Analysis of KATP Channels Opening Probability of Hippocampus Cells Treated with Kainic Acid

Background: Kainic acid (KA)-induced seizures may be a valuable tool in the assessment of anti-epileptic drug efficacy in complex partial seizures. This study investigated the effects of KA on ATP-sensitive K+ (KATP) channels opening probability (NPo), which plays a crucial role in neuronal activities. Methods: For the optimisation and validation protocol, β-cells were plated onto 35 mm plastic petri dishes and maintained in RPMI-1640 media supplemented with 10 mM glucose, 10% FCS and 25 mM of N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES). The treatment effects of 10 mM glucose and 30 μM fluoxetine on KATP channels NPo of β–cells were assessed via cell-attached patch-clamp recordings. For hippocampus cell experiments, hippocampi were harvested from day 17 of maternal Lister-hooded rat foetus, and then transferred to a Ca2+ and Mg2+-free HEPES-buffered Hank’s salt solution (HHSS). The dissociated cells were cultured and plated onto a 25 mm round cover glasses coated with poly-d-lysine (0.1 mg/mL) in a petri dish. The KATP channels NPo of hippocampus cells when perfused with 1 mM and 10 mM of KA were determined. Results: NPo of β-cells showed significant decreasing patterns (P < 0.001) when treated with 10 mM glucose 0.048 (0.027) as well as 30 μM fluoxetine 0.190 (0.141) as compared to basal counterpart. In hippocampus cell experiment, a significant increase (P < 0.001) in mean NPo 2.148 (0.175) of neurons when applied with 1 mM of KA as compared to basal was observed. Conclusion: The two concentrations of KA used in the study exerted contrasting effects toward the mean of NPo. It is hypothesised that KA at lower concentration (1 mM) opens more KATP channels, leading to hyperpolarisation of the neurons, which may prevent neuronal hyper excitability. No effect was shown in 10 mM KA treatment, suggesting that only lower than 10 mM KA produced significant changes in KATP channels. This implies further validation of KA concentration to be used in the future.

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Relationship Between the Pyroptosis Pathway and Epilepsy: A Bioinformatic Analysis

Frontiers in Neurology ◽

10.3389/fneur.2021.782739 ◽

2022 ◽

Vol 12 ◽

Author(s):

Lu Xia ◽

Lu Liu ◽

Qiang Wang ◽

Jing Ding ◽

Xin Wang

Keyword(s):

Correlation Analysis ◽

Kainic Acid ◽

Sham Group ◽

Differentially Expressed Gene ◽

Bioinformatic Analysis ◽

Go Annotation ◽

Protein Levels ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Anti Epileptic Drug

PurposeThis study aimed to analyse the correlation between the pyroptosis pathway and epilepsy using bioinformatics analysis technology. We analyzed the expression of gasdermin D (GSDMD) and gasdermin E (GSDME), the key molecules of pyroptosis, in kainic acid-induced epileptic mice.MethodsWeighted gene co-expression network analysis (WGCNA) was used to construct a signed co-expression network from expression data to screen gene sets closely related to epilepsy. The correlation between the module and epilepsy was verified through module conservative analysis, gene ontology (GO) annotation analysis, and correlation analysis with known epilepsy genes. We obtained currently recognized pyroptosis-related molecules through literature review, and correlation analysis was used to evaluate their correlation with epilepsy. Differentially expressed gene (DEG) analysis was used to analyse expression changes of pyroptosis-related molecules at the transcriptome level, compared to the sham group. We subsequently established a kainic acid-induced status epilepticus (SE) model in mice and validated the mRNA and protein expression of GSDMD and GSDME, the key molecules of pyroptosis, by quantitative reverse transcription PCR (qRT-PCR) and western blotting (WB).ResultsUsing WGCNA, module conservative analysis, and correlation analysis with known epilepsy genes, we screened out a module (a gene set of interest) closely related to epilepsy that was prominently enriched in immune and inflammatory-related biological processes. Correlation analysis results suggest that pyroptosis-related molecules are closely related to this module, but have no obvious correlation with others. DEG analysis of molecules associated with pyroptosis suggests that most of the pyroptosis-related molecules had significantly increased expression after SE, such as IL1b, Casp1, Casp4, Pycard, Gsdmd, Nlrp3, Aim2, Mefv, Tlr2, Tlr3, and Tlr4. qRT-PCR and WB analysis confirmed that the mRNA and protein levels of GSDMD in the mouse hippocampus were significantly upregulated after SE. The mRNA expression of GSDME was not different between the epilepsy group and sham group. However, the WB results showed that the expression of full-length GSDME was decreased and GSDME-N-terminus were significantly increased after SE.ConclusionsOur study highlights that the pyroptosis pathway may be closely related to epilepsy. GSDMD and GSDME, the key executive molecules of pyroptosis, will help to understand the pathogenesis of epilepsy and aid in discovering new targets for anti-epileptic drug treatments.

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Pharmacological modulation of KATP channels

Biochemical Society Transactions ◽

10.1042/bst0300333 ◽

2002 ◽

Vol 30 (2) ◽

pp. 333-339 ◽

Cited By ~ 43

Author(s):

F. M. Gribble ◽

F. Reimann

Keyword(s):

Type 2 Diabetes ◽

Cardiac Muscle ◽

Katp Channel ◽

Katp Channels ◽

Β Cells ◽

Pancreatic Β Cells ◽

Tissue Specific ◽

Pharmacological Modulation ◽

Clinical Conditions

Pharmacological modulation of ATP-sensitive K+ (KATP) channels is used in the treatment of a number of clinical conditions, including type 2 diabetes and angina. The sulphonylureas and related drugs, which are used to treat type 2 diabetes, stimulate insulin secretion by closing KATP channels in pancreatic β-cells. Agents used to treat angina, by contrast, act by opening KATP channels in vascular smooth and cardiac muscle. Both the therapeutic KATP channel inhibitors and the KATP channel openers target the sulphonylurea receptor (SUR) subunit of the KATP channel, which exists in several isoforms expressed in different tissues (SUR1 in pancreatic β-cells, SUR2A in cardiac muscle and SUR2B in vascular smooth muscle). The tissue-specific action of drugs that target the KATP channel is attributed to the properties of these different SUR subtypes. In this review, we discuss the molecular basis of tissue-specific drug action, and its implications for clinical practice.

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Diabetes induced by gain-of-function mutations in the Kir6.1 subunit of the KATP channel

The Journal of General Physiology ◽

10.1085/jgp.201611653 ◽

2016 ◽

Vol 149 (1) ◽

pp. 75-84 ◽

Cited By ~ 4

Author(s):

Maria S. Remedi ◽

Jonathan B. Friedman ◽

Colin G. Nichols

Keyword(s):

Insulin Secretion ◽

Katp Channel ◽

Vascular System ◽

Wild Type Mouse ◽

Neonatal Diabetes ◽

Katp Channels ◽

Β Cells ◽

Pancreatic Β Cells ◽

Gain Of Function ◽

Insulin Promoter

Gain-of-function (GOF) mutations in the pore-forming (Kir6.2) and regulatory (SUR1) subunits of KATP channels have been identified as the most common cause of human neonatal diabetes mellitus. The critical effect of these mutations is confirmed in mice expressing Kir6.2-GOF mutations in pancreatic β cells. A second KATP channel pore-forming subunit, Kir6.1, was originally cloned from the pancreas. Although the prominence of this subunit in the vascular system is well documented, a potential role in pancreatic β cells has not been considered. Here, we show that mice expressing Kir6.1-GOF mutations (Kir6.1[G343D] or Kir6.1[G343D,Q53R]) in pancreatic β cells (under rat-insulin-promoter [Rip] control) develop glucose intolerance and diabetes caused by reduced insulin secretion. We also generated transgenic mice in which a bacterial artificial chromosome (BAC) containing Kir6.1[G343D] is incorporated such that the transgene is only expressed in tissues where Kir6.1 is normally present. Strikingly, BAC-Kir6.1[G343D] mice also show impaired glucose tolerance, as well as reduced glucose- and sulfonylurea-dependent insulin secretion. However, the response to K+ depolarization is intact in Kir6.1-GOF mice compared with control islets. The presence of native Kir6.1 transcripts was demonstrated in both human and wild-type mouse islets using quantitative real-time PCR. Together, these results implicate the incorporation of native Kir6.1 subunits into pancreatic KATP channels and a contributory role for these subunits in the control of insulin secretion.

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Electrophysiological Characterization of Pancreatic Islet Cells in the Mouse Insulin Promoter-Green Fluorescent Protein Mouse

Endocrinology ◽

10.1210/en.2005-0803 ◽

2005 ◽

Vol 146 (11) ◽

pp. 4766-4775 ◽

Cited By ~ 53

Author(s):

Yuk M. Leung ◽

Ishtiaq Ahmed ◽

Laura Sheu ◽

Robert G. Tsushima ◽

Nicholas E. Diamant ◽

...

Keyword(s):

Fluorescent Protein ◽

Katp Channels ◽

Β Cells ◽

Β Cell ◽

Mouse Islet ◽

Channel Density ◽

Na Currents ◽

Insulin Promoter ◽

Green Fluorescent ◽

Ca2 Channels

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet β-cells to enable convenient identification of β-cells as green cells. The GFP-expressing β-cells of the MIP-GFP mouse were functionally indistinguishable from β-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet β- and α-cells. β-Cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. α-Cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at −70 mV holding potential. α-Cells had ATP-sensitive K+ channel (KATP) channel density as high as that in β-cells, and, surprisingly, α-cell KATP channels were more sensitive to ATP inhibition (IC50 = 0.16 ± 0.03 mm) than β-cell KATP channels (IC50 = 0.86 ± 0.10 mm). Whereas α-cells were rather uniform in size [2–4.5 picofarad (pF)], β-cells varied vastly in size (2–12 pF). Of note, small β-cells (<4.5 pF) showed little exocytosis, whereas medium β-cells (5–8 pF) exhibited vigorous exocytosis, but large β-cells (>8 pF) had weaker exocytosis. We found no correlation between β-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in β-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine α-cell and β-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

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Validation of the Zebra-fish Pentylene Tetrazol Seizure Model: Behaviour Assay for Assessing Anti-Epileptic Drug Efficacy

Biochemistry & Analytical Biochemistry ◽

10.4172/2161-1009.1000270 ◽

2016 ◽

Vol 5 (2) ◽

Cited By ~ 1

Author(s):

Chongbin Liu ◽

Juan Zhou

Keyword(s):

Drug Efficacy ◽

Zebra Fish ◽

Anti Epileptic Drug ◽

Seizure Model

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Glucose elicits cephalic-phase insulin release in mice by activating KATP channels in taste cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00433.2016 ◽

2017 ◽

Vol 312 (4) ◽

pp. R597-R610 ◽

Cited By ~ 23

Author(s):

John I. Glendinning ◽

Yonina G. Frim ◽

Ayelet Hochman ◽

Gabrielle S. Lubitz ◽

Anthony J. Basile ◽

...

Keyword(s):

Signaling Pathway ◽

Insulin Release ◽

Katp Channels ◽

Glucose Sensing ◽

Oral Glucose ◽

Β Cells ◽

Oral Stimulation ◽

Cephalic Phase ◽

Taste Cells ◽

A Subunit

The taste of sugar elicits cephalic-phase insulin release (CPIR), which limits the rise in blood glucose associated with meals. Little is known, however, about the gustatory mechanisms that trigger CPIR. We asked whether oral stimulation with any of the following taste stimuli elicited CPIR in mice: glucose, sucrose, maltose, fructose, Polycose, saccharin, sucralose, AceK, SC45647, or a nonmetabolizable sugar analog. The only taste stimuli that elicited CPIR were glucose and the glucose-containing saccharides (sucrose, maltose, Polycose). When we mixed an α-glucosidase inhibitor (acarbose) with the latter three saccharides, the mice no longer exhibited CPIR. This revealed that the carbohydrates were hydrolyzed in the mouth, and that the liberated glucose triggered CPIR. We also found that increasing the intensity or duration of oral glucose stimulation caused a corresponding increase in CPIR magnitude. To identify the components of the glucose-specific taste-signaling pathway, we examined the necessity of Calhm1, P2X2+P2X3, SGLT1, and Sur1. Among these proteins, only Sur1 was necessary for CPIR. Sur1 was not necessary, however, for taste-mediated attraction to sugars. Given that Sur1 is a subunit of the ATP-sensitive K+ channel (KATP) channel and that this channel functions as a part of a glucose-sensing pathway in pancreatic β-cells, we asked whether the KATP channel serves an analogous role in taste cells. We discovered that oral stimulation with drugs known to increase (glyburide) or decrease (diazoxide) KATP signaling produced corresponding changes in glucose-stimulated CPIR. We propose that the KATP channel is part of a novel signaling pathway in taste cells that mediates glucose-induced CPIR.

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Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00016.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. E293-E301 ◽

Cited By ~ 15

Author(s):

Xuehui Geng ◽

Lehong Li ◽

Rita Bottino ◽

A. N. Balamurugan ◽

Suzanne Bertera ◽

...

Keyword(s):

Plasma Membrane ◽

Insulin Secretion ◽

Calcium Influx ◽

Katp Channels ◽

Second Phase ◽

Β Cells ◽

High Potassium ◽

Transient Rise ◽

Atp Inhibition ◽

Promising Treatment

Understanding mechanisms by which glibenclamide stimulates insulin release is important, particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Antidiabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high-affinity ATP-sensitive potassium (KATP) channel receptors at the plasma membrane of β-cells. This normally occurs during glucose stimulation, where ATP inhibition of plasmalemmal KATP channels leads to voltage activation of L-type calcium channels for rapidly switching on and off calcium influx, governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas, including glibenclamide, have additional KATP channel receptors within β-cells at insulin granules. We tested nonpermeabilized β-cells in mouse islets for glibenclamide-stimulated insulin secretion mediated by granule-localized KATP channels by using conditions that bypass glibenclamide action on plasmalemmal KATP channels. High-potassium stimulation evoked a sustained rise in β-cell calcium level but a transient rise in insulin secretion. With continued high-potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule-localized KATP channels that functionally contribute to sustained second-phase insulin secretion.

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The effect of gliquidone on KATP channels in pancreatic β-cells, cardiomyocytes, and vascular smooth muscle cells

Diabetes Research and Clinical Practice ◽

10.1016/j.diabres.2015.05.036 ◽

2015 ◽

Vol 109 (2) ◽

pp. 334-339 ◽

Cited By ~ 5

Author(s):

Shu-Yi Liu ◽

Hao-Min Tian ◽

Da-Qing Liao ◽

Yan-Fang Chen ◽

Zhong-Ping Gou ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Katp Channels ◽

Muscle Cells ◽

Β Cells ◽

Pancreatic Β Cells

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Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating

eLife ◽

10.7554/elife.24149 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 96

Author(s):

Gregory M Martin ◽

Craig Yoshioka ◽

Emily A Rex ◽

Jonathan F Fay ◽

Qing Xie ◽

...

Keyword(s):

Symmetry Axis ◽

Transmembrane Domain ◽

Katp Channels ◽

Structural Basis ◽

Channel Activity ◽

Membrane Excitability ◽

Β Cells ◽

Pancreatic Β Cells ◽

Binding Domains ◽

Channel Assembly

KATP channels are metabolic sensors that couple cell energetics to membrane excitability. In pancreatic β-cells, channels formed by SUR1 and Kir6.2 regulate insulin secretion and are the targets of antidiabetic sulfonylureas. Here, we used cryo-EM to elucidate structural basis of channel assembly and gating. The structure, determined in the presence of ATP and the sulfonylurea glibenclamide, at ~6 Å resolution reveals a closed Kir6.2 tetrameric core with four peripheral SUR1s each anchored to a Kir6.2 by its N-terminal transmembrane domain (TMD0). Intricate interactions between TMD0, the loop following TMD0, and Kir6.2 near the proposed PIP2 binding site, and where ATP density is observed, suggest SUR1 may contribute to ATP and PIP2 binding to enhance Kir6.2 sensitivity to both. The SUR1-ABC core is found in an unusual inward-facing conformation whereby the two nucleotide binding domains are misaligned along a two-fold symmetry axis, revealing a possible mechanism by which glibenclamide inhibits channel activity.

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