Tetrodotoxin-resistant (TTX-r) Na+ currents are involved in signal transduction and transmission in small diameter axons (e.g. reviewed by Baker & Wood, 2001). Small diameter dorsal root ganglion (DRG) neurones generate TTX-r Na+ currents that can be discriminated by their biophysical characteristics (e.g. Rush et al. 1998; Cummins et al. 1999). The NaV1.8 channel underlies high-threshold currents, and another TTX-r current (attributed to NaV1.9) can be functionally isolated in neurones from NaV1.8 null-mutant animals, although it can also be recorded in wild-type. This kinetically slow current has a voltage threshold close to -65 mV (Cummins et al. 1999), and generates a large persistent component.
DRG cultures were prepared from wild-type and NaV1.8 null-mutant mice, and from 3-week-old rats. The animals were killed humanely and the neurones prepared by enzymatic dissociation of the isolated ganglia. Voltage-clamp and current-clamp recordings were made within 2 days using the whole-cell patch-clamp technique, from neurones less than 25 mm in apparent diameter. TTX-r persistent Na+ current amplitude was up-regulated over 5 min by the inclusion of 500 mM GTP or GTP-λ-S in the recording pipette, up to one order of magnitude, but not by GDP. Following up-regulation, the voltage threshold fell by 15.74 ± 2.24 mV (n = 6) when measured in current-clamp from a holding potential of -90 mV. In neurones not generating the current, threshold increased by 3.72 ± 1.29 mV (n = 27; P < 0.0001, Student’s two-tailed, unpaired t test) over a similar period and threshold fell by 1.05 ± 0.46 mV (n = 15; P < 0.001, Student’s two-tailed, unpaired t test) with GDP internal. All values are given as means ± S.E.M., and data from NaV1.8 null-mutant and wild-type neurons are pooled. The fall in voltage threshold led to a breakdown of accommodation with just supra-threshold depolarization (Fig. 1). We suggest that up-regulation of the persistent current could induce sustained firing in pain fibres and may represent a way in which the excitability of C-fibres can be changed by inflammatory mediators.
We thank the MRC and The Wellcome Trust for support.
All procedures accord with current UK legislation.
