Studies were performed using high standard gigaseal automated patch clamp technology (Bell & Fermini, 2021) as detailed before (Le Marois et al., 2020). Briefly, CHO cells expressing hERG or KV7.1/minK were obtained from B’Sys GmbH (Switzerland), and CaV1.2/β2/α2δ1, Kir2.1 or KV4.3/KChIP2.2 from Charles River Laboratories (USA), respectively. HEK cells expressing NaV1.5 were constructed in house. All expressions were constitutive except Kir2.1, CaV1.2 and NaV1.5 which were induced overnight by exposure to 1 µg/mL doxycycline (in the presence of 3 µM verapamil for CaV1.2). Patch-clamp recordings were performed in whole-cell patch-clamp mode at room temperature on a Qpatch® 48X or Model II workstation (Sophion, Denmark). The voltage-protocols, holding potentials and triggering frequencies used for each current are schematized as insets in their respective panel in the figure. Extracellular buffers for potassium channels contained (in mM) NaCl, 150 ; KCl, 4 ; CaCl2, 2 ; MgCl2, 1 and HEPES, 10. For the hERG channels, glucose (10 mM) was added. The pH was adjusted to 7.4 with NaOH. The Intracellular buffers contained (in mM): KF, 120 ; KCl, 20; EGTA, 10 ; MgCl2, 1; HEPES, 10. For KV7.1 channels, 10 mM EDTA was added as a second chelator, EGTA was decreased to 5 mM and no MgCl2 was added. The pH was adjusted to 7.2 with KOH. The extracellular buffer for CaV1.2 channels contained (in mM): NaCl, 145 ; KCl, 4 ; BaCl2, 10 and HEPES, 10, and the pH was adjusted to 7.4 with NaOH. The intracellular buffer contained: CsF, 27 ; CsCl, 112 ; EGTA, 8.2 ; NaCl, 2 ; HEPES, 10 and Mg-ATP, 4. The pH was adjusted to 7.2 with CsOH. For NaV1.5 channels, the extracellular buffer contained: NaCl, 137 ; KCl, 4 ; CaCl2, 2 ; MgCl2, 1 and HEPES, 10, and the pH was adjusted to 7.4 with NaOH. The intracellular buffer contained: CsF, 150 ; EGTA/CsOH, 1/5 ; NaCl, 10 ; MgCl2, 1 ; CaCl2, 1 ; HEPES, 10, and the pH was adjusted to 7.2 with CsOH. For the recording of the late component of NaV1.5, intracellular CsF was reduced to 130 mM and the extracellular buffer was supplemented with 10 nM of the sea anemone toxin ATX-II to slow channel inactivation (Wu et al., 2019). All channels were exposed to 6 concentrations of Naringenin applied cumulatively in an ascending order up to 300 µM. At the end of each recording, a reference inhibitor was added at a maximally active concentration to isolate leak currents if any. E-4031 (10 µM) was used for the hERG currents, lidocaine (3 mM) for the NaV1.5 currents, CdCl2 (0.2 mM) for CaV1.2, SKF-96365 (30 µM) for KV4.3, HMR-1556 (30 µM) for KV7.1 and BaCl2 (3 mM) for the Kir2.1 channels.
Racemic naringenin (CAS Number 67604-48-2) was obtained from Sigma-Aldrich (catalog # N5893). Concentrated stock solutions prepared in pure DMSO were diluted in extracellular buffer containing 1% Pluronic F-68 so as to obtain the following six final concentrations which were applied to the cells in ascending order : 1.2 µM, 3.7 µM, 11 µM, 33 µM, 100 µM and 300 µM. Final DMSO was kept below 1%. All current inhibitions were quantified as change in normalized peak current amplitude except for KV4.3 currents which were quantified as change in normalized integral charge transferred calculated as the area under the current trace vs time. Half-maximal inhibitory concentrations (IC50) and Hill slope values (nH) were estimated by fitting a sigmoidal curve to the current measurements normalized with respect to pre-drug baseline. Minimal and maximal inhibition were constrained to zero and 100%, respectively. Calculations and graphs were done with Prism 8.3.0 (GraphPad Software, San Diego, CA, USA).