3.4 | Identification of the ropivacaine binding site in the central cavity and ‘side pockets´ of Kv1.5 channels
Next we probed the binding site of ropivacaine (which is the S-enantiomer) that also blocks Kv1.5 channels (Valenzuela, Delponet al. , 1997), albeit with a reduced stereoselectivity of inhibition (θ = 2.5-fold) (Longobardo, Delpon et al. , 1998). The following experiments were performed to probe whether the differential binding sites for these two local anesthetics actually determine the reduced stereoselectivity and affinity of ropivacaine compared to bupivacaine. First, ropivacaine had an IC50 of 141.6 ± 6.7 µM with a Hill coefficient of 1.1 ± 0.02 (Figure 3a,b) and thus was, as previously reported, less potent than bupivacaine. Ropivacaine caused a voltage-independent inhibition in the depolarized voltage range of 0 to +70 mV (Figure 3d) and blocked Kv1.5 channels with an open state affinity (Figure 3e,f), similar as bupivacaine.
The potency of ropivacaine was reduced for the S6 segment mutants T480A, L510A, V512A and V516A that either face the pore or the ‘side pockets´, similar as for bupivacaine (Figure 3g versus Figure 1g). For the I502A mutant that faces into fenestrations that connect the central cavity with the ‘side pockets´, the effects were somewhat more pronounced and with L506A and I508A we identified two additional pore facing residues to be relevant for the inhibition by ropivacaine. Thus, also for ropivacaine the results support the idea that the drug interacts with the classical binding site in the central cavity and the selectivity filter, as well as with parts of the ‘side pockets´ of Kv1.5.
Strikingly, we also identified for ropivacaine L436, F439, F440 and I443 in the S5 segment, residues that face into the ‘side pockets´ (Figure 3g). However, with L437 we identified one additional residue in the S5 segment that faces into fenestrations connecting the ‘side pockets´ to the central cavity, similar as I502 does (Marzian, Stansfeld et al. , 2013). Most importantly, ropivacaine requires interactions with residues of the S4 segment (L413) and the S4-S5 linker (L420), residues that were previously reported to interact with Psora-4 bound to the ‘side pockets´ (Marzian, Stansfeld et al. , 2013). Thus, ropivacaine binds to the central cavity and the ‘side pockets´ of Kv1.5 channels. In addition, ropivacaine, which has a reduced stereoselectivity and affinity, must utilize a different binding site or mode in the ‘side pockets´.
Also for ropivacaine, plotting the extent of C-type inactivation of single mutants against their respective inhibition, did not reveal a correlation between the inactivation properties of the mutants and their ropivacaine affinity (Figure 3h). Thus, the residues identified by our alanine scan most likely exhibit a reduced affinity, due to an impaired drug binding.