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Tta a2

Manufactured by Alomone
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Sourced in Israel, United Kingdom, United States

TTA-A2 is a small molecule that acts as a selective antagonist for the T-type calcium channel. It is a useful tool for the study of T-type calcium channel function in various biological systems.

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Lab products found in correlation

Thapsigargin, by Bio-Techne (2 mentions) Isradipine, by Bio-Techne (2 mentions) Ryanodine, by Bio-Techne (2 mentions) Nnc 55 0396, by Alomone (2 mentions) Xestospongin c xec, by Cayman Chemical (2 mentions) Nicardipine, by Merck Group (2 mentions) Z 944, by Bio-Techne (1 mentions) Pinacidil, by Merck Group (1 mentions) Gsk 7975a, by Aobious (1 mentions) Retigabine, by Alomone (1 mentions) Tetrodotoxin ttx, by Abcam (1 mentions) Zd7288, by Bio-Techne (1 mentions) Xe991, by Abcam (1 mentions) Nnc 55 0396, by Merck Group (1 mentions) Mgso4, by Thermo Fisher Scientific (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Tetrodotoxin ttx, by Bio-Techne (1 mentions) Tta p2, by Alomone (1 mentions)

7 protocols using tta a2

1

Perfusion of Tissues with Oxygenated KRB Solution

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All tissues were perfused continuously with KRB solution containing (mmol/L): NaCl, 5.9; NaHCO3, 120.35; KCl, 1.2; MgCl2, 15.5; NaH2PO4,1.2; CaCl2, 2.5; and glucose, 11.5. The KRB solution was warmed to a physiological temperature of 37 ± 0.3°C and bubbled with a mixture of 97% O2 – 3% CO2. For experiments utilizing external solutions with 0 [Ca2+]o, CaCl2 was omitted and 0.5 mM ethylene glycol-bis (β-aminoethyl ether)-N, N, N’, N’–tetraacetic acid (EGTA) was added to the solution. NNC 55–0396 and TTA-A2 were purchased from Alomone Labs (Jerusalem, Israel). 2-aminoethyl-diphenylborinate (2-APB), tetracaine, nicardipine, pinacidil was purchased from Millipore-Sigma (St. Louis, Missouri, USA). Thapsigargin, isradipine, Z-944, CPA and ryanodine were purchased from Tocris Bioscience (Ellisville, Missouri, USA). GSK 7975A was purchased from Aobious (Aobious INC, MA, USA), and xestospongin C (XeC) was purchased from Cayman Chemical (Michigan, USA).
Baker S.A., Leigh W.A., Del Valle G., De Yturriaga I.F., Ward S.M., Cobine C.A., Drumm B.T, & Sanders K.M. (2021). Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon. eLife, 10, e64099.
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2

In Vitro and In Vivo Drug Preparation

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TTA-A2 and TTX were purchased from Alomone Labs and Tocris Bioscience (Bristol, UK), respectively; other chemicals were from Sigma-Aldrich. All drugs were dissolved in distilled water for in vitro experiments or in saline for in vivo experiments, except for TTA-A2, which was dissolved in dimethyl sulfoxide at high times first and then diluted to the final concentration in saline.
Feng X.J., Ma L.X., Jiao C., Kuang H.X., Zeng F., Zhou X.Y., Cheng X.E., Zhu M.Y., Zhang D.Y., Jiang C.Y, & Liu T. (2019). Nerve injury elevates functional Cav3.2 channels in superficial spinal dorsal horn. Molecular Pain, 15, 1744806919836569.
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3

Voltage Clamp Analysis of Ion Currents

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Voltage clamp experiments to define steady-state Kv7 current, persistent sodium current, and T-type calcium current used slow (20 mV/s) ramps from −95 to +5 mV. Voltage clamp experiments to define steady-state HCN current used ramps from −105 to −55 mV (20 mV/s), delivered after a 2- second holding period at −105 mV to activate HCN channels. We isolated the Kv7 potassium current by subtracting the whole current before and after bath application of 10–20 μM XE-991 (Abcam), sodium current by subtraction using 1 μM tetrodotoxin (TTX, Abcam), T-type calcium current by subtraction using 2 μM TTA-A2 (Alomone), and HCN current by subtraction using 100 μM ZD-7288 (Tocris). 10 μM retigabine (Alomone) was applied to enhance Kv7 potassium current. In experiments comparing local application of ZD-7288 to the soma versus bath application to the entire cell, we used 1 mM ZD-7288 in the puffer pipette to ensure that the lesser effect of somatic application was not due to failure to saturate block of somatic HCN channels.
Hu W, & Bean B.P. (2018). Differential control of axonal and somatic resting potential by voltage-dependent conductances in cortical layer 5 pyramidal neurons. Neuron, 97(6), 1315-1326.e3.
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4

Perfused Tissue Maintenance Protocol

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All tissues were perfused and maintained with KRB solution containing (mmol/liter): 120.35 NaCl, 15.5 NaHCO3, 5.9 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.5 CaCl2, and 11.5 glucose. The KRB solution warmed to a physiological temperature of 37 ± 0.2°C and bubbled with a mixture of 97% O2–3% CO2. For experiments using external solutions with zero [Ca2+]o, CaCl2 was omitted and 1 mM EGTA was added to the solution. NNC 55-0396 and TTA-A2 were purchased from Alomone Labs, nicardipine was purchased from Sigma-Aldrich, thapsigargin, isradipine, and ryanodine were purchased from Tocris Bioscience, and multiple batches of Xestospongin C (XeC) were purchased from Cayman Chemical.
Drumm B.T., Hennig G.W., Battersby M.J., Cunningham E.K., Sung T.S., Ward S.M., Sanders K.M, & Baker S.A. (2017). Clustering of Ca2+ transients in interstitial cells of Cajal defines slow wave duration. The Journal of General Physiology, 149(7), 703-725.
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5

Evaluation of Monoamine Release by SAK3

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SAK3 was synthesized by Shiratori pharmaceutical Ltd (Chiba, Japan; Fig 1A) according to a previous study [18 (link)]. As SAK3 (0.5 mg/kg, p.o.) shows maximal effects of ACh release and significant cognitive enhancement in several animal models including APPNL-F KI mice [18 (link),19 (link),22 ], we chose dose of SAK3 at 0.5 mg/kg to evaluate monoamine release. SAK3 was dissolved in distilled water. T-type calcium channel inhibitor, NNC 55–0396 (1 μM: Sigma-Aldrich, St-Louis, MO) [23 (link)], TTA-A2 (1 μM: Alomone Labs, Jerusalem, Israel), α7 nAChR antagonist MLA (1 nM: Sigma-Aldrich), and α4β2 nAChR antagonist DhβE (100 μM: Tocris, Bristol, UK) were dissolved in Ringer’s solution.
Wang S., Yabuki Y., Matsuo K., Xu J., Izumi H., Sakimura K., Saito T., Saido T.C, & Fukunaga K. (2018). T-type calcium channel enhancer SAK3 promotes dopamine and serotonin releases in the hippocampus in naive and amyloid precursor protein knock-in mice. PLoS ONE, 13(12), e0206986.
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6

Pressure Myography of Lymphatic Vessels

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Lymphatic vessels were isolated and cannulated in 0.5% albumin-supplemented Krebs buffer; the cannulating pipettes contained the same solution. Pressure myograph experiments were conducted in Krebs buffer without albumin. Krebs buffer contained (in mM): NaCl, 146.9; KCl, 4.7; CaCl2, 2; MgSO4, 1.2; NaH2PO4.H2O, 1.2; NaHCO3, 3; Na-HEPES, 1.5; D-glucose, 5 (pH 7.4, 37 °C). For patch clamp recordings, the physiological Ca2+ bath solution contained (in mM): 1.8 CaCl2, 110 NaCl, 1 CsCl, 1.2 MgCl2, 10 HEPES, 10 D-glucose (pH adjusted to 7.4 with NaOH). Ba2+ bath solution contained (in mM): 10 or 20 BaCl2, 110 NaCl, 1 CsCl, 1.2 MgCl2, 10 HEPES, 10 D-glucose (pH adjusted to 7.4 with NaOH). Patch pipettes contained (in mM): 135 CsCl, 10 HEPES, 10 EGTA, 5 GTP·Mg, (pH adjusted to 7.2 with CsOH). All chemicals were obtained from Sigma (St. Louis, MO, USA), except for BSA (US Biochemicals; Cleveland, OH, USA), MgSO4, HEPES (Fisher Scientific; Pittsburgh, PA, USA), and TTA-A2 (Alomone, Israel). TTA-A2, nifedipine and Bay K8644 were dissolved in DMSO. Ni2+, mibefradil and acetylcholine were dissolved in Krebs buffer (pre-warmed for Ni2+) without BSA. DMSO by itself, at its maximal concentration (0.033%) had no effect on contractions.
To K.H., Gui P., Li M., Zawieja S.D., Castorena-Gonzalez J.A, & Davis M.J. (2020). T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions. Scientific Reports, 10, 70.
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7

Electrophysiological Experiment Reagents

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TTA-A2 and TTA-P2 were obtained from Alomone Labs. Tetrodotoxin (TTX) was obtained from Tocris Bioscience (Bristol, UK). All the other drugs for electrophysiological experiment were obtained from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were dissolved in distilled water except that TTA-A2 and TTA-P2 was dissolved in DMSO and were stored at −20°C unless otherwise mentioned.
Wu J., Peng S., Xiao L., Cheng X., Kuang H., Zhu M., Zhang D., Jiang C, & Liu T. (2018). Cell-Type Specific Distribution of T-Type Calcium Currents in Lamina II Neurons of the Rat Spinal Cord. Frontiers in Cellular Neuroscience, 12, 370.
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