scholarly journals Novel 384-Well Population Patch Clamp Electrophysiology Assays for Ca2+-Activated K+ Channels

2006 ◽  
Vol 12 (1) ◽  
pp. 50-60 ◽  
Author(s):  
Victoria H. John ◽  
Tim J. Dale ◽  
Emma C. Hollands ◽  
Mao Xiang Chen ◽  
Leanne Partington ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Rank Order ◽  
Large Cell ◽  
Activator Concentration ◽  
Response Curves ◽  
Channel Expression ◽  
Planar Array ◽  
Patch Clamp Electrophysiology ◽  
Assay Precision ◽  
Voltage Gated Ion Channels

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK—expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 μM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z′ values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorksHT) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non—voltage-gated ion channels.

scholarly journals An assessment of KIR channel function in human cerebral arteries

2019 ◽  
Vol 316 (4) ◽  
pp. H794-H800 ◽  
Author(s):  
Maria Sancho ◽  
Yuan Gao ◽  
Bjorn O. Hald ◽  
Hao Yin ◽  
Melfort Boulton ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Patch Clamp ◽  
Cerebral Arteries ◽  
Channel Expression ◽  
Expression Activity ◽  
Patch Clamp Electrophysiology ◽  
Resting Tone ◽  
The Impact ◽  
Arterial Tone

In the rodent cerebral circulation, inward rectifying K+ (KIR) channels set resting tone and the distance over which electrical phenomena spread along the arterial wall. The present study sought to translate these observations into human cerebral arteries obtained from resected brain tissue. Computational modeling and a conduction assay first defined the impact of KIR channels on electrical communication; patch-clamp electrophysiology, quantitative PCR, and immunohistochemistry then characterized KIR2.x channel expression/activity. In keeping with rodent observations, computer modeling highlighted that KIR blockade should constrict cerebral arteries and attenuate electrical communication if functionally expressed. Surprisingly, Ba2+ (a KIR channel inhibitor) had no effect on human cerebral arterial tone or intercellular conduction. In alignment with these observations, immunohistochemistry and patch-clamp electrophysiology revealed minimal KIR channel expression/activity in both smooth muscle and endothelial cells. This absence may be reflective of chronic stress as dysphormic neurons, leukocyte infiltrate, and glial fibrillary acidic protein expression was notable in the epileptic cortex. In closing, KIR2.x channel expression is limited in human cerebral arteries from patients with epilepsy and thus has little impact on resting tone or the spread of vasomotor responses. NEW & NOTEWORTHY KIR2.x channels are expressed in rodent cerebral arterial smooth muscle and endothelial cells. As they are critical to setting membrane potential and the distance signals conduct, we sought to translate this work into humans. Surprisingly, KIR2.x channel activity/expression was limited in human cerebral arteries, a paucity tied to chronic brain stress in the epileptic cortex. Without substantive expression, KIR2.x channels were unable to govern arterial tone or conduction.

scholarly journals Population Patch-Clamp Electrophysiology Analysis of Recombinant GABAA α1β3γ2 Channels Expressed in HEK-293 Cells

2009 ◽  
Vol 14 (7) ◽  
pp. 769-780 ◽  
Author(s):  
Emma C. Hollands ◽  
Tim J. Dale ◽  
Andrew W. Baxter ◽  
Helen J. Meadows ◽  
Andrew J. Powell ◽  
...  
Keyword(s):  
Patch Clamp ◽  
High Throughput Screening ◽  
Rational Design ◽  
Rank Order ◽  
Hek293 Cells ◽  
Small Scale ◽  
Single Shot ◽  
Hek 293 ◽  
Planar Array ◽  
Receptor Pharmacology

γ-Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human α1β3γ2 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a series of agonists, the rank order of potency was muscimol > GABA > isoguvacine. A range of known positive modulators, including diazepam and pentobarbital, produced concentration-dependent augmentation of the GABA EC 20 response (1 µM). The competitive antagonists bicuculline and gabazine produced concentration-dependent, parallel, rightward displacement of GABA curves with pA2 and slope values of 5.7 and 1.0 and 6.7 and 1.0, respectively. In contrast, picrotoxin (0.2-150 µM) depressed the maximal GABA response, implying a non-competitive antagonism. Overall, the pharmacology of human α1β3γ2 GABAA determined by PPC was highly similar to that obtained by conventional patch-clamp methods. In small-scale single-shot screens, Z′ values of >0.5 were obtained in agonist, modulator, and antagonist formats with hit rates of 0% to 3%. The authors conclude that despite the inability of the method to resolve the peak agonist responses, PPC can rapidly and usefully quantify pharmacology for the α1β3γ2 GABAA isoform. These data suggest that PPC may be a valuable approach for a focused set and secondary screening of GABAA receptors and other slow ligand-gated ion channels. ( Journal of Biomolecular Screening 2009:769-780)

scholarly journals Progress in automating patch clamp cellular physiology

2018 ◽  
Vol 2 ◽  
pp. 239821281877656 ◽  
Author(s):  
Luca A. Annecchino ◽  
Simon R. Schultz
Keyword(s):  
Patch Clamp ◽  
Cellular Mechanisms ◽  
Biologically Relevant ◽  
Synaptic Physiology ◽  
Complex Information ◽  
Electrophysiological Recordings ◽  
Planar Array ◽  
Patch Clamp Electrophysiology ◽  
Neuronal Computation

Patch clamp electrophysiology has transformed research in the life sciences over the last few decades. Since their inception, automatic patch clamp platforms have evolved considerably, demonstrating the capability to address both voltage- and ligand-gated channels, and showing the potential to play a pivotal role in drug discovery and biomedical research. Unfortunately, the cell suspension assays to which early systems were limited cannot recreate biologically relevant cellular environments, or capture higher order aspects of synaptic physiology and network dynamics. In vivo patch clamp electrophysiology has the potential to yield more biologically complex information and be especially useful in reverse engineering the molecular and cellular mechanisms of single-cell and network neuronal computation, while capturing important aspects of human disease mechanisms and possible therapeutic strategies. Unfortunately, it is a difficult procedure with a steep learning curve, which has restricted dissemination of the technique. Luckily, in vivo patch clamp electrophysiology seems particularly amenable to robotic automation. In this review, we document the development of automated patch clamp technology, from early systems based on multi-well plates through to automated planar-array platforms, and modern robotic platforms capable of performing two-photon targeted whole-cell electrophysiological recordings in vivo.

scholarly journals Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Chai-Ann Ng ◽  
Jessica Farr ◽  
Paul Young ◽  
Monique J Windley ◽  
Matthew D Perry ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Lines ◽  
High Throughput ◽  
Rapid Assessment ◽  
Cost Effective ◽  
Missense Variant ◽  
Loss Of Function ◽  
Channel Expression ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology

Abstract KCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bi-cistronic vector to co-express Wild-type (WT) and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch clamp electrophysiology analysis of channel function. Stable cell lines take 3–4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 h of channel expression. This high-throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

scholarly journals Heterozygous KCNH2 variant phenotyping using Flp-In HEK293 and high-throughput automated patch clamp electrophysiology

2021 ◽  
Author(s):  
Chai-Ann Ng ◽  
Jessica Farr ◽  
Paul Young ◽  
Monique J. Windley ◽  
Matthew D. Perry ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cell Lines ◽  
High Throughput ◽  
Rapid Assessment ◽  
Cost Effective ◽  
Missense Variant ◽  
Loss Of Function ◽  
Channel Expression ◽  
Automated Patch Clamp ◽  
Patch Clamp Electrophysiology

AbstractKCNH2 is one of the 59 medically actionable genes recommended by the American College of Medical Genetics for reporting of incidental findings from clinical genomic sequencing. However, half of the reported KCNH2 variants in the ClinVar database are classified as variants of uncertain significance. In the absence of strong clinical phenotypes, there is a need for functional phenotyping to help decipher the significance of variants identified incidentally. Here, we report detailed methods for assessing the molecular phenotype of any KCNH2 missense variant. The key components of the assay include quick and cost-effective generation of a bicistronic vector to co-express WT and any KCNH2 variant allele, generation of stable Flp-In HEK293 cell lines and high-throughput automated patch-clamp electrophysiology analysis of channel function. Stable cell lines take 3-4 weeks to produce and can be generated in bulk, which will then allow up to 30 variants to be phenotyped per week after 48 hours of channel expression. This high throughput functional genomics assay will enable a much more rapid assessment of the extent of loss of function of any KCNH2 variant.

scholarly journals Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1716
Author(s):  
Kun Tong ◽  
Ruotian Zhang ◽  
Fengzhi Ren ◽  
Tao Zhang ◽  
Junlin He ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Formalin Test ◽  
Sodium Ion ◽  
Inhibitory Potency ◽  
Voltage Gated ◽  
Patch Clamp Electrophysiology ◽  
Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

scholarly journals Isolating Nuclei from Cultured Cells for Patch-Clamp Electrophysiology of Intracellular Ca 2+ Channels

2013 ◽  
Vol 2013 (9) ◽  
pp. pdb.prot073056 ◽  
Author(s):  
Don-On Daniel Mak ◽  
Horia Vais ◽  
King-Ho Cheung ◽  
J. Kevin Foskett
Keyword(s):  
Patch Clamp ◽  
Cultured Cells ◽  
Intracellular Ca ◽  
Patch Clamp Electrophysiology

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology

2000 ◽  
Vol 278 (5) ◽  
pp. C1055-C1061 ◽  
Author(s):  
Tim Robinson ◽  
Lars Thomsen ◽  
Jan D. Huizinga
Keyword(s):  
Patch Clamp ◽  
Data Acquisition ◽  
Front Panel ◽  
Analysis Program ◽  
Digital To Analog Converter ◽  
Analog Converter ◽  
Data Acquisition Card ◽  
System Requirements ◽  
User Need ◽  
Patch Clamp Electrophysiology

An acquisition and analysis program, “LabPatch,” has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/ .

scholarly journals Involvement of NO in Antinociception of NSAIDS in Murine Formalin Hind Paw Assay

Drug Research ◽  
2020 ◽  
Vol 70 (04) ◽  
pp. 145-150 ◽  
Author(s):  
Viviana Noriega ◽  
Hugo F. Miranda ◽  
Juan Carlos Prieto ◽  
Ramón Sotomayor-Zárate ◽  
Fernando Sierralta
Keyword(s):  
Phase I ◽  
Phase Ii ◽  
Dose Response ◽  
Rank Order ◽  
Linear Regression Analysis ◽  
Second Phase ◽  
Response Curves ◽  
Before And After ◽  
Two Phases ◽  
Phase Phase

AbstractThere are different animal models to evaluate pain among them the formalin hind paw assay which is widely used since some of its events appear to be similar to the clinical pain of humans. The assay in which a dilute solution of formalin is injected into the dorsal hindpaw of a murine produces two ‘phases’ of pain behavior separated by a inactive period. The early phase (Phase I) is probably due to direct activation of nociceptors and the second phase (Phase II) is due to ongoing inflammatory input and central sensitization. Mice were used to determine the potency antinociceptive of piroxicam (1,3,10,and 30 mg/kg), parecoxib (0.3, 1,3,10 and 30 mg/kg), dexketoprofen (3,10,30 and 100 mg/kg) and ketoprofen (3,10,30 and 100 mg/kg). Dose-response for each NSAIDs were created before and after 5 mg/kg of L-NAME i.p. or 5 mg/kg i.p. of 7-nitroindazole. A least-squares linear regression analysis of the log dose–response curves allowed the calculation of the dose that produced 50% of antinociception (ED50) for each drug. The ED50 demonstrated the following rank order of potency, in the phase I: piroxicam > dexketoprofen > ketoprofen > parecoxib and in the phase II: piroxicam > ketoprofen > parecoxib > dexketoprofen. Pretreatment of the mice with L-NAME or 7-nitroindazol induced a significant increase of the analgesic power of the NSAIDs, with a significant reduction of the ED50. It is suggested that NO may be involved in both phases of the trial, which means that nitric oxide regulates the bioactivity of NSAIDs.

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