SCN9A Loss of Function Mutations E1677 Figure 3. Electrophysiological characterisation of HEK293 cells transiently transfected with wild-type and mutant SCN9A. (A) Phase contrast, EGFP and DsRed2 fluorescence of HEK293 cells transiently co- transfected with plasmids expressing SCN9A+DsRed2 and SCN1B+SCN2B+EGFP under a CMV promoter. Note that in the phase contrast with the patch pipette one of the fluorescently positive cells is missing as it was used in the previous patch experiment. (B) 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 cells co-expressing either WT or mutant NaV1.7 (as indicated) with the β-subunits, identified by their fluorescence as shown in (A). The inset shows the voltage pulse protocol. (C) Current-voltage relationship of peak currents as shown in (B) normalised for cell size (pA/pF). NaV1.7+NaVβ1+NaVβ2 (n=5), NaV1.7-ΔR1370- L1374+NaVβ1+NaVβ2 (n=5), NaV1.7-R896Q+NaVβ1+NaVβ2 (n=7), NaVβ1+NaVβ2 only (n=5). WT data was fitted with a Boltzmann equation y = (A2+(A1-A2)/(1+exp((V0.5- x)/k)(x-Vrev), where V0.5 = -33 mV, k= 2 mV Vrev= 65 mV. Mutations cause a complete loss of function of Nav1.7 Given that plasma membrane staining was occasionally seen when the mutant channels were overexpressed, we decided to investigate how the mutations affected the biophysical properties of the sodium channel. To do this, plasmids bearing either WT SCN9A or the mutant sequences were co-expressed in HEK293A cells with the auxiliary sodium channel β1 and β2 subunits (encoded by SCN1B and SCN2B). Whole cell voltage clamp recordings from cells co-expressing wild-type Nav1.7 with the β1β2 subunits, revealed a voltage-gated Na+ current with a peak amplitude of -685 ± 134 pA/pF at -20 mV (n=5), compared with a background current of -13 ± 2 pA/pF (n=5) in cells transfected with the β1β2 construct alone (p=0.001 for wild-type Nav1.7 vs control) (Figure 3). The voltage dependence of activation of Nav1.7 could be described by a Boltzmann function, with half maximum activation (V0.5) of -33± 2 mV, k = 2 ± 1 mV and a reversal potential (Vrev) of +65 ± 4 mV (n=5). Voltage dependent inactivation could also be described by a Boltzmann function with half maximal inactivation at -74 ± 2 mV and k = 4.3 ± 0.6 mV (n=5) (Supp. Figure S5). These properties are similar to those described previously for Nav1.7 current [Klugbauer et al., 1995; Cummins et al, 2004; Herzog et al., 2003]. In contrast, cells co-transfected