At the bottom of the ion permeation pathway,
side chains of opposing Ile1040 residues in S6
form a tight seal at the lower restriction site,
signifying that both structures are in closed conformations (Fig. 3, A and B).
The pore helix of h TRPM4 has an additional
turn compared with that in other TRP channels
(Fig. 3C) (9–13). The middle of the pore helix
features a single-turn p helix (Arg964-Arg969) followed by a proline residue, Pro970, conserved in
all human TRPM channels except TRPM2. Likewise, similar to what was first identified in
TRPV1 (10) and subsequently observed in other
TRP channels (7, 11), there is a one-turn p helix
(Val1030-Leu1035) in the middle of the S6 helix
(Fig. 3C). Although the exact role of these single-turn p helices is unclear, they could potentially
facilitate helix bending under different functional states, leading to movement of the lower
gate or modulation of the upper pore.
Outside of the pore, h TRPM4 has an extensive
extracellular loop comprising more than 30 res-
idues that connect the pore loop to the S6 helix.
The loop is clearly resolved, likely stabilized by a
disulfide bond between two cysteine residues
(Cys993 and Cys1011) (Fig. 3C and fig. S8, F and
G), a feature predicted to exist in the majority
of TRPM channels. This disulfide bond is not
seen in the CaCl2 structure (fig. S7H), where
radiation damage may have caused disulfide bond
breakage (24). The loop also contains a glycosyl-
ation site (Asn992) with the attached glycan point-
ing toward the extracellular space (Fig. 3C and
fig. S7, G and H).
In addition to the transmembrane S1-S6 domain, h TRPM4 has distinct membrane-embedded
a-helical segments (Fig. 4A). These segments
surround the exterior of the S1-S4 domain within the inner leaflet of the membrane and mediate extensive interactions with the soluble domain.
Preceding the S1 helix, there are two short helices
shaped as an inverted “V” embedded within the
inner leaflet of the membrane (Fig. 4B). We term
this the “pre-S1 elbow,” similar to that observed
in the mechanotransduction channel NOMPC
(9). A disordered loop, unresolved in both structures, connects the pre-S1 elbow to an amphipathic helix, which we term the “pre-S1 shoulder,”
positioned at the inner surface of the membrane
(Fig. 4C). The pre-S1 shoulder helix contains
large hydrophobic residues buried in the membrane and a number of charged residues facing
the cytosol. Among these charged residues,
Arg767 was identified as important for interactions with the phosphatidylinositol lipids PIP2
and PIP3 (25).
An extended S2-S3 linker distinct to the TRPM
subfamily forms a short amphipathic helix, positioned at the membrane surface, near the Ca2+-
binding site (Fig. 2B). The loop that connects S2
with the S2-S3 linker is ~12 residues longer in
h TRPM4 compared with other human TRPM
channels and extends beyond the membrane
bilayer, interacting with MHR4. Ser839 in the
linker is a predicted phosphorylation site important for trafficking TRPM4 to the plasma membrane (26); however, this linker is only weakly
resolved in the CaCl2 structure. In addition, the
S2-S3 linker also interacts loosely with the cytoplasmic domain MHR3 (Fig. 4D).
The last segment is positioned between the
pre-S1 shoulder and the S2-S3 linker helix (Fig.
4, A and F). Here, a loop fills the gap between S1
and S2 and connects the conserved TRP domain
to the C-terminal helix 1 (CH1), which is located
at a steep angle relative to S1, extending downward from the middle of the membrane bilayer
to the cytoplasmic surface more than 20 Å away
from the end of the TRP domain (Fig. 4E). The
C terminus of CH1 contains five arginine residues, similar to the pre-S1 shoulder mentioned
above, which likely constitute interaction partners for negatively charged lipid headgroups at
the membrane interface. CH1 connects to CH2
SCIENCE sciencemag.org 12 JANUARY 2018 • VOL 359 ISSUE 6372 231
CH1 Pre-S1 elbow CHS
Fig. 4. Membrane-embedded helical segments surround the S1-S4
domain. (A) Side view of the h TRPM4 monomer, with membrane-
embedded helical segments labeled and in solid color. (B) The pre-S1
elbow (gold) and pre-S1 shoulder (yellow). Lipid density between the
pre-S1 elbow and S1 is modeled with CHS. Side chains interacting with
CHS are shown as sticks. (C) Same as (B), viewed from the opposite
direction. (D) Cytoplasmic domain MHR3 interacts with the S2-S3 linker.
(E) The CH1 helix after the TRP domain. Charged residues in the pre-S1
shoulder (C) and CH1 (E) are shown in sticks. Missing links are shown in
dashed lines in (A) and (D).