Structural basis for pore blockade of the human cardiac sodium channel Na_v1.5 by tetrodotoxin and quinidine

Abstract

Among the nine subtypes of human voltage-gated Na + (Na v ) channels, tetrodotoxin (TTX)-resistant Na v 1.5 is the primary one in heart. More than 400 point mutations have been identified in Na v 1.5 that are associated with cardiac disorders exemplified by type 3 long QT syndrome and Brugada syndrome, making Na v 1.5 a common target for class I antiarrythmic drugs. Here we present the cryo-EM structures of Na v 1.5 bound to quinidine and TTX with resolutions at 3.2 Å and 3.3 Å, respectively, for the pore domain. The structures allow mapping of 278 disease-related mutations in the resolved region, establishing the framework for mechanistic investigation of these pathogenic mutations. Quinidine is positioned right below the selectivity filter and coordinated by residues from repeats I, III, and IV. Pore blockade is achieved through both direct obstruction of the ion permeation path and induced rotation of a Tyr residue that leads to intracellular gate closure. Structural comparison of Na v 1.5 with our previously reported Na v 1.2/1.4/1.7 reveals the molecular determinant for the different susceptibility to TTX by TTX-resistant and -sensitive Na v subtypes, and highlights the functional significance of the underexplored and least conserved extracellular loops.

Modeling the Interactions Between Sodium Channels Provides Insight Into the Negative Dominance of Certain Channel Mutations

“…In such frameworks, interactions between channels can be explored by adding a Hamiltonian term to the energy profile, as commonly done in physics for multi-body dynamic problems. As the structure of the Na v 1.5 channel is presently established (Li et al, 2019;Jiang et al, 2020), such approaches can in the future also be applied to this channel. Furthermore, molecular studies of channel dimerization and its allosteric interactions with other channels and proteins could be conducted, as done for instance for the epidermal factor growth receptor (Tsai and Nussinov, 2019).…”

Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the Long QT Syndrome

“…Despite the change of the channel properties, the structure of Na v 1.5-E1784K is nearly identical to that of quinidine-bound WT Na v 1.5 (Fig. 1 C ) (16). More importantly, the last resolved residue is Glu1781 that marks the end of S6 IV .…”

“…4 C ). In the structures of EeNa v 1.4 and all resolved human Na v channels (16, 32, 36-38), the IFM motif undergoes a pronounced displacement to plug into a cavity that is constituted by the S6 helices and the S4-S5 segments in repeats III and IV (Fig. 4 C ).…”

“…Among all the structures of eukaryotic Na v channels, only two major conformations were captured. The structure of Na v PaS is featured with closed PD without fenestration, a CTD-sequestered III-IV linker, and VSDs in less polarized conformations (33, 34); while all the human, electric eel, and rat Na v channels exhibit similar conformations characterized by the insertion of the IFM motif into a cavity outside the S6 tetrahelical bundle (16, 32, 36-38, 43). Although the functional state of the Na v PaS structure cannot be defined due to its resistance to electrophysiological recording, the conformations of the PD, CTD, and the III-IV linker are consistent with those expected in a resting or pre-open channel.…”

“…To further the molecular understanding of Na v 1.5 channelopathies, we sought to solve structures of human Na v 1.5 with disease mutations in addition to our previous structural resolution of wild type (WT) human Na v 1.5 bound to pore blockers (16). We started with Na v 1.5-E1784K, which is the most common mutation shared by LQT3 and BrS.…”