Effect of GTPγS on Na_V1.9 single-channel activity. Single Na_V1.9 channel openings were elicited by 300-ms pulses to −40 mV from a holding potential of −120 mV with 150 nM TTX present in the bath solution. (A) Single-channel traces from excised, outside-out patches dialyzed with control intracellular solution. Dotted lines indicate the zero current level, and individual single-channel amplitudes are marked by horizontal lines. The illustrated patch recordings contain at least six channels. (B) Single-channel traces from excised, outside-out patches dialyzed with pipette solution containing 200 µM GTPγS. The illustrated patch recordings contain at least four channels. (C) Single-channel amplitudes measured in the absence (control, n = 4) or presence (+GTPγS, n = 4) of 200 µM GTPγS. (D) Values for nP_o calculated from single-channel recordings from control and +GTPγS patches. Intracellular GTPγS increased nP_o ∼2.5 (*, P = 0.004). (E) Open time histograms plotted for single-channel recordings performed in the absence or presence of GTPγS. The mean open times calculated for control solution were τ₁ = 0.09 ± 0.01 ms (A₁ = 95.7 ± 1.8%) and τ₂ = 0.47 ± 0.07 ms (A₂ = 4.3 ± 1.8%; n = 4). In the presence of GTPγS, mean open times were τ₁ = 0.24 ± 0.05 ms (A₁ = 91.5 ± 2.4%) and τ₂ = 1.55 ± 0.35 ms (A₂ = 8.5 ± 2.4%; n = 4). The differences in τ₁ and τ₂ values between control and +GTPγS were significant, P = 0.026 and P = 0.023, respectively. Data are represented as means ± standard error of the mean.