3.6 The specificity of binding.
Next, we investigated how specific the binding was, by testing the
F1579A binding site mutant channels. We expected that if the binding was
indeed specific, then we would see neither inhibition of the amplitude
nor modulation of gating. Interestingly, mutation of the binding site
did not prevent inhibition of the amplitude (provided that channels
assumed inactivated conformation), but the modulation of gating was
radically decreased (Fig. 6A, B, C). This suggests that when the
high-affinity binding site is disrupted, inhibition is still possible,
probably by binding to secondary binding sites, but these binding sites
are less potent in conferring gating modulation. The involvement of this
phenylalanine in the mechanism of modulation has been shown before
(Ahern et al., 2008; Arcisio-Miranda et al., 2010; Hanck et al., 1994;
Hanck et al., 2009; Liu et al., 2003; Muroi & Chanda, 2009), therefore
the lack of significant modulation is not unexpected. However, the fact
that mutant channels could still be effectively inhibited in inactivated
state, was indeed unexpected, and it suggests the existence of one or
more additional binding sites. Association to these lower affinity
binding sites, which too were only available in the inactivated
conformation, caused predominantly channel block, and less modulation.