharmacological investigations including hit validation, trafficking correction, potentiation, thermal stability restoration, and the functional characterization of mutant CFTR. AB and LF performed experiments. AB and LF analyzed the data. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Becq, F. Phosphatase inhibitors activate normal and defective CFTR chloride channels. Berger, H. Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases.

Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes. Malfunctioning of these channels has been implicated in human diseases such as lysosomal storage disorders, neurodegenerative diseases and metabolic pathologies, as well as in the progression of certain infectious diseases.

be widely considered the gold standard method to record ion channel activity. Patch-Clamp Methods and Protocols, Second Edition, provides a comprehens.

Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function

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Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes

Patch-Clamp Methods and Protocols

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The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells , tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells such as neurons , cardiomyocytes , muscle fibers , and pancreatic beta cells , and can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts. Patch clamping can be performed using the voltage clamp technique. In this case, the voltage across the cell membrane is controlled by the experimenter and the resulting currents are recorded. Alternatively, the current clamp technique can be used. In this case the current passing across the membrane is controlled by the experimenter and the resulting changes in voltage are recorded, generally in the form of action potentials.

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Patch-Clamp Methods and Protocols, Second Edition, provides a comprehensive collection of new techniques for the development of automated.

Patch clamp

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 - Он еще раз оглядел комнату. - Вас подбросить в аэропорт? - предложил лейтенант - Мой Мото Гуччи стоит у подъезда. - Спасибо, не стоит. Я возьму такси.  - Однажды в колледже Беккер прокатился на мотоцикле и чуть не разбился. Он больше не хотел искушать судьбу, кто бы ни сидел за рулем.

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Успокойся, Дэвид. Спокойно. Он оглядел пустой зал. Ни души. Продала кольцо и улетела.

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В связи с чрезвычайной обстановкой, в которой обычно осуществляется антитеррористическая деятельность АНБ, ТРАНСТЕКСТ станет независимым инструментом дешифровки, использование которого будет регулироваться исключительно самим АНБ. Энсей Танкадо был возмущен. Получалось, что АНБ фактически получило возможность вскрывать всю почту и затем пересылать ее без какого-либо уведомления. Это было все равно что установить жучки во все телефонные аппараты на земле. Стратмор попытался убедить Танкадо, что ТРАНСТЕКСТ - это орудие охраны правопорядка, но безуспешно: Танкадо продолжал настаивать на том, что это грубейшее нарушение гражданских прав.

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 О Боже, - прошептал.  - Ну и мерзавец этот Танкадо. ГЛАВА 110 Невидящими глазами Джабба смотрел на распечатку, которую ему вручила Соши. Он побледнел и вытер рукавом пот со лба. - Директор, у нас нет выбора.

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У нас… - Он нас сделал, - сказал Стратмор, не поднимая головы.  - Танкадо обманул всех. По его тону ей стало ясно, что он все понял. Вся ложь Танкадо о невскрываемом алгоритме… обещание выставить его на аукцион - все это было игрой, мистификацией. Танкадо спровоцировал АНБ на отслеживание его электронной почты, заставил поверить, что у него есть партнер, заставил скачать очень опасный файл.

Они сцепились.


16.05.2021 at 22:50 - Reply

It seems that you're in Germany.

JocelГ­n G.
20.05.2021 at 17:00 - Reply

Since its inception, patch-clamp has continued to be widely considered the gold standard method to record ion channel activity.

Alisa M.
23.05.2021 at 04:43 - Reply

The chloride Cl - channel cystic fibrosis transmembrane conductance regulator CFTR is defective in cystic fibrosis CF , and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature.

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