Patch-Clamp Recording

All ionic currents were recorded with automated whole-cell, patch-clamp technique. The inhibitory effects of 30–1000 μM of oseltamivir phosphate on Kir3.1/3.4 and K_V1.5‐derived currents were assessed using port-a-patch automated patch-clamp technology (Nanion Technologies, Munich, Germany) at TOA EIYO LTD., whereas those of 10–1,000 μM on hERG, Na_V1.5 and Ca_V1.2-derived currents were evaluated using Qube 384 automated patch-clamp system (Sophion Bioscience A/S, Ballerup, Denmark) at Sophion Bioscience K.K. Each current was recorded at room temperature (20–25°C) except for Kir3.1/3.4-derived current which was done at 30°C. The recording data was analyzed by PatchMaster (ver. 2 × 69, HEKA Electronik GmbH, Lambrecht, Germany) for Kir3.1/3.4 and K_V1.5-derived currents, or by Sophion Analyzer (ver. 6.5, Sophion Bioscience A/S) for hERG, Na_V1.5 and Ca_V1.2-derived ones. The cells for recording Kir3.1/3.4 or K_V1.5-derived currents were exposed to oseltamivir phosphate solution in the order of rising concentration using a perfusion system. The cells for recording hERG, Na_V1.5 or Ca_V1.2-derived currents were exposed to the three test solutions; namely, vehicle, oseltamivir phosphate at either concentration of 10, 30, 100, 300 or 1,000 μM, and selective ionic channel blockers or sodium-free bath solution with an interval of the replacement time of > 14 min. The IC₅₀ values were calculated by fitting the data to Hill equation using GraphPad prism 8 (ver. 8.31; GraphPad Software, Inc. CA, United States).

The protocol for recording Kir3.1/3.4-derived K⁺ current consisted of a 500 ms prepulse at –120 mV, followed by a 1,000 ms ramp pulse from –120 mV to + 60 mV with an interval of 10 s at a holding potential of −40 mV. The current amplitude was measured at + 60 mV for analyzing the concentration-response relationship (n = 3). The bath solution was composed of (in mM) 100 NaCl, 40 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 D (+)-glucose and 10 HEPES at pH 7.4 with NaOH. The internal recording solution was composed of (in mM) 50 KCl, 10 NaCl, 60 KF, 2 MgCl₂, 20 EGTA and 10 HEPES at pH 7.2 titrated with KOH. The K_V1.5-derived K⁺ current was evoked by a 500 ms step pulse at 0 mV with an interval of 20 s at a holding potential of –70 mV. The peak amplitude was measured for analyzing the concentration-response relationship (n = 3). The external recording solution was composed of (in mM) 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 5 D (+)-glucose and 10 HEPES at pH 7.4 titrated with NaOH, whereas the internal recording solution was the same as that used for recording Kir3.1/3.4 current. Tail current of hERG-derived K⁺ current was elicited by a 2,000 ms step pulse at –50 mV after a 1,000 ms prepulse at +40 mV with an interval of 30 s at a holding potential of −70 mV (n = 6). The peak amplitude of the tail currents was normalized with the following equation (I_peak–I_b)/(I_f–I_b); I_peak is the peak amplitude in the presence of oseltamivir; I_b is that with vehicle (1% PBS containing bath solution, basal); and I_f is that in the presence of 100 μM quinidine (fully blocked). The bath solution was composed of (in mM) 145 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 D (+)-glucose and 10 HEPES at pH 7.4 titrated with NaOH. The internal recording solution was composed of (in mM) 120 KF, 20 KCl, 10 EGTA and 10 HEPES at pH 7.2 titrated with KOH. The Na_V1.5‐derived Na⁺ current was evoked by a 20 ms step pulse at −10 mV every 10 s at a holding potential of −90 mV (n = 6). The peak amplitude in the presence of oseltamivir was measured and normalized using that with vehicle and that obtained in a sodium-free bath solution. To assess the frequency-dependent block of Na_V1.5 by oseltamivir, a train of 30 step pulses at −10 mV for 20 ms at 1 Hz, 5 Hz or 10 Hz was applied, and the peak amplitude at the 30th test pulse (P30) was normalized by that at the first pulse (P1). The steady-state inactivation curves of Na_V1.5 channels before and after application of vehicle and oseltamivir phosphate were obtained by the following protocol; a 500 ms prepulse ranging from –120 mV to –10 mV in 10 mV increments followed by a 20 ms test pulse at –10 mV with an interval of 20 s at a holding potential of –120 mV. The peak amplitude was normalized with the maximum peak amplitude for each cell in the same condition and was fitted with Boltzmann equation to calculate the half-maximum voltage of inactivation (V_{1/2, inact}). The bath solution was composed of (in mM) 145 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 D (+)-glucose and 10 HEPES at pH 7.4 titrated with NaOH. The sodium-free bath solution was composed of (in mM) 145 NMDG-Cl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 D (+)-glucose and 10 HEPES at pH 7.4 titrated with NaOH. The internal recording solution was composed of (in mM) 10 NaCl, 140 CsF, 1 EGTA and 10 HEPES at pH 7.3 titrated with CsOH. The Ca_V1.2-derived inward Ca²⁺ current was evoked by a 200 ms step pulse at 0 mV every 30 s at a holding potential of –80 mV (n = 6). The peak amplitude in the presence of oseltamivir was normalized using that with vehicle and that in the presence of 30 μM nifedipine. The bath solution was composed of (in mM) 145 NaCl, 4 KCl, 10 CaCl₂ and 10 HEPES at pH 7.4 with NaOH. The internal recording solution was composed of (in mM) 112 CsCl, 2 NaCl, 28 CsF, 8.2 EGTA, 4 MgATP and 10 HEPES at pH 7.2 titrated with CsOH.