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Outward stabilization of the S4 segments in domains III and IV enhances lidocaine block of sodium channels
Author(s) -
Sheets Michael F.,
Hanck Dorothy A.
Publication year - 2007
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2007.134262
Subject(s) - chemistry , sodium channel , gating , biophysics , sodium , intracellular , lidocaine , cysteine , extracellular , stereochemistry , biochemistry , anesthesia , biology , medicine , organic chemistry , enzyme
The anti‐arrhythmic drug lidocaine has been shown to have a lower affinity for block of voltage‐gated sodium channels at hyperpolarized potentials compared to depolarized potentials. Concomitantly, lidocaine reduces maximum gating charge ( Q max ) by 40% resulting from the complete stabilization of the S4 in domain III in an outward, depolarized position and partial stabilization of the S4 in domain IV in wild‐type Na + channels (Na V 1.5). To investigate whether the pre‐positioning of the S4 segments in these two domains in a depolarized conformation increases affinity for lidocaine block, a cysteine residue was substituted for the 3rd outermost charged residue in the S4 of domain III (R3C‐DIII) and for the 2nd outermost Arg in S4 of domain IV (R2C‐DIV) in Na V 1.5. After biotinylation by exposure to extracellular MTSEA‐biotin the mutated S4s became stabilized in an outward, depolarized position. For Na + channels containing both mutations (R3C‐DIII + R2C‐DIV) the IC 50 for rested‐state lidocaine block decreased from 194 ± 15 μ m in control to 28 ± 2 μ m after MTSEA‐biotin modification. To determine whether an intact inactivation gate (formed by the linker between domains III and IV) was required for local anaesthetic drugs to modify Na + channel gating currents, a Cys was substituted for the Phe in the IFM motif of the inactivation gate (ICM) and then modified by intracellular MTSET (WT‐ICM MTSET ) before exposure to intracellular QX‐222, a quarternary amine. Although WT‐ICM MTSET required higher concentrations of drug to block I Na compared to WT, Q max decreased by 35% and the V 1/2 shifted leftward as previously demonstrated for WT. The effect of stabilization of the S4s in domains III and IV in the absence of an intact inactivation gate on lidocaine block was determined for R3C‐DIII + ICM, R2C‐DIV + ICM and R3C‐DIII + R2C‐DIV + ICM, and compared to WT‐ICM. IC 50 values were 1360 ± 430 μ m , 890 ± 70 μ m , 670 ± 30 μ m and 1920 ± 60 μ m , respectively. Thermodynamic mutant‐cycle analysis was consistent with additive (i.e. independent) contributions from stabilization of the individual S4s in R3C‐DIII + ICM and R2C‐DIV + ICM. We conclude that the positions of the S4s in domains III and IV are major determinants of the voltage dependence of lidocaine affinity.