Sodium channels and mammalian sensory mechanotransduction

Abstract

Background: Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. β and γENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results: Here we show that deleting β and γENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion: Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.

Figure 1

Acute pain thresholds are normal in β and γ ENaC KO mice. a) The Randall-Selitto test between βENaC^flox/flox and Nav1.8-Cre/βENaC^flox/floxmice does not show any difference in behavior (t test, p = 0.6, n = 8 and 9 respectively). b, c and d) comparison of βENaC^flox/flox and Advillin-Cre/βENaC^flox/flox mice in the Von Frey test (t test, p = 0.64, n = 6 and 7 respectively), the Hargreaves test (t test, p = 0.6, n = 8 and 7 respectively) and the rotarod test (t test, p = 0.88, n = 4 and 4 respectively). e) Randall-Selitto test between γENaC^flox/flox and Nav1.8-Cre/γENaC^flox/flox mice (t test, p = 0.57, n = 10 and 10 respectively). f,g and h) comparison of γENaC^flox/flox and Advillin-Cre/γENaC^flox/flox mice in the Von Frey test (t test, p = 0.13, n = 11 and 13 respectively), the Hargreaves test (t test, p = 0.26, n = 13 and 12 respectively) and the rotarod test (t test, p = 0.56, n = 4 and 6 respectively).

Figure 2

Mechanically-activated currents are normal in Nav1.7/Nav1.8 DKO neurons. a) Three types of mechanically activated currents are evoked in cultures of DKO DRG neurons Left to right, examples of SA, RA and IA currents are depicted. The mechanical stimulation protocol is shown below each trace. b) The bar graph depicts percentage of each type of current kinetics observed in DKO and wild type littermates. The percentage of each type of MA currents in DKO neurons was similar to that of the WT littermates (Chi square test, p = 0.9268). c) The mean amplitude of peak SA/IA and RA currents measured at 8 μm were not significantly different between the DKO and wild type neurons (one way ANOVA, p = 0.8167).

Figure 3

Comparison of the properties of action potentials obtained in the three genotypes. The properties of wide and narrow action potentials obtained from each genotype are shown. See materials and methods for calculation of the parameters. a, b, c, d) There was no significant difference between parameters of narrow action potentials in WT and DKO (ANOVA, p > 0.05, n = 4 and 5). c) The wide action potentials were slower in DKO and Nav1.7 KO compared to WT (One way ANOVA, p < 0.001, n = 18, 7 and 18 respectively). d) DKO action potentials had lower amplitudes than WT or Nav1.7KO (One way ANOVA, p = 0.02, n = 18, 17 and 7 respectively).

Figure 4

Nav1.7/Nav1.8 DKO DRG neurons are deficient in their ability to generate action potentials. a,b) Representative traces of action potentials (upper traces) evoked in response to depolarizing current injections (lower traces) are shown. Wide (a) and narrow (b) action potentials are observed in DKO neurons. c) Illustrates a representative example of graded responses to increasing depolarizing currents in a DKO neuron. d) Same cell as c, holding the neuron at -90 mV prior to depolarizing current injections resulted in generation of an all- or-none like action potential with a fast overshoot. e) Mean resting membrane potential for neurons with wide, narrow and no elicited action potentials (no AP) are shown. f) The bar graph shows the total percentage of neurons capable of firing action potentials in WT, Nav1.7KO and Nav1.7/Nav1.8 knock out mouse DRG neurons. Both DKO and Nav1.7KO groups are different from WT in having percentage of neurons not firing AP (Chi-square test p < 0.001 and p = 0.0072 respectively). Proportion of neurons firing action potentials in DKO or Nav1.7 KO are not significantly different from each other (Chi-square test p = 0.4306).

Figure 5

Current-voltage relationship in cells that can generate all or none action potentials and those responding with small graded potentials. a, b) Representative current-voltage (I-V) relationships in WT cell responding to increasing step current injections by eliciting all-or-none action potentials (a) is depicted in b (arrows). Generation of an action potential is marked by the non-linearity in the relationship at the point where additional stimulation does not result in significantly increased potentials (arrow). b, c) examples of I-V relationship for wide and narrow action potentials from WT and DKO neurons. d, e) I-V relationship for a DKO cell eliciting graded responses to step current injections depicted in e (arrows). Note the linear I-V relationship (e, closed squares) where no significant overshoot is present (d). Holding the same cell at -90 mV results in appearance of an overshoot with non-linear I-V relationship (d, open squares). f)In a Nav1.7 KO cell responding with small graded potentials the I-V relationship is linear hence all-or-none action potentials are not generated compared to a Nav1.7KO cell capable of generating action potentials.

Figure 6

Cre-mediated deletion of β and γ ENaC subunits in DRG neurons in mice. a) PCR detection of the Cre recombinase band in genomic DNA from heterozygous Nav1.8-Cre (left) and Advillin-Cre (right) mice. b) Detection of floxed and WT bands in WT, heterozygous and homozygous βENaC^flox/flox mice (top) and of the KO band in the DRG but not the ear tissue of Nav1.8-Cre mice crossed to homozygous βENaC^flox/flox (bottom). c) detection of floxed and WT bands in WT, heterozygous and homozygous γENaC^flox/flox mice (top) and of the KO band in the DRG but not the ear tissue of Advillin-Cre mice crossed to homozygous γENaC^flox/flox (bottom).