E1684 Sheynin et al. Supp. Figure S5. Voltage dependent activation and inactivation of wild-type and FLAG-tagged NaV1.7 channels. (A) (left) Current responses to 50 ms voltage steps in 5 mV increments between -110 and +60 mV from a holding potential of -100 mV, in a whole cell voltage clamp recording applied at 0.5 Hz for a cell co- expressing FLAG-tagged NaV1.7 with the β-subunits. The inset shows the voltage pulse protocol. (right) Current-voltage relationship of peak currents as shown on the left normalised for cell size (pA/pF). NaV1.7+NaVβ1+NaVβ2 (n=5), FLAG-tagged NaV1.7+NaVβ1+NaVβ2 (n=5). The data was fitted with a Boltzmann equation y = (A2+(A1-A2)/(1+exp((V0.5-x)/k)(x-Vrev). There was no significant difference between currents from wild-type and FLAG-tagged NaV1.7-expressing cells, with V0.5 = -33±1 mV and -33±2 mV; k= 2±1 mV and 3±1 mV; and Vrev= 65±4 mV and 67±4 mV respectively. The peak current for FLAG-tagged NaV1.7 at -20 mV was 591±115 pA/pF which was not significantly different from wild-type expressing cells. (B) (left) Current responses of a FLAG-tagged NaV1.7+NaVβ1+NaVβ2 expressing cell in whole cell voltage clamp to a 50 ms test pulse to a potential of -10 mV applied immediately after 500 ms conditioning voltage steps to potentials from -120 to 0 mV (at 5 mV increments). The inset shows the voltage pulse protocol applied at 0.5 Hz. (right) Voltage dependence of the steady-state inactivation assessed as on the left for NaV1.7+NaVβ1+NaVβ2 (n=5), FLAG-tagged NaV1.7+NaVβ1+NaVβ2 (n=5). The data was fitted with a Boltzmann equation y = (A1-A2)/(1+exp(x-V0.5)/k)) + A2. There was no significant difference between currents from wild-type and FLAG-tagged NaV1.7-expressing cells, with V0.5 = -74±5 mV and 76±1 mV and k= 4±1 mV and 6±1 mV respectively.