Fig. 3.

Some VGNs expressed TTX-insensitive Na⁺ current (I_NaI) with the properties of Na_V1.5 channels, in addition to I_NaS. A and B: current remaining in 300 nM TTX (I_NaI) had more negative voltage dependence than total Na_V current. Data are from P2 VGN. A1: inactivation: as prepulse voltages (bottom) became less negative, the peak current evoked by the step to −30 mV decreased in both control and 300 nM TTX solutions but with different voltage dependence: a prepulse to −102 mV (red traces) decreased total I_NaV only slightly (top) relative to the peak value (blue traces, prepulse to −130 mV) but inactivated approximately half of I_NaI (bottom). A2: activation: at −58 mV (red traces), I_NaV was one-quarter maximal but I_NaI was nearly half-maximal. A3: current-voltage relation: peak inward current in control and 300 nM TTX solutions (top) for the VGN in A1 and A2 shows a small TTX-insensitive component (red) that activated at relatively negative potentials (y-scale expanded, bottom). B: conductance-voltage (G-V) relations for activation and inactivation. B1: normalized plots of activation (peak current vs. test step, open symbols) and inactivation (peak current vs. prepulse, filled symbols) and Boltzmann function fits (curves); for the VGN in A. Gray triangles, total I_NaV; red circles, I_NaI. B2: population data. Thin curves, all fits for room temperature, ruptured-patch data. Thin gray curves, total I_NaV for VGNs with both I_NaS and I_NaI or just I_NaS. Thin red curves, I_NaI, obtained in 300 nM TTX. Thick black and red curves, curves generated with average fit parameters, Table 2. B3: I_NaI voltage range was still negative when recorded at body temperature (red curves) or at room temperature with the perforated patch method (dark cyan curves; inactivation curves only). Thick gray curves, population averages for I_NaI from B2 (ruptured patch, room temperature). C: I_NaI in 300 nM TTX (black) was strongly blocked by 5 μM TTX (blue) and abolished by replacing external Na⁺ with NMDG⁺ (red, no TTX present), demonstrating that it is carried by Na⁺ ions flowing through Na_V channels (K⁺ internal, K⁺ external, P7). D: voltage range of inactivation recorded in 300 nM TTX did not shift negatively with time, as illustrated by inactivation curves taken at different times since patch rupture. Negative inactivation ranges were recorded immediately post rupture: inverted red open triangles, 300 nM TTX, 10 s after rupture. In 0 TTX, inactivation ranges of total I_NaV did not shift negatively with time: cyan triangles and purple diamonds, 2 VGNs, 7.5 min since rupture; green triangles, a 3rd VGN, 13 min since rupture. Removal of TTX shifted inactivation curves positively despite increasing time since rupture: black squares, in 300 nM TTX; gray circles, the same VGN later, after stopping TTX application. E: I_NaI (isolated in 300 nM TTX) was partially and reversibly blocked by 200 μM external Cd²⁺. The Cd²⁺-blocked (difference) current (E1, P4) had similar time course (E2, normalized E1 data) and voltage dependence (E3, different VGN, P2) to the current in 300 nM TTX, consistent with them being one and the same.