(fig. S7). Our structure suggests that the VSLD
cavity is unlikely to bind PIP2, because the net
charge of its interior is negative and this cavity is
not large enough to accommodate both menthol
and PIP2. The reported effects of mutations to
Arg1008 may reflect downstream conformational
changes following PIP2 binding. Notably, we found
that the interface between the TRP domain, the
pre-S1 domain, and MHR4 contains many basic
amino acid residues, including the previously
identified Lys994 and Arg997 (fig. S7). We speculate that PIP2 binds at this interfacial site, where
it could modulate the position of the TRP domain to enable a nondesensitized state. The distinct location of this putative PIP2 binding site
compared to TRPV1 and TRPML reflects the diverse effects of PIP2 on TRP channels (25, 26).
In the context of previous mutagenesis data,
our structural analyses suggest that the ligand-
dependent gating mechanism of TRPM8 differs
substantially from TRPV1. In TRPV1, the binding
of vanilloid to the S4b and the S4-S5 linker is
thought to induce a “swivel” motion in the TRP
domain, which would pull on S6 to open the S6
gate (23, 30). We suggest that menthol binds in the
VSLD cavity, which is distinct from the vanilloid-
binding site in TRPV1. Whereas vanilloid-mediated
gating involves non–a-helical elements (310 and p
helices) and an S4-S5 linker, neither of these struc-
tural features appear to be present in the confor-
mational state of our TRPM8FA structure. Thus, we
speculate that ligand-induced repositioning of the
TRP domain in TRPM8 may directly lead to the
opening of the S6 gate.
Our observation of the extensive intersubunit
interactions between the TMD and the top layer
of the CD ring leads us to speculate that the
gating-related TRP domain motion may also in-
volve the top layer of the CD ring (Fig. 3F and
fig. S6B). Furthermore, the CTDH2, with its cen-
tral position within the CD and its link with the
TRP domain, may be important for coupling
the movements of the TRP domain to those of
the MHR elements and especially MHR4 (Fig. 3G
and fig. S6D). In addition, the tetrameric coiled
coil located in the bottom layer ring may play a
role in regulating the position of CTDH2.
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Cryo-EM data were collected at The Scripps Research Institute
(TSRI) electron microscopy facility. We thank Y. Zhang and H. Yang
(Duke University) for providing access to their calcium imaging
apparatus and guidance to calcium imaging experiments. We thank
J.-C. Ducom (TSRI High Performance Computing facility) for
computational support, B. Anderson for microscope support, and
M. Herzik and S. Chowdhury for helpful discussion and training. We
thank Z. Johnson for advice on sample freezing. This work was
supported by the National Institutes of Health (grants R35NS097241
to S.-Y.L., DP2EB020402 to G.C.L.). G.C.L is supported as a Searle
Scholar and a Pew Scholar. Computational analyses of EM data
were performed using shared instrumentation funded by NIH
grant S10OD021634. The coordinates are deposited in the
Protein Data Bank with the PDB ID 6BPQ, and the electron density
maps have been deposited in EMDB with the ID EMD-7127.
Materials and Methods
Figs. S1 to S7
3 November 2017; accepted 27 November 2017
Published online 7 December 2017
Fig. 4. The voltage-sensor–like domain (VSLD) and the putative menthol-binding site in the
VSLD cavity. (A) Comparison of the VSLD in TRPM8 (blue) with the canonical voltage-sensor
domain in the Kvchim channel (green) (PDB ID: 2R9R) and the VSLD of TRPV1 (purple) (PDB ID:
3J5P). A gating charge Arg841 in the VSLD of TRPM8 is near three negatively charged amino acids
below a large hydrophobic seal (spheres in bright orange) in TRPM8 (left). Many gating charge
arginines in S4 of Kvchim are located above and below a small hydrophobic seal (spheres in bright
orange) and interact with negatively charged amino acids (middle). The interior of the VSLD of
TRPV1 is lined with hydrophobic and polar amino acids. (B) Residues critical for the sensitivity
of TPRM8 to menthol (shown in green stick representation) were mapped to S1 (Tyr745), S4 (Arg841),
and the TRP domain (Tyr1004). Residues implicated in icilin sensitivity of TRPM8 (yellow stick
representation) were mapped to S3 (Asn799 and Asp802). All of these residues point toward the
VSLD cavity. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp;
E, Glu; K, Lys; L, Leu; N, Asn; R, Arg; and Y, Tyr.