NaV1.8 is a tetrodotoxin-resistant (TTX-r) Na+ channel that normally functions only in small diameter primary sensory neurones, and it is known to play a role in pain-pathways. The heterologous expression of peripheral nerve TTX-r Na+ channels in mammalian systems has proved problematic, with low probabilities of functional expression and abnormal channel voltage-dependence and kinetics. We now know that poor expression may result from a requirement for accessory proteins, including the chaperone protein p11 (Okuse et al. 2002). However, Na+ channel α-subunits in nerve are normally associated with β-subunits (e.g. Chen et al. 2002), and there is evidence that co-expression of NaV1.8 with the β1 subunit increases functional expression when mRNA is injected into Xenopus oocytes (Vijayaragavan et al. 2001).
We have used lipofectamine to co-transfect green fluorescent protein (GFP, 0.4 Ág) and an excess of cDNA for NaV1.8 in pRK7 vector (0.7 Ág), with and without one of 3 different β-subunits, 1 (0.7 Ág), 1A (0.7 Ág) and 3 (0.7 Ág) in 7 µl lipofectamine into a COS-7 cell line cultured in 35 mm Petri dishes. The lipofectamine DNA suspension was in contact with the cells for 2 h before washing, and 48 h elapsed before recording from fluorescent cells began.
We found functional expression of NaV1.8 in only 15.6 % of fluorescent cells (total n = 96), when the α-subunit alone was transfected with GFP. There was no significant change in the probability of functional expression with β3 and β1A subunits, but co-transfection with β1 reduced the probability to 2.2 % (total n = 45, P < 0.05 Chi Square test, Yates corrected). The maximal Na+ current density was unaffected by co-transfection of β3 (17.9 ± 5.73 pA pF-1 α-subunit alone, n = 7, versus16.85 ± 1.85 pA pF-1 α + β3 subunits, n = 6, means ± S.E.M.) however the average maximal current density was reduced by β1A to 5.84 pA pF-1 (n = 3), although the effect was not statistically significant. The current voltage-dependence and kinetics appeared unchanged by the co-transfection of β-subunits, although the slope of the voltage-dependence of inactivation was non-significantly increased.
Our findings suggest that co-transfection of Na+ channel β-subunits does not appear to be sufficient to recapitulate neuronal NaV1.8 channel behaviour in a COS-7 heterologous expression system.
We thank Lori Isom for the β-subunit clones. This work was supported by the MRC and the Wellcome Trust