b1 at more negative membrane potentials (Fig. 2C). The
voltage of half-maximal activation (V1=2) shifted towards more
hyperpolarised voltages with -
12.0 mV when 5 lM BTX was
applied ðn ¼ 6Þ and with -
14.8 mV in the presence of 10 lM
BTX ðn ¼ 4Þ (Table 1). As can be seen in part IV of Fig. 2A,
Nav1.8/b1 activates at )20 mV under the influence of BTX,
whereas in control conditions the channels are still closed. As a
result of the total loss of inactivation and the leftward shift of
the activation curve, the Naþ ion influx increases dramatically.
A clear marker to demonstrate this is the area under the I–V
curve (AUC) (Fig. 2B). Compared to the control situation, the
AUC increases with -
427% when 10 lM BTX is present
(n ¼ 4, Table 1). BTX also changes the ion selectivity as in-
dicated by the shift in the reversal potentials (Erev) (Fig. 2B).
An average hyperpolarising shift of 5.0 mV is present when 5
lM BTX is added ðn ¼ 6Þ and a shift of 11.1 mV was noted in
the presence of 10 lM BTX ðn ¼ 4Þ (Table 1). So both the
gating and selectivity of Nav1.8 are affected by BTX, with the
effect on inactivation being the most drastic. Lower concen-
trations were also tested and we observed that 1 lM of BTX,
measured after a 900 pulse train experiment at a frequency of 1
Hz, already starts to affect Nav1.8/b1. At this concentration, a
small initial block of the current is seen, which is soon followed
by an influx of Naþ ions caused by the inhibition of the in-
activation. In addition, the activation is clearly shifted to more
hyperpolarised voltages. In order to simulate neurons in living
organisms, which have a higher firing rate than 1 Hz (e.g., 20–
50 Hz), we also tested higher frequency pulse trains. At 10 Hz,
we noticed that lower concentrations (500 nM) cause effects
that are related to higher concentrations at 1 Hz (data not
shown). Hence, we believe that in vivo the effects of BTX may
be as catastrophic at even lower concentrations