Effect of holding potential on inhibition by isoflurane of three major voltage-gated Na⁺ channel isoforms. Na⁺ currents were evoked from holding potentials of −100 or −70 mV by 25-ms test steps to V_max: 0 mV for Na_v1.2, −10 mV for Na_v1.4, and −30 mV for Na_v1.5. (A) Isoflurane (approximately 0.8 mm) significantly inhibited all three isoforms from both holding potentials, but to different degrees. Normalized peak I_Na was fitted to the Hill equation. All three isoforms were more sensitive to isoflurane from a holding potential of −70 mV, which is near normal resting potential. (B) Representative current traces. (C) Na_v1.5 was most sensitive to isoflurane at a holding potential of −70 mV, but least sensitive to isoflurane at −100 mV, whereas Na_v1.2 was most sensitive to isoflurane at −100 mV and least sensitive at −70 mV. Data are expressed as mean ± SEM, n = 3–17. **P< 0.01; ***P< 0.001 by one-way analysis of variance with Newman-Keulspost hoctest (C) or by unpairedttest (A).