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.