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.