Fig. 4.

NAN and IAV could form functional channels when expressed simultaneously in Drosophila S2 cells. (A) Representative traces of spontaneous channel activities in control and Inactive-mCherry– and Nanchung-Flag–coexpressing S2 cells at −60 mV (holding potential) in Na⁺/K⁺-based solutions. (B) Representative traces of single-channel activities in control and Inactive-mCherry– and Nanchung-Flag–coexpressing S2 cells at −60 mV (holding potential) in Na⁺/K⁺-based solutions with 2 μM NAM in internal solution. (C) Histogram showing activity of NAN-IAV single-channel currents with NAM. The black line indicates the Gaussian fit for the histogram. (D) The open probability of NAN-IAV channels with different concentrations of NAM in internal solution (mean ± SEM; n = 12, 8, 7, 9, 13, and 5 in each group). NP_o indicates the total open probability of active NAN-IAV channels. (E) Comparison of the current amplitude (absolute value) of spontaneously active NAN-IAV channels and NAM-induced channels at −60 mV (holding potential) in Na⁺/K⁺-based solutions. NAN-IAV, spontaneous current (n = 9); NAN-IAV+ 2 μM NAM (n = 10). Mean ± SEM, unpaired t test. NS, not statistically significant. (F) Permeability ratios of different cation combinations (n = 8, 6, and 7; mean ± SEM). “X” means Na⁺, K⁺, or Ca²⁺. (G) Average current–voltage relationship of NAN-IAV single-channel currents in the bi-ionic extracellular Na⁺/intracellular Cs⁺ condition with NAM (n = 8; mean ± SEM). (H) Single-channel I–V curves of NAN-IAV in extracellular K⁺/intracellular Cs⁺ condition with NAM (n = 6; mean ± SEM). (I) Single-channel I–V curves of NAN-IAV in extracellular Ca²⁺/intracellular Cs⁺ conditions with NAM (n = 7; mean ± SEM).