A conserved ring of charge in mammalian Na+ channels: a molecular regulator of the outer pore conformation during slow inactivation

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Abstract
The molecular mechanisms underlying slow inactivation in sodium channels are elusive. Our results suggest that EEDD, a highly conserved ring of charge in the external vestibule of mammalian voltage‐gated sodium channels, undermines slow inactivation. By employing site‐directed mutagenesis, we found that charge alterations in this asymmetric yet strong local electrostatic field of the EEDD ring significantly altered the kinetics of slow inactivation gating. Using a non‐linear Poisson–Boltzmann equation, quantitative computations of the electrostatic field in a sodium channel structural model suggested a significant electrostatic repulsion between residues E403 and E758 at close proximity. Interestingly, when this electrostatic interaction was eliminated by the double mutation E403C + E758C, the kinetics of recovery from slow inactivation of the double‐mutant channel was retarded by 2500% compared to control. These data suggest that the EEDD ring, located within the asymmetric electric field, is a molecular motif that critically modulates slow inactivation in sodium channels.