3.3 | Binding to both, the central cavity and the ‘side
pockets´ is an essential prerequisite for stereoselective inhibition of
Kv1.5 by bupivacaine
Bupivacaine causes a strong stereoselective inhibition of Kv1.5 (Arias,
Guizy et al. , 2007; Franqueza, Longobardo et al. , 1997;
Valenzuela, Delpon et al. , 1995) with the R- enantiomer being
9-fold (θ) more potent (Figure 2a,f). Mutants at T507, L510 and V514
were previously reported to modulate the stereoselectivity of
bupivacaine inhibition (Franqueza, Longobardo et al. , 1997). Due
to the Kv1.2 crystal structure we know by now that these residues are
located at the backside of S6 and face into the ‘side pockets´.
Strikingly, we found that the S5 mutants F440A and I443A drastically
reduced the stereoselectivity of bupivacaine (Figure 2b-f) with an
almost complete loss of stereoselectivity for I443A (Figure 2d,f).
Mutants at T479 located in the pore signature sequence facing the
central cavity did not affect the stereoselectivity of bupivacaine
inhibition (Franqueza, Longobardo et al. , 1997). In addition, we
found that T479A does not cause a major reduction in bupivacaine
inhibition (Figure 1g), presumably as the threonine side chain is not
perfectly facing into the central cavity. In contrast, mutating the
neighbouring residue T480 caused a drastic reduction in bupivacaine
affinity (Figure 1g) and an almost complete loss of stereoselectivity
inhibition of Kv1.5 by bupivacaine (Figure 2e,f). These experiments show
that residues in the central cavity and the ‘side pockets´ are crucial
determinants for the stereoselectivity inhibition of Kv1.5 by
bupivacaine. The fact that residues in the ‘side pockets´ determine
bupivacaine affinity and stereoselectivity strongly argues for a binding
of this local anesthetic to the ‘side pockets´ of Kv1.5 channels.