Volume 13 Supplement 1
New structural determinants of charged local anaesthetic block of voltage-gated sodium channels
© Lukács et al; licensee BioMed Central Ltd. 2012
Published: 17 September 2012
Some blockers of voltage-gated Na+ and Ca2+ channels are assumed to pass through the membrane and then bind to amino acids in the internal vestibule by access from the internal side of the membrane. However, in the heart isoform of the voltage-gated Na+ channel, in L-type calcium channels and in T-type calcium channels an additional external access pathway (EAP) through the protein has been suggested. Furthermore, in voltage-gated Na+ channels (NaV) mutations at a specific site in the middle of the domain IV transmembrane segment 6 (site 1575 in rNaV1.4, 1760 in rNaV1.4) open an EAP for QX-222, a permanently charged, hydrophilic lidocaine analogue. Recently, the first crystal structure of a NaV was published . In this bacterial channel structure (NaVAb) the side chain homologous to rNaV1.4 I1575 (I202 in NaVAb) is in close contact with a pore-loop sidechain, homologous to rNaV1.4 W1531 (W179 in NaVAb). In contrast, in all currently available structural homology models of NaV, W1531 is not in contact with I1575. If W1531 were positioned as suggested in the NaVAb structure then a reduction in the length of the side chain at this site would be predicted to open the EAP. To test this hypothesis we generated the mutations W1531A and W1531G and tested these constructs for block by external QX-222.
Whole-cell patch clamp measurements were done on TsA 201 cells transiently transfected with plasmids coding the rNaV1.4 α subunit and its mutants, the sodium channel β1 subunit and GFP. Block levels were derived at 2 Hz stimulation frequency from a holding potential of −120 mV.
Mutations W1531A and W1531G were found to be sensitive to extracellular QX-222 (block: 20.6 ± 2% and 17.7 ± 3.5%, respectively).
Our results indicate that position 1531 is an important part of the EAP in rNaV1.4, as predicted from the crystal structure of NaVAb. Thus the bacterial channel NaVAb appears to share important structural motifs with eukaryotic sodium channels.
This study was funded by the Austrian Science Fund (FWF, grants P210006-B11 and W1232-B11).
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.