Charge at the lidocaine binding site residue Phe‐1759 affects permeation in human cardiac voltage‐gated sodium channels

Abstract

Our homology molecular model of the open/inactivated state of the Na + channel pore predicts, based on extensive mutagenesis data, that the local anaesthetic lidocaine docks eccentrically below the selectivity filter, such that physical occlusion is incomplete. Electrostatic field calculations suggest that the drug's positively charged amine produces an electrostatic barrier to permeation. To test the effect of charge at this pore level on permeation in hNa V 1.5 we replaced Phe-1759 of domain IVS6, the putative binding site for lidocaine's alkylamino end, with positively and negatively charged residues as well as the neutral cysteine and alanine. These mutations eliminated use-dependent lidocaine block with no effect on tonic/rested state block. Mutant whole cell currents were kinetically similar to wild type (WT). Single channel conductance (γ) was reduced from WT in both F1759K (by 38%) and F1759R (by 18%). The negatively charged mutant F1759E increased γ by 14%, as expected if the charge effect were electrostatic, although F1759D was like WT. None of the charged mutations affected Na + /K + selectivity. Calculation of difference electrostatic fields in the pore model predicted that lidocaine produced the largest positive electrostatic barrier, followed by lysine and arginine, respectively. Negatively charged glutamate and aspartate both lowered the barrier, with glutamate being more effective. Experimental data were in rank order agreement with the predicted changes in the energy profile. These results demonstrate that permeation rate is sensitive to the inner pore electrostatic field, and they are consistent with creation of an electrostatic barrier to ion permeation by lidocaine's charge. Local anaesthetic (LA) drugs such as lidocaine interfere with impulse conduction in nerve and muscle by binding to the inner pore of voltage-gated Na + channels and blocking current (Hille, 2001). The major drug mechanism of action is not resolved, with experimental evidence variously favouring steric block, stabilization of a closed state, or some combination of the two. Extensive site-directed mutagenesis experiments have provided strong evidence that lidocaine-like drugs (LA) bind in the inner pore. S6 segment residues in domains I, III and IV (but not II) have been shown to be important for use-dependent LA block (Ragsdale et al. 1994;Wright et al. 1998;Yarov-Yarovoy et al. 2001;Yarov-Yarovoy et al. 2002). Two residues in domain IV S6 are of particular importance -Phe-1759 (following the heart Na V 1.5 isoform numbering, corresponding to Phe-1579 in skeletal Na V 1.4 and Phe-1764 in brain Na V 1.2) and Tyr-1766 (Tyr-1586 in Na V 1.4; Tyr-1771 in Na V 1.2), because their alanine mutants exhibit the greatest changes in LA affinity. Open/inactivated state block of the brain isoform Na V 1.2 by etidocaine was reduced by 130-and 35-fold for the alanine substitutions of the phenylalanine and tyrosine, respectively (Ragsdale et al. 1994). Cysteine accessibility experiments with methanethiosulphonate (MTS) reagents confirm th...

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