Permeation, Gating, and Modulation of the TRPA1 Channel in Long-Timescale Molecular Dynamics Simulations

Abstract

The cation channel TRPA1, a member of the transient receptor potential (TRP) ion channel superfamily, is a promising drug target in pain and inflammatory diseases, but its function and dynamics—including ion permeation and channel gating—are not well understood at the structural level. We have used the recently published cryo-EM structure of the closed, non-conducting state of TRPA1 (Paulsen et al., Nature, 2015) as a starting point to study the functional states and activation of this channel using long-timescale, all-atom molecular dynamics simulations enabled by special-purpose hardware. A key functional region of TRPA1—the channel's selectivity filter (SF)—was unstable in simulations started from the cryo-EM model of the closed state, even when using extensive restraints. We constructed an alternative model of the SF region based on a high-resolution crystal structure of a closely related ion channel. This SF model, which is compatible with experimental data, remained stable on simulation timescales of 100 µs or longer, allowing us to study ion permeation in TRPA1 at the single-ion level. To study ion permeation across the open channel, we introduced a mutation (V961R) within the TRPA1 internal gate, leading to a rapid pore opening. Upon subsequent back mutation to the wild-type protein, the pore remained in an open, conducting conformation. Additional simulations under an applied membrane potential, moreover, led to direct observation of inward and outward ionic currents and suggested a novel structural determinant of channel gating within the SF region. We also studied modulation of the open and conducting channel by performing free binding simulations with the TRPA1 inhibitor, A-967079, which, intriguingly, was observed to bind in a location previously hypothesized to be a potential binding site.