SCN9A (NM_002977) Stable Cell Line

Figure 1. qPCR data on specific over-expression of SCN9A. In a SYBR green qPCR experiment, specific over-expression of SCN9A was determined using gene specific primers. Data are shown as fold of over-expression after normalization against GAPDH.

Figure 2. Current-Voltage Relationship: Activation. A) Representative currents obtained by a family of depolarising pulses between -50 and +60 mV from a holding potential of -90 mV; B) Peak I-V relationship (mean ± SEM, n= 67); C) Normalised G (conductance) -V plot. To ensure appropriate voltage-clamp only cells with peak current amplitudes ranging from 0.4 to 1.2 nA were included. The threshold for activation was -30 mV and peak currents were obtained between 0 and +10 mV). The reversal potential was +60mV, close to the theoretical value of +66mV, calculated using the Nernst equation. From the G-V plot the Boltzmann parameters for activation were: V½ -7.5 mV, slope of 5.4 (mV/e-fold)

Figure 3. Current-Voltage Relationship: Inactivation. The voltage-dependence of inactivation was measured by applying long (1 s) conditioning pulses to varying potentials (-110 to -30 mV) followed by a test pulse to 0 mV. The Boltzmann parameters for inactivation were: V½ -60mV and slope 5.4(mV/e-fold). Currents are normalised to the current evoked by the test pulse following a conditioning potential of -110 mV. Data is presented as mean of 50 cells. SEM are too small to be clearly visible.

Figure 4. Expression Profile. At a step potential of 0mV, peak inward currents of >0.4 nA were observed in 324 of 370 cells (87%), with a mean amplitude of 2.02 ± 1.00 nA (n=324; mean ± S.D.). The maximum current evoked from a depolarising pulse to 0 mV was divided into 0.2 nA bins.

Figure 5. Expression Stability. Stable expression of hNav1.7 over multiple cell passages. The ordinate shows the mean (±SD) Na+ current amplitudes obtained from population patch clamp recordings from a Vh of -80mV. Note the stable expression for >35 passages.

Figure 6. Pharmacology. Inhibition of hNav1.7 Na+ currents by known Na+ channel blockers. The IC50 values are close to those previously described: tetrodotoxin (15nM), ProTX-II (4.1nM), tetracaine (6.5?M) and lidocaine (262?M). Currents were evoked by a 20 ms test pulse to 0mV from a holding potential of -90mV in the absence (control) and presence of inhibitor.

Figure 7. Cell Growth Properties. Images of cells at 60% confluency at low and high (inset) magnification. Growth curve (confluency) for HEK hNav1.7 cells seeded at 10% confluency at 37?C measured every 3 h for a total of 72 h.