Pain perception is altered by a nucleotide polymorphism in SCN9A

Abstract

The gene SCN9A is responsible for three human pain disorders. Nonsense mutations cause a complete absence of pain, whereas activating mutations cause severe episodic pain in paroxysmal extreme pain disorder and primary erythermalgia. This led us to investigate whether single nucleotide polymorphisms (SNPs) in SCN9A were associated with differing pain perception in the general population. We first genotyped 27 SCN9A SNPs in 578 individuals with a radiographic diagnosis of osteoarthritis and a pain score assessment. A significant association was found between pain score and SNP rs6746030; the rarer A allele was associated with increased pain scores compared to the commoner G allele (P = 0.016). This SNP was then further genotyped in 195 pain-assessed people with sciatica, 100 amputees with phantom pain, 179 individuals after lumbar discectomy, and 205 individuals with pancreatitis. The combined P value for increased A allele pain was 0.0001 in the five cohorts tested (1277 people in total). The two alleles of the SNP rs6746030 alter the coding sequence of the sodium channel Nav1.7. Each was separately transfected into HEK293 cells and electrophysiologically assessed by patch-clamping. The two alleles showed a difference in the voltage-dependent slow inactivation (P = 0.042) where the A allele would be predicted to increase Nav1.7 activity. Finally, we genotyped 186 healthy females characterized by their responses to a diverse set of noxious stimuli. The A allele of rs6746030 was associated with an altered pain threshold and the effect mediated through C-fiber activation. We conclude that individuals experience differing amounts of pain, per nociceptive stimulus, on the basis of their SCN9A rs6746030 genotype.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Schematic of the SCN9A gene showing the position of known mutations (arrows) that result in human disorders of pain appreciation. SCN9A encodes the voltage-gated sodium channel, Nav1.7, which is composed of four repeating domains, each comprising six transmembrane segments. The regions of the gene that encode the transmembrane segments and the inactivation domain are indicated (see colored key).

Fig. 2.

Biophysical properties of NaV1.7–1150R and NaV1.7–1150W currents. (A) Current responses to 50-ms voltage steps of 5-mV increments between −70 and +40 mV from a holding potential of −100 mV in a whole-cell voltage clamp recording applied at 0.5 Hz for a HEK293 cell expressing Nav1.7–1150W. (Inset) The voltage pulse protocol. (B) Current–voltage relationship of the peak currents normalized for cell size [picoamperes per picofarad (pA/pF)] obtained using the experimental setup shown in A. Lines represent fits of the data with a Boltzmann equation: ■, NaV1.7–1150W [V0.5 = −26.0 ± 2 mV, k = 4.8 ± 0.4 mV, Vrev = 64 ± 2 mV (n = 17)]; ○, Nav1.7–1150R [V0.5 = −29.0 ± 1 mV, k = 4.4 ± 0.3 mV, Vrev = 62 ± 2 mV (n = 10)]. (C) Voltage dependence of the activation time constant τm (left) and inactivation time constant τh (right), obtained by fitting current traces elicited by depolarizing pulses from −40 to +40 mV of 50 ms duration from a holding potential of −100 mV (as shown in A) with a Hodgkin–Huxley m3h model (for voltage gated sodium channels). ■, NaV1.7–1150W, O, Nav1.7–1150R. (D) Voltage dependence of the steady-state inactivation of NaV1.7–1150W currents was measured by holding the membrane potential for 500 ms at conditioning voltages from −120–0 mV (at 5-mV increments) before stepping to a test pulse at −10 mV for 50 ms. (Inset) The voltage pulse protocol, which was applied at 0.5 Hz. Only the current responses to the test pulse are shown. (E) Peak currents obtained as in D were normalized to the maximum peak current and plotted against the holding potential applied during the conditioning pulse. Lines represent a fit of the data with a Boltzmann equation: ■, NaV1.7–1150W [V0.5 = −73.0 ± 2 mV, k = 5.5 ± 0.2 mV (n = 17)]; O, Nav1.7–1150R [V0.5 = −73.0 ± 2 mV, k = 5.4 ± 0.2 mV (n = 10)]. (F) Voltage dependence of the slow steady-state inactivation of NaV1.7–1150W currents was measured by holding the membrane potential for 10 s at conditioning voltages from −120 mV to 0 mV (at 5-mV increments) before stepping up for 100 ms to −120 mV to recover channels in the fast inactivated state followed by a test pulse at −10 mV for 20 ms. (Inset) The voltage pulse protocol. Only the current responses to the test pulse are shown. (G) Peak currents obtained as in f were normalized to the maximum peak current and plotted against the holding potential applied during the conditioning pulse. Lines represent a fit of the data with a Boltzmann equation: ■, NaV1.7–1150W [V0.5 = −35 ± 3 mV, k = 11 ± 1 mV, A2 = 15 ± 2% (n = 7)]; O, Nav1.7–1150R [V0.5 = -37 ± 5 mV, k = 14 ± 1 mV; A2 = 18 ± 3% (n = 7)].