3.1 | Analysis of Kv1.5 inhibition by bupivacaine
First, we analyzed the inhibition
of Kv1.5 channels by racemic R/S-bupivacaine using the Xenopus
laevis oocyte expression system (Figure 1a). The IC50of bupivacaine was 16.9 ± 3.7 µM with a Hill coefficient of 0.76 ± 0.08
(Figure 1b). Bupivacaine did not cause a major shift in the
voltage-dependence of the Kv1.5 activation curve (Figure 1c). The
inhibition of Kv1.5 by bupivacaine was voltage-dependent, as previously
described (Gonzalez, Longobardo et al. , 2001; Valenzuela, Delponet al. , 1997). However, in the voltage range of 0 to +70 mV when
the inner gate is primarily in the open state, the block was
voltage-independent (Figure 1d). To probe whether the block by
bupivacaine requires repetitive channel openings or has a closed state
dependence, we measured the currents in the absence of bupivacaine
(I ctrl) and after 12 minutes of drug application
while the channels were held at -80 mV in the closed state
(I bupi) (Figure 1e,f). These recordings revealed
that Kv1.5 channels have to be opened before bupivacaine can block the
channels and that a rapid open channel block already reaches steady
state inhibition within the first test pulse (Figure 1e,f).