The TTX-resistant sodium channel Nav1.8 (SNS/PN3): expression and correlation with membrane properties in rat nociceptive primary afferent neurons

Abstract

We have examined the distribution of the sensory neuron-specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8-like immunoreactivity (Nav1.8-LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C-, Adelta- or Aalpha/beta. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8-LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60% of C-, Adelta- and Aalpha/beta-fibre nociceptive units respectively and 88% of C-unresponsive units were positive. C-unresponsive units had electrical membrane properties similar to C-nociceptors and were considered to be nociceptive-type neurons. Weak positive Nav1.8-LI was also present in some LTM units including a C LTM, all Adelta LTM units (D hair), about 10% of cutaneous LTM Aalpha/beta-units, but no muscle spindle afferent units. Nav1.8-LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A- but not C-fibre neurons. Nav1.8-LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A-fibre neurons and with AP rise time only in positive C-fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A-fibre neurons) and larger AP overshoots that are typical of nociceptors.

Figure 1. Western blot analysis and relationship of subjective score to relative intensity of Na_v1.8-LI

A, Western blot analysis. A single band of strong immunoreactivity at approximately 240 kDa in the membrane fraction of rat DRG is shown. Note that membrane fractions of rat whole brain, cerebellum, skeletal muscle, heart, superior cervical ganglia, liver and kidney showed no detectable immunoreactivity. Membrane extract from CHO-SNS22 (a cell line stably transfected with rat Na_V1.8 cDNA) showed strong immunoreactivity at the same size with DRG. Non-transfected CHO cells did not show any bands. B, relationship of subjective score to relative intensity of Na_v1.8-LI. The x-axis shows the subjective score (0 for the most negative, 1 for just positive and 5 for the most intense Na_v1.8-LI). The y-axis shows the percentage of relative intensity of Na_v1.8-LI from 0 to 100 %, ranging from the most negative (0 %) to the most intense Na_v1.8-LI (100 %).

Figure 2. Cell area and Na_v1.8-LI relative intensity

The cross-sectional areas (x-axis) through each neuronal profile containing a nucleus (A) and through the largest section of each identified dye-injected neuron (B) are plotted against the relative intensity (percentage maximum intensity). In both cases, all neurons are shown as hatched histograms. Superimposed in grey are neurons with Na_v1.8-LI relative intensity > 20 % and superimposed in black are those with > 50 % maximum intensity. B, histograms for neurons with dorsal root C-, Aδ- and Aα/β-fibres are displayed separately. C and D, relative intensity is plotted against cell size. The lines (y-axis) show 20 and 50 % relative intensity, and those on the x-axis show the borderlines between small, medium and large neurons. C and D, regression lines, r² and P values are given where a significant correlation exists (P < 0.05). LTM, low threshold mechanoreceptors; NOC, nociceptors; UNR, unresponsive units.

Figure 3. Na_v1.8-LI intensity and conduction velocity

Na_v1.8-LI intensity plotted against dorsal root CVs for all DRG neurons examined (A) and for all A-fibre neurons (B). The P and r² values are given only if the correlation is significant (P < 0.05). Both the borderline between Na_v1.8-LI-positive and -negative neurons (20 % maximum intensity) and 50 % maximum intensity are shown by lines on the y-axis. The lines from the x-axes show the C (0.8 m s⁻¹), C/Aδ (1.4 m s⁻¹) and the Aδ/Aα/β (6.5 m s⁻¹) borderlines. NOC, nociceptive neurons; LTM, low threshold mechanoreceptive neurons; UNR, unresponsive neurons.

Figure 4. Na_v1.8-LI intensity in different neuronal types

A, scattergraph of the relative intensities of nociceptive and LTM units in all CV groups together shows a much greater median relative intensity in nociceptors (Mann-Whitney test, P < 0.0001). B, the proportions of positive (< 20 %, black) and negative (white) nociceptive and LTM units from all CV groups together are compared (χ² test, P < 0001). C, neurons are subdivided by sensory modality within C, Aδ and Aα/β CV groups. The borderline between negative (< 20 %) and positive (≥ 20 %) neurons is indicated by the line at 20 % relative intensity. Abbreviations: NOCI, nociceptive neurons; LTM, low threshold mechanoreceptive neurons; UNR, unresponsive neurons; F/G, field or guard hair LTM neurons; RA, rapidly adapting LTM neurons; SA, slowly adapting LTM neurons; MS, muscle spindle neurons; +ve, Na_v1.8-LI-positive (≥ 20 %) neurons; -ve, Na_v1.8-LI-negative (< 20 %) neurons.

Figure 5. Examples of Na_v1.8-LI in identified neurons

Shown on the left are 5 DRG neurons injected with a fluorescent dye (marked with arrowheads) and on the right are the same neurons stained for Na_v1.8-LI. Their sensory properties, dorsal root CV (bottom left) and Na_v1.8-LI relative intensity (bottom right) are given. Neurons were considered positive if their Na_v1.8-LI was ≥ 20 % and negative if it was < 20 %. HTM, high threshold mechanoreceptive neurons; CV, conduction velocity; LY, Lucifer yellow; CB, Cascade blue.

Figure 6. Na_v1.8-LI intensity and action potential shape

Only units with membrane potentials of at least −50 mV except the sole C-LTM (−48 mV) were included. Plots for AP duration at base (A), AP rise time (B) and AP fall time (C) are shown. Linear regressions were calculated for A-fibre nociceptors, A-fibre LTMs and all A-fibres together. Where regressions reached significance (P < 0.05) regression lines are shown, except for all units together (omitted for clarity). P and r² values for these and for other correlations are given in Table 1. In addition, there was a significant correlation for C-fibre nociceptor AP rise times (P < 0.05, r² = 0.91, n = 4). For abbreviations see legend to Fig. 4.

Figure 7. Na_v1.8-LI intensity and membrane potential

Membrane potentials of C-fibre neurons are plotted against Na_v1.8-LI intensity. A significant correlation was found for C-fibre nociceptive neurons but not for C-fibre unresponsive neurons. For abbreviations see legend to Fig. 4.

Figure 8. Na_v1.8-LI relative intensity versus action potential overshoot

Only units with membrane potentials of at least −50 mV except the sole C-LTM (−48 mV) and somatic APs with peaks that reached at least −20 mV at peak were included. Regression lines are shown where there was a significant (P < 0.05) linear regression. P and r² values for these correlations were: for all A-fibre units, P < 0.0001, r² = 0.56; for all positive units, P < 0.0001, r² = 0.56; for A-fibre nociceptive units P < 0.0001, r² = 0.42. For abbreviations see legend to Fig. 4.