Background:

Lidocaine, a frequently used local anaesthetic drug is believed to reach its binding site in the intracellular vestibule of voltage-gated sodium channel (Na_V) via the cell membrane. QX-222 is a permanently charged, quaternary amine analogue of lidocaine that can access this binding site via a hydrophilic route across the channel protein in the wild-type heart sodium channel (Na_V1.5). In muscle type channel (Na_V1.4) this route is closed. In these channels, mutation I1575A in the upper part of the S6 segment in domain IV have been shown to open such an external access pathway (Sunami et al. Mol. Pharmacol. 59:684).

Question:

In the first crystal structure of a Na_V (NavAb, Payandeh et al. Nature 475:353) a tryptophan (W179, homologous to W1531 in rNav1.4) of the P-loop is positioned in close proximity to I202 in S6 segment (homologous to I1575 of rNav1.4). If this interaction is present in the mammalian channels, then mutations at site W1531 may also open the EAP. Methods: Whole-cell patch clamp measurements were performed on tsA201 cells transiently transfected with Na_V1.4 its mutants. Development of block was assessed by application of 25 ms pulses to 0 mV at 2 Hz stimulation frequency from a holding potential of -120 mV.

Results:

QX-222 blocked currents through W1531A and W1531G by 21±3% and 15±2%, respectively. In both constructs block development was extremely fast (time constants: ~3s and ~2s for W1531A and W1531G), i.e. ~10-20 fold more rapid than I1575A (~40s). Thus, mutations at site 1531 open an access pathway allowing for rapid block by QX-222, as predicted from the crystal structure of Na_VAb.

Conclusions:

Our results showing that W1531 and I1575 are in close proximity in Na_V1.4 similar to Na_VAb. W1531 seems be the hydrophobic barrier between the inner and outer vestibule of the sodium channel.

Funded by Austrian Science Fund (FWF, P210006-B11 and W1232-B11)