Human Nav1.7 Stable Cell Line
Human Nav1.7 (SCN9A) is a voltage-gated Na+ channel preferentially expressed in sensory neurones, which plays a key role in the depolarisation phase (upstroke) of the action potential. Mutations in this gene have been associated with primary erythermalgia, channelopathy-associated insensitivity to pain, and paroxysmal extreme pain disorder. Nav1.7 is of interest as a target for novel analgesics.
Essen Bioscience and OriGene have co-developed a stable human Nav1.7 alpha-subunit cell line in human embryonic kidney (HEK293) cells for various functional screening assays.
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|Description:||Human Nav1.7 Stable Cell Line – HEK293|
Ref seq. NM_002977.1.
Other names: ETHA; FEB3B; Nav1.7; NE-NA; NENA; PN1
|cDNA Clone:||OriGene TrueClone SC309017 in pCMV-6-Neo|
|Format:|| 2 x 1 mL frozen cell vials each containing 1 x 106 cells|
|Mycoplasma:||Negative by DNA staining and direct culture methods (ATTC – detailed information available upon request)|
|Cell Line Validation:
- Gene expression: qPCR experiments determined specific over-expression of SCN9A.
- Functional validation
- Current-Voltage relationship: activation
The basic biophysical properties, expression levels, and pharmacology of Nav1.7-HEK293 cells were assessed using the IonWorks planar array electro-physiology platform. Figure 2, Figure 3
- Expression stability
Stable expression of hNav1.7 over multiple cell passages is confirmed. Figure 4, Figure 5
Inhibition of hNav1.7 Na+ currents by known Na+ channel blockers such as tetrodotoxin, ProTX-II, tetracaine and lidocaine. Figure 6
|Growth Properties:||Growth rates and percentage confluency were determined using an IncuCyte (Essen Bioscience) from images taken every 3 h over a 72 h period. The doubling time for the cell line was approximately 20 h for the linear section of the growth curve. Figure 7|
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.
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)
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.
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.
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.
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 (6.5M) and lidocaine (262M). 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.
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 37C measured every 3 h for a total of 72 h.