scholarly journals Application of Machine-Learning Methods to Recognize mitoBK Channels from Different Cell Types Based on the Experimental Patch-Clamp Results

2021 ◽  
Vol 22 (2) ◽  
pp. 840
Author(s):  
Monika Richter-Laskowska ◽  
Paulina Trybek ◽  
Piotr Bednarczyk ◽  
Agata Wawrzkiewicz-Jałowiecka
Keyword(s):  
Machine Learning ◽  
Patch Clamp ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Cell Types ◽  
Inner Mitochondrial Membrane ◽  
Patch Clamp Recording ◽  
K Nearest Neighbors ◽  
Channel Protein ◽  
Different Cell Types

(1) Background: In this work, we focus on the activity of large-conductance voltage- and Ca2+-activated potassium channels (BK) from the inner mitochondrial membrane (mitoBK). The characteristic electrophysiological features of the mitoBK channels are relatively high single-channel conductance (ca. 300 pS) and types of activating and deactivating stimuli. Nevertheless, depending on the isoformal composition of mitoBK channels in a given membrane patch and the type of auxiliary regulatory subunits (which can be co-assembled to the mitoBK channel protein) the characteristics of conformational dynamics of the channel protein can be altered. Consequently, the individual features of experimental series describing single-channel activity obtained by patch-clamp method can also vary. (2) Methods: Artificial intelligence approaches (deep learning) were used to classify the patch-clamp outputs of mitoBK activity from different cell types. (3) Results: Application of the K-nearest neighbors algorithm (KNN) and the autoencoder neural network allowed to perform the classification of the electrophysiological signals with a very good accuracy, which indicates that the conformational dynamics of the analyzed mitoBK channels from different cell types significantly differs. (4) Conclusion: We displayed the utility of machine-learning methodology in the research of ion channel gating, even in cases when the behavior of very similar microbiosystems is analyzed. A short excerpt from the patch-clamp recording can serve as a “fingerprint” used to recognize the mitoBK gating dynamics in the patches of membrane from different cell types.

scholarly journals Gnrh1-induced responses are indirect in female medaka Fsh cells

2019 ◽  
Author(s):  
Kjetil Hodne ◽  
Romain Fontaine ◽  
Eirill Ager-Wick ◽  
Finn-Arne Weltzien
Keyword(s):  
Patch Clamp ◽  
Adult Female ◽  
Reproductive Function ◽  
Cell Types ◽  
Gonadal Development ◽  
Pituitary Cells ◽  
Tissue Slices ◽  
Lh Cells ◽  
Different Cell Types

ABSTRACTReproductive function in vertebrates is stimulated by gonadotropin-releasing hormone (GnRH) that controls the synthesis and release of the two pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH, which regulates different stages of gonadal development, are produced by two different cell types in the fish pituitary, in contrast to mammals and birds, thus allowing the investigation of their differential regulation. In the present work, we show by fluorescentin situhybridization that Lh cells in adult female medaka express Gnrh receptors, whereas Fsh cells do not. This is confirmed by patch clamp recordings and cytosolic Ca2+measurements on dispersed pituitary cells, where Lh cells, but not Fsh cells, respond to Gnrh1 by increased action potential frequencies and cytosolic Ca2+levels. In contrast, both Fsh and Lh cells are able to respond electrically and by elevating the cytosolic Ca2+levels to Gnrh1 in brain-pituitary tissue slices. Using Ca2+uncaging in combination with patch clamp recordings and cytosolic Ca2+measurements, we show that Fsh and Lh cells form homo- and heterotypic networks in the pituitary. Taken together, these results show that the effects of Gnrh1 on Fsh release in adult female medaka is indirect, likely mediated via Lh cells.

Persistence: a new statistic for characterizing ion-channel activity

1995 ◽  
Vol 350 (1334) ◽  
pp. 353-367 ◽  
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Single Channel ◽  
Statistical Technique ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Conductance Level

We introduce and illustrate by examples a new statistical technique, the persistence function, for characterizing ion-channel activity in a single-channel patch-clamp recording. Persistence is a function of both current and time. It is the probability that the current is at a given level (conditional on it having been at that level at an earlier time). Viewed as a function of current it exhibits the prominent conductance levels present in the recording, and viewed as a function of time for a conductance level it portrays the kinetics at that level.

Recording of single γ-aminobutyrate- and acetylcholine-activated receptor channels translated by exogenous mRNA in Xenopus oocytes

1983 ◽  
Vol 218 (1213) ◽  
pp. 481-484 ◽  
Keyword(s):  
High Resolution ◽  
Patch Clamp ◽  
Xenopus Oocytes ◽  
Optic Lobe ◽  
Single Channel ◽  
Patch Clamp Recording ◽  
Chick Optic Lobe ◽  
Single Channel Currents ◽  
Channel Currents

High resolution (‘giga-seal’) patch clamp recording in Xenopus oocytes was used to measure single channel currents from ACh- and GABA-activated receptors. The proteins that make up these receptors had been translated from mRNA derived from, respectively, denervated cat muscle and chick optic lobe.

scholarly journals Q&A: using Patch-seq to profile single cells

BMC Biology ◽  
2017 ◽  
Vol 15 (1) ◽  
Author(s):  
Cathryn R. Cadwell ◽  
Rickard Sandberg ◽  
Xiaolong Jiang ◽  
Andreas S. Tolias
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
Patch Clamp ◽  
Single Cell ◽  
Single Cells ◽  
Cell Types ◽  
Patch Clamp Recording ◽  
Electrophysiological Properties ◽  
Whole Cell Patch Clamp ◽  
Single Cell Rna Sequencing

Abstract Individual neurons vary widely in terms of their gene expression, morphology, and electrophysiological properties. While many techniques exist to study single-cell variability along one or two of these dimensions, very few techniques can assess all three features for a single cell. We recently developed Patch-seq, which combines whole-cell patch clamp recording with single-cell RNA-sequencing and immunohistochemistry to comprehensively profile the transcriptomic, morphologic, and physiologic features of individual neurons. Patch-seq can be broadly applied to characterize cell types in complex tissues such as the nervous system, and to study the transcriptional signatures underlying the multidimensional phenotypes of single cells.

scholarly journals Regulation by Light of Plant Potassium Uptake through K Channels: Biochemical, Physiological and Biophysical Study

1995 ◽  
Author(s):  
Nava Moran ◽  
Richard Crain ◽  
Wolf-Dieter Reiter
Keyword(s):  
Patch Clamp ◽  
Red Light ◽  
Cell Types ◽  
Cell Swelling ◽  
Signaling Cascade ◽  
Patch Clamp Recording ◽  
Independent Manner ◽  
K Channels ◽  
Biophysical Study ◽  
Samanea Saman

The swelling of plant motor cells is regulated by various signals with almost unknown mediators. One of the obligatory steps in the signaling cascade is the activation of K+-influx channels -K+ channels activated by hyperpolarization (KH channels). We thus explored the regulation of these channels in our model system, motor cell protoplasts from Samanea saman, using patch-clamp in the "whole cell" configuration. (a) The most novel finding was that the activity of KH channels in situ varied with the time of the day, in positive correlation with cell swelling: in Extensor cells KH channels were active in the earlier part of the day, while in Flexor cells only during the later part of the day; (b) High internal pH promoted the activity of these channels in Extensor cells, opposite to the behavior of the equivalent channels in guard cells, but in conformity with the predicted behavior of the putative KH channel, cloned from S. saman recently; (c) HIgh external K+ concentration increased (KH channel currents in Flexor cells. BL depolarized the Flexor cells, as detected in cell-attached patch-clamp recording, using KD channels (the K+-efflux channels) as "voltage-sensing devices". Subsequent Red-Light (RL) pulse followed by Darkness, hyperpolarized the cell. We attribute these changes to the inhibition of the H+-pump by BL and its reactivation by RL, as they were abolished by an H+-pump inhibitor. BL increased also the activity KD channels, in a voltage-independent manner - in all probability by an independent signaling pathway. Blue-Light (BL), which stimulates shrinking of Flexor cells, evoked the IP3 signaling cascade (detected directly by IP3 binding assay), known to mobilize cytosolic Ca2+. Nevertheless, cytosolic Ca2+ . did not activate the KD channel in excised, inside-out patches. In this study we established a close functional similarity of the KD channels between Flexor and Extensior cells. Thus the differences in their responses must stem from different links to signaling in both cell types.

Effects of a spider toxin and its analogue on glutamate-activated currents in the hippocampal CA1 neuron after ischemia

1995 ◽  
Vol 74 (1) ◽  
pp. 218-225 ◽  
Author(s):  
H. Tsubokawa ◽  
K. Oguro ◽  
T. Masuzawa ◽  
T. Nakaima ◽  
N. Kawai
Keyword(s):  
Nmda Receptor ◽  
Patch Clamp ◽  
Pyramidal Neurons ◽  
Single Channel ◽  
Patch Clamp Recording ◽  
Ca1 Neurons ◽  
Ca1 Pyramidal Neurons ◽  
Spider Toxin ◽  
Single Channel Currents ◽  
Channel Currents

1. We studied the effects of polyamine toxins derived from a spider venom on CA1 pyramidal neurons in gerbil hippocampal slices by patch-clamp recording. Joro spider toxin (JSTX) and its synthetic analogue, 1-naphthyl acetyl spermine (Naspm), which are known to block non-N-methyl-D-aspartate (non-NMDA) receptor in a subunit specific manner, were used. 2. Naspm depressed the excitatory postsynaptic currents (EPSCs) mediated by non-NMDA receptor channels. A further reduction of EPSCs occurred with addition of 6-cyano-7-nitroquin-oxaline-2,3- dione (CNQX). Conversely, when CNQX was applied first, no further depression of EPSCs occurred on addition of Naspm, indicating that Naspm blocks a fraction of the CNQX-sensitive non-NMDA-receptor-mediated currents. 3. After ischemia, the time course of EPSCs of CA1 pyramidal neurons was slowed and Naspm depressed the slow EPSCs more strongly than those in control neurons. 4. Analysis of single-channel currents by outside-out patch-clamp recording from ischemic CA1 neurons revealed that Naspm blocked a subpopulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate- and kainate-induced single-channel currents. 5. Because the EPSCs in CA1 neurons after ischemia are mediated by Ca(2+)-permeable non-NMDA receptor-mediated conductances, the present results indicate that Naspm and JSTX are effective at blocking abnormal EPSCs that may induce Ca2+ accumulation leading to delayed neuronal death after transient ischemic insult.

Calcium-dependent regulation of cholecystokinin secretion and potassium currents in STC-1 cells

1993 ◽  
Vol 264 (6) ◽  
pp. G1031-G1036 ◽  
Author(s):  
A. W. Mangel ◽  
N. D. Snow ◽  
M. A. Misukonis ◽  
S. Basavappa ◽  
J. P. Middleton ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Reversal Potential ◽  
Barium Chloride ◽  
Cytosolic Calcium ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Electrophysiological Properties ◽  
Calcium Dependent ◽  
Control Value

Secretory and electrophysiological properties of STC-1 cells, a cholecystokinin-secreting cell line, were examined with a radioimmunoassay and patch-clamp recording techniques. Stimulation of cholecystokinin secretion was seen after exposure to agents anticipated to increase the level of intracellular calcium, including thapsigargin (8 microM), bombesin (50 nM), potassium-induced depolarization (50 mM), or after blockade of potassium channels with barium chloride (2 mM). The secretory effects of these agents were blocked by pretreatment with the calcium channel blocker diltiazem (1 microM). Whole cell patch-clamp recordings showed a hyperpolarizing shift in reversal potential after exposure to either thapsigargin (8 microM) or bombesin (50 nM) from a control value of -27 +/- 3 to -57 +/- 7 or -48 +/- 6 mV, respectively. This shift was in the direction of the reversal potential for potassium and was blocked by barium chloride (5 mM). Single-channel recordings from cell-attached membrane patches showed an inwardly rectifying potassium channel with channel open probability modulated by bombesin. These results indicate that in STC-1 cells a potassium current is increased by agents that stimulate CCK secretion, presumably by increasing the level of cytosolic calcium. STC-1 cells may serve as a model system to study the electrophysiological and secretory mechanisms involved in the release of cholecystokinin.

scholarly journals Cooperative Gating between Single HCN Pacemaker Channels

2006 ◽  
Vol 128 (5) ◽  
pp. 561-567 ◽  
Author(s):  
John P. Dekker ◽  
Gary Yellen
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Noise Analysis ◽  
Cell Types ◽  
Hcn Channels ◽  
Voltage Range ◽  
Channel Current ◽  
Central Neurons ◽  
Pacemaker Channels ◽  
Single Channel Analysis

HCN pacemaker channels (If, Iq, or Ih) play a fundamental role in the physiology of many excitable cell types, including cardiac myocytes and central neurons. While cloned HCN channels have been studied extensively in macroscopic patch clamp experiments, their extremely small conductance has precluded single channel analysis to date. Nevertheless, there remain fundamental questions about HCN gating that can be resolved only at the single channel level. Here we present the first detailed single channel study of cloned mammalian HCN2. Excised patch clamp recordings revealed discrete hyperpolarization-activated, cAMP-sensitive channel openings with amplitudes of 150–230 fA in the activation voltage range. The average conductance of these openings was ∼1.5 pS at −120 mV in symmetrical 160 mM K+. Some traces with multiple channels showed unusual gating behavior, characterized by a variable long delay after a voltage step followed by runs of openings. Noise analysis on macroscopic currents revealed fluctuations whose magnitudes were systematically larger than predicted from the actual single channel current size, consistent with cooperativity between single HCN channels.

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