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Paxilline

Manufactured by Merck Group
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Sourced in United States, Italy

Paxilline is a lab equipment product manufactured by Merck Group. It is a chemical compound that functions as a potent inhibitor of large-conductance calcium-activated potassium (BK) channels. The core function of Paxilline is to serve as a research tool for investigating the role of BK channels in various biological processes.

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Lab products found in correlation

Ns1619, by Merck Group (6 mentions) Dimethyl sulfoxide (dmso), by Merck Group (6 mentions) Thapsigargin, by Merck Group (5 mentions) Nifedipine, by Merck Group (4 mentions) Pertussis toxin, by Merck Group (3 mentions) Apamin, by Merck Group (3 mentions) Tram 34, by Merck Group (3 mentions) Ska 31, by Merck Group (3 mentions) Bp1600 100, by Thermo Fisher Scientific (3 mentions) Tea cl, by Merck Group (2 mentions) Ruthenium red, by Merck Group (2 mentions) Heparin, by Merck Group (2 mentions) Digitonin, by Merck Group (2 mentions) Mabn70, by Merck Group (2 mentions) Phospho creb, by Cell Signaling Technology (2 mentions) Phospho erk, by Cell Signaling Technology (2 mentions) Sto 609, by Merck Group (2 mentions) X rhod 1, by Thermo Fisher Scientific (2 mentions) Phospho camkiv, by Santa Cruz Biotechnology (2 mentions) Sc 28443 r, by Santa Cruz Biotechnology (2 mentions)

29 protocols using paxilline

1

Paxilline and MTS reagent handling

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Salts and paxilline were obtained from Sigma-Aldrich. The details of the handling of paxilline were as described previously (Zhou et al., 2010 (link)). 20-mM stock solutions of paxilline were prepared in 100% DMSO and stored at −20°C. paxilline-containing physiological solutions were prepared the day of an experiment and only used up to 4 h after preparation. [2-(trimethylammonium)ethyl] MTS bromide (MTSET), 2-aminoethyl MTS (MTSEA), and sodium (2-sulfanoethyl) MTS (MTSES) were obtained from Toronto Research Chemicals.
Zhou Y, & Lingle C.J. (2014). Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism. The Journal of General Physiology, 144(5), 415-440.
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2

Reconstitution of Bioactive Compounds

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AVP, Leu8-OT, and Pro8-OT Anaspec 58863 were reconstituted in DMSO Sigma-Aldrich D4540. NS-1619 Sigma-Aldrich N170, Paxilline Sigma-Aldrich P2928, SKA-31 Sigma-Aldrich S5573, thapsigargin Sigma-Aldrich T9033, and TRAM-34 Sigma-Aldrich T6700 were reconstituted in DMSO. Pertussis toxin Sigma-Aldrich P7208 was reconstituted in ultrapure water with 5 mg/mL bovine serum albumin Fisher Scientific BP1600-100. Dynorphin A (1–13) amide (American Peptide 26-4-51A) was dissolved in 25 mM Tris at pH 7.4. Apamin (Sigma-Aldrich A1289) was reconstituted in 0.05 M acetic acid.
Pierce M.L., French J.A, & Murray T.F. (2020). Comparison of the pharmacologic profiles of arginine vasopressin and oxytocin analogs at marmoset, titi monkey, macaque, and human oxytocin receptors. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 125, 109832.
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3

Measuring BK Channel-Mediated AHP Currents

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The IAHP currents were evoked in voltage-clamp by holding the cell at −50 mV then depolarizing to +25 mV for 20 and 50 ms in order to elicit a maximum level of BK channel-mediated current. We determined the absolute IAHP amplitude as the difference between the pre-pulse baseline and the peak of the tail current following the step offset. Peak IAHP was compared between control and drug, either paxilline (10 μM, Sigma-Aldrich), iberiotoxin (100 nM, Sigma-Aldrich), rapamycin (400 nM, Tocris) or TBS using a paired t-test (control and drug).
Springer S.J., Burkett B.J, & Schrader L.A. (2015). Modulation of BK channels contributes to activity-dependent increase of excitability through MTORC1 activity in CA1 pyramidal cells of mouse hippocampus. Frontiers in Cellular Neuroscience, 8, 451.
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4

Reconstitution of Bioactive Compounds

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AVP, Leu8-OT, and Pro8-OT Anaspec 58863 were reconstituted in DMSO Sigma-Aldrich D4540. NS-1619 Sigma-Aldrich N170, Paxilline Sigma-Aldrich P2928, SKA-31 Sigma-Aldrich S5573, thapsigargin Sigma-Aldrich T9033, and TRAM-34 Sigma-Aldrich T6700 were reconstituted in DMSO. Pertussis toxin Sigma-Aldrich P7208 was reconstituted in ultrapure water with 5 mg/mL bovine serum albumin Fisher Scientific BP1600-100. Dynorphin A (1–13) amide (American Peptide 26-4-51A) was dissolved in 25 mM Tris at pH 7.4. Apamin (Sigma-Aldrich A1289) was reconstituted in 0.05 M acetic acid.
Pierce M.L., French J.A, & Murray T.F. (2020). Comparison of the pharmacologic profiles of arginine vasopressin and oxytocin analogs at marmoset, titi monkey, macaque, and human oxytocin receptors. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 125, 109832.
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5

Ion Channel Modulation Assay

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External solutions were exchanged as previously reported [8 ]. Nifedipine, paxilline, TEA-OH, and TEACl were purchased from Sigma Aldrich. Tetrodotoxin (TTX) citrate was purchased from Tocris (Northpoint, Fourth Way Avonmouth, UK) and apamin from Alomone Labs (Jerusalem, Israel).
Marcantoni A., Chiantia G., Tomagra G., Hidisoglu E., Franchino C., Carabelli V, & Carbone E. (2022). Two firing modes and well-resolved Na+, K+, and Ca2+ currents at the cell-microelectrode junction of spontaneously active rat chromaffin cell on MEAs. Pflugers Archiv, 475(2), 181-202.
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6

Calcium Signaling Regulation by BK Channels

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Paxilline, IbTx, STO-609, thapsigargin, EGTA, ruthenium red, heparin, digitonin, 2-APB, U0126, bicuculline, DMSO, DAPI and all the other reagents to make solutions were from Sigma-Aldrich. Fluo-4/AM, Fluo-4/dextran, X-Rhod-1 were from Life Technologies. Antibodies specific for BK channel α-subunit (L6/60, Millipore MABN70, immunocytochemisty (ICC) 20 μg/ml, western blot (WB) 10 μg/ml, reference (Ref.) PMID 16566008), BK channel β4-subunit (Alomone Lab APC-061, ICC 1:100, Ref. PMID 21697543), Lamin B (Abcam ab16048, WB 1:1000, ICC 1:500, Ref. PMID 24265311) GAPDH (Cell Signalling 3683S, WB 1:1000, Ref. PMID 22895339), Calnexin (Cell Signalling 2433S, WB 1:1000, Ref. PMID 19815548), COX IV (Cell Signalling 4850S, WB 1:1000, Ref. PMID 19737931), phospho-CREB (Cell Signalling 9198S, WB 1:1000, ICC 1:500, Ref. PMID 17906627), CREB (Cell Signalling 9197S, WB 1:1000, Ref. PMID 20864036), phospho-CaMKIV (Santa Cruz Biotechnology sc-28443-R, WB 1:200, Ref. PMID 18559891), CaMKIV (Cell Signalling 4032S, WB 1:1000, Ref. PMID 21784978), phospho-ERK (Cell Signalling 9101S, WB 1:1000, Ref. PMID 11782377), and ERK (Cell Signalling 9102S, WB 1:1000, Ref. PMID 21669876), actin (Cell Signalling 4967S, WB 1:1000, Ref. PMID 17525163), RyR (Thermo MA3-925, WB 1:1000, Ref. PMID 1645737), Na/K ATPase (Cell Signalling 3010S, WB 1:1000, Ref. PMID 21724843) were used.
Li B., Jie W., Huang L., Wei P., Li S., Luo Z., Friedman A.K., Meredith A.L., Han M.H., Zhu X.H, & Gao T.M. (2014). Nuclear BK Channels Regulate Gene Expression via the Control of Nuclear Calcium Signaling. Nature neuroscience, 17(8), 1055-1063.
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7

Protein Degradation Mechanisms Analysis

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MG132 (S2619, Selleck Chemicals), bortezomib (S1013, Selleck Chemicals), MLN4924 (M2189, Abmol), Cycloheximide (R750107, Sigma-Aldrich), Paxilline (P2928, Sigma-Aldrich) and BMS-204352 (SML1313, Sigma-Aldrich) were dissolved in DMSO. Anti-CRBN (SAB045910, Sigma-Aldrich), anti-DDB1 (37–6200, Invitrogen), anti-actin (1844–1, Epitomics), anti-β-actin (4967, cell Signaling Technology; 5779–1, Epitomics), anti-β-tubulin (32–2600, Invitrogen), anti-Pan-Cadherin (4068, Cell Signaling Technology), anti-ubiquitin (sc-271289, Santa Cruz), anti-Cul1 (AP16324b, Abgent), anti-Fbxo7 (ab84129, Abcam), anti-Cul4A (A300-739A, Bethyl Laboratories), anti-Cul4B (2527–1,Epitomics), anti-Slo1 (NBP1-48250, Novus Biologicals), anti-mSlo1 (Millipore, MABN70), anti-HA (H6908; H3663, Sigma-Aldrich), anti-Flag (F3165, Sigma-Aldrich), anti-Myc (2272, Cell Signaling Technology), anti-GFP (sc-8334, Santa Cruz), anti-HA agarose (E6779, Sigma-Aldrich), anti-Flag M2 agarose (A2220, Sigma-Aldrich), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz) and goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz) were used following the manufacturers’ protocol.
Song T., Liang S., Liu J., Zhang T., Yin Y., Geng C., Gao S., Feng Y., Xu H., Guo D., Roberts A., Gu Y, & Cang Y. (2018). CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory. PLoS Genetics, 14(1), e1007165.
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8

Comprehensive Pharmacological Compound Protocol

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2,6-Bis(2-benzimidazolyl)pyridine (BBP), dofetilide, bicuculline methiodide, paxilline, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) and 4-aminopyridine were purchased from Sigma-Aldrich (Germany). 2-(4-Chlorophenoxy)-2-methyl-N-[5-[(methylsulfonyl)amino]tricyclo[3.3.1.13,7]dec-2-yl]-propanamide (JNJ 303) was purchased from Tocris (United Kingdom) and N-(2-{[(1R)-1-[3-(trifluoromethyl)phenyl]ethyl]amino}-1H-1,3-benzodiazol-4-yl)acetamide (AP14145) was synthetized by Syngene Inc. (Bangalore, India). All compounds were dissolved in pure dimethyl sulfoxide (Sigma-Aldrich, Germany) into 10 mM stock solutions and stored at −20°C. On the day of the experiment, the stock solution was thawed and the corresponding amount of compound was added to the working solution.
Simó-Vicens R., Bomholtz S.H., Sørensen U.S, & Bentzen B.H. (2018). 2,6-Bis(2-Benzimidazolyl)Pyridine (BBP) Is a Potent and Selective Inhibitor of Small Conductance Calcium-Activated Potassium (SK) Channels. Frontiers in Pharmacology, 9, 1409.
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9

Membrane Protein Purification and Analysis

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All reagents were dissolved and stored in DMSO or water. DQ-BSA-red was from Life Technologies; anti-SLO-1 antibodies were purchased from NeuroMab; vacuolin-1 was from Calbiochem; ML-SA1 was from Princeton BioMolecular Research Inc.; quinidine, paxilline, clofilium tosylate, NS1619, IBTX, TEA chloride, and filipin complex were purchased from Sigma-Aldrich.
Wang W., Zhang X., Gao Q., Lawas M., Yu L., Cheng X., Gu M., Sahoo N., Li X., Li P., Ireland S., Meredith A, & Xu H. (2017). A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores. The Journal of Cell Biology, 216(6), 1715-1730.
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10

Investigating BK Channel Modulation

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All chemicals were bath applied through a peristaltic pump system at a steady perfusion and suction rate. The time required for the solution to flow to the recording slice through stopcocks was approximately 1 minute. All drugs, including paxilline (a specific BK channel blocker28, 29), were purchased from Sigma‐Aldrich (St. Louis, MO, USA), except NS11021 (a specific BK channel opener27, 30, 31) and TTX (a sodium channel blocker), which were bought from Tocris Bioscience (Bristol, UK). All chemicals were dissolved in dimethyl sulfoxide (except TTX that was dissolved in distilled water) at 1000 times final concentration, stored at −20°C, and diluted to the final concentration in an oxygenated ASCF solution immediately before use.
Liu N., Xie H., Xiang‐wei W., Gao K., Wang T, & Jiang Y. (2019). The absence of NIPA2 enhances neural excitability through BK (big potassium) channels. CNS Neuroscience & Therapeutics, 25(8), 865-875.
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