No abstract

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here, we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane helices S5–S6 and the intervening pore loop, which is flanked by S1–S4 voltage sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including N-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

In recent work with large high symmetry viruses, single particle electron cryomicroscopy (cryoEM) has reached the milestone of determining near atomic resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains extraordinarily challenging. Using a newly developed single electron counting detector, we confirm that electron beam induced motion significantly degrades resolution and, importantly, show how the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy. Thus, intrinsic image information can be restored to high resolution (Thon rings visible to ~3 Å). Using this approach we determined a 3.3 Å resolution structure of a ~700 kDa protein with D7 symmetry showing clear side chain density. Our method greatly enhances image quality and data acquisition efficiency - key bottlenecks in applying near atomic resolution cryoEM to a broad range of protein samples.

TRP channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (S1-S4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiologic modulation exhibited by TRPV1 and other TRP channels.