Structural basis for severe pain caused by mutations in the S4-S5 linkers of voltage-gated sodium channel Na V 1.7

Figures

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  Fig. 1.. IEM mutations in voltage sensors of NaV1.7. (A) Topology diagram of NaV1.7 with seven IEM mutations (red circles) in the VSDs and the S4-S5 linkers of DI and DII selected for this study. Equivalent IEM mutations were introduced into the homotetrameric bacterial channel NaVAb (cyan) for electrophysiological and structural characterization. (B) Structure of human NaV1.7 (PDB: 6J8I) with the locations of three IEM mutations mapped onto the model (red spheres). Each domain is colored as in A. (C) Structure of NaVAb (PDB: 3RVY) with the locations of three IEM mutations mapped onto the model (red spheres). For clarity, the IEM mutations are shown only in one subunit (teal). Due to the tetrameric nature of NaVAb, the mutations are also present in the remaining subunits (cyan). Residues are labeled with domain colors of their equivalent mutations in NaV1.7 as in B. (D) View of IEM mutations (red spheres) in NaV1.7 from the cytosolic side. (E) View of IEM mutations (red spheres) in NaVAb from the cytosolic side. The structure of NaV1.7 from panel D is superimposed (with the IEM mutations shown as pink spheres) with an rmsd of ~2.0 Å for Cα atoms in the S4-S5 linkers and PM.

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  Fig. 2.. Functional properties of NaVAb with IEM mutations. (A) Families of sodium currents. NaVAb/Δ28 WT and mutants were expressed in Hi5 insect cells and studied by whole-cell voltage clamp recording as described in Materials and Methods. (B) Time to reach peak current is plotted as a function of stimulus voltage as calculated from the results of panel A. (C) Mean time constants for inactivation calculated from the results of experiments similar to those in panel A as described in Materials and Methods. (D) Voltage dependence of activation of WT and the indicated IEM mutants in the S4-S5 linker. Current/voltage (I/V) relationships of the peak sodium currents were recorded in response to steps to voltages ranging from −160 mV to +60 mV in 10-mV increments from a holding potential of −160 mV or −180 mV. Conductance/voltage (G/V) relationships were calculated as described in Materials and Methods. (E) Voltage-dependent activation curves determined from peak tail current amplitudes measured at −180 mV. Short depolarizing pulses, 10 ms, were applied from a holding potential of −180 mV in 10-mV steps. Tail currents were normalized to the highest amplitudes. (Inset) Representative traces of…

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  Fig. 3.. Structure of NaVAb/I119T. (A) Crystal structure of NaVAb/I119T with the mutation shown. (B) Close-up view of the NaVAb/I119T structure with the VS in the activated state (teal and cyan for different subunits). Side chains of residues I119T (red), I217 (teal) and key side chains in S5 and S6 of a neighboring subunit (cyan) including Ser132′ and Asn211′ are shown as sticks with the σA-weighted 2FO-FC electron density map contoured at 1.0σ level overlaid (mesh). (C) Comparison of NaVAb/I119T structure with the structure of NaVAb WT (light orange). Side chain of I119T forms a hydrogen bond (black dashes) with side chain of Ser132′. Conformational changes in the C-terminal end of the S6 segments starting from Asn211′ in the mutant structure abolish the CHAPSO binding site present in the WT structure. (D) Homology model of NaVAb/I119T structure in the resting state. The van der Waals hemispheres are shown as dots. NaVAbΔ28 (PDB: 6MWA) was used for the comparison.

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  Fig. 4.. Structure of NaVAb/L123T. (A) Crystal structure of NaVAb/L123T with the mutation shown. (B) Close-up view of NaVAb/L123T structure with the VS in the activated state (teal and cyan for different subunits). Side chains of residues L123T (red) and Asn211′ (cyan) are shown as sticks with the σA-weighted 2FO-FC electron density map contoured at 1.0σ level overlaid (mesh). (C) Comparison of NaVAb/L123T structure with the structure of NaVAb WT (light orange). Side chain of N211′ makes a van der Waals contact with the side chain of V126 in the WT structure but rotates to form a hydrogen bond (black dashes) with side chain of L123T in the mutant structure. The van der Waals hemispheres are shown as dots. (D) Homology model of NaVAb/L123T structure in the resting state.

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  Fig. 5.. Structure of NaVAb/V126T. (A) Crystal structure of NaVAb/V126T with the mutation shown. (B) Close-up view of NaVAb/V126T structure with the VS in the activated state (teal and cyan for different subunits). Side chains of residues V126T (red), Ile216 (teal), and Asn211’ (cyan) are shown as sticks with the σA-weighted 2FO-FC electron density map contoured at 1.0σ level overlaid (mesh). (C) Comparison of NaVAb/V126T structure with the structure of NaVAb WT (light orange). Side chain of V126T rotates to form a hydrogen bond (black dashes) with side chain of Asn211’. (D) Homology model of NaVAb/L123T structure in the resting state. The van der Waals hemispheres are shown as dots.

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  Fig. 6.. Potential hydrogen bond formation between mutant IEM residues in the S4-S5 linker and the PM of NaV1.7. (A) NaV1.7/I234T. (B) NaV1.7/S241T. (C) NaV1.7/I848T.