Molecular basis for pore blockade of human Na ⁺ channel Na _v 1.2 by the μ-conotoxin KIIIA

Abstract

The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo–electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the μ-conotoxin KIIIA, in the presence of an auxiliary subunit, β2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of β2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.

Molecular Determinants of μ-Conotoxin KIIIA Interaction with the Human Voltage-Gated Sodium Channel NaV1.7

“…We also found strong coupling energies between K7 on KIIIA and E919 on hNav1.7 (2.5 kcalmol -1 ) ( Table 4). These results are consistent with the interactions observed in our model ( Figure 4A) and the recent structure of KIIIA -Nav1.2 complex (18).…”

“…The backbone root mean square deviation (RMSD) of the KIIIA -hNav1.7 model and the KIIIA -hNav1.2 structure over the toxin binding region (formed by KIIIA, P1-helix, P2-helix, and extracellular loops) is ~1.0 Å. Specific pairwise contacts between K7-E919 and H12-D923 in our KIIIA -hNav1.7 model validated by mutant cycle analysis appeared to be exact in the KIIIA -hNav1.2 complex structure with the corresponding pairwise contacts between K7-E945 and H12-D949 ( Figure 4A and B) (31). Our KIIIA -hNav1.7 model also correctly predicted other non-tested contacts, including pairwise interactions between KIIIA N3 and W8 with E307 and Y339, respectively, on the extracellular S5-P1 loop of DI ( Figure 4A and B).…”

“…Interestingly, the KIIIA residues D11 and R10 in our KIIIA -hNav1.7 model are positioned similarly in the KIIIA -hNav1.2 structure, but details of toxinchannel interactions are different ( Figure 4A). D11 forms a hydrogen bond with T1398 on the P2-helix in DIII in our KIIIA -hNav1.7 model ( Figure 4A), but the substitution of Thr (T1398) hNav1.7 to Met (M1425) on the P2-helix in DIII in hNav1.2 removes this potential interaction and promoting a new hydrogen bond to be formed with the nearby residue Y1429 (31). Remarkably, residues T1398 and I1399 on the P2-helix in DIII of hNav1.7 are Met and Asp, respectively, in all other human Nav channels.…”

“…The cryo-EM structure of the KIIIA -hNav1.2 complex was published in January of 2019 (18) while this manuscript was in preparation. Our computational model of hNav1.7, docking of KIIIA to the hNav1.7 model, and functional testing of KIIIA and hNav1.7 mutations presented here were completed prior to the publication of KIIIA -hNav1.2 complex structure and the availability of the PDB coordinates.…”

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

“…For instance, we sought to understand the pore blocker effect of μ‐conotoxin KIIIA (a peptide toxin isolated from Conus kinoshitai ) on sodium channels. The structure of the complex of KIIIA and human sodium channel hNa v 1.2 has been solved, informing the development of subtype‐specific pore blockers . However, we found that native KIIIA ( 1 ) is prone to disulfide scrambling, complicating biochemical and pharmacological experiments.…”