The IKACh current in atrial myocytes was measured with standard whole-cell voltage-clamp technique. The Multiclamp 700B (Molecular Devices) amplifier, an A/D converter (Digidata 1550B plus Hum Silencer, Molecular Devices), and the pClamp 10.6 PC software (Molecular Devices) were used for current acquisition. Clampfit 10.6 (pClamp, Molecular Devices) and OriginPro 2018b software packages (OriginLab Corp) were used for data analysis. Whole-cell measurements were performed at room temperature using borosilicate glass microelectrodes with tip resistances of 2.5–3 MΩ filled with pipette solution (in mM): KCl 140, MgCl2 1, HEPES 10, EGTA 5, Mg2ATP 5, and GTP 0.1, pH adjusted to 7.2 with KOH. Basal current was recorded by holding the atrial myocyte at −40 mV, followed by 1000 ms steps from −140 mV to +40 mV in 20 mV increments. The extracellular solution contained (in mM): NaCl 100, KCl 50, MgCl2 1, HEPES 5, and D-glucose 5.5, adjusted to pH 7.4. To measure the constitutively active IKACh current in atrial cardiomyocytes, 100 nM Tertiapin-Q (TPQ, Alomone Labs) was applied to the extracellular solution.
Tertiapin q tpq
Manufactured by Alomone
Sourced in Palestine, State of
Tertiapin-Q (TPQ) is a selective inhibitor of G protein-coupled inwardly-rectifying potassium (GIRK) channels. It acts by binding to and blocking the GIRK channels, thereby modulating their activity. TPQ is a useful tool for studying the role of GIRK channels in various biological processes.
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2 protocols using tertiapin q tpq
1
Measuring IKACh Currents in Atrial Myocytes
2
Selective Expression of Gi/o-DREADDs in β-Cells
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Unless otherwise noted all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Thermo Fisher (Waltham, MA). Clozapine N-oxide (CNO) was purchased from Hello Bio (Princeton, NJ). Clonidine hydrochloride (Clon), exendin-3 (9–39) amide (Ex9), forskolin (FSK), L-054,264, ouabain (Oua), and myristoylated PKI 14–22 amide (PKI) were purchased from R&D Systems (Minneapolis, MN). Tertiapin-Q (TPQ) was purchased from Alomone Labs (Jerusalem, Israel). Dasatinib (Dasa), H-89, and NSC 87877 were purchased from Cayman Chemical (Ann Arbor, MI). Liraglutide (Lira) was purchased from Novo Nordisk (Plainsboro, NJ). An optimized rat insulin promoter (RIP)60 (link) and the coding sequence of hM4D(Gi)-mCherry (Gi/o-DREADD-mCherry fusion protein; Plasmid #75033; Addgene, Watertown, MA)95 (link) were cloned into a pLenti6 lentiviral transfer plasmid and utilized to produce 3rd-generation lentiviruses (LVs) that express Gi/o-DREADDs selectively in β-cells (βGi/o-DREADDs) as previously described96 (link).
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