Structure of the human lipid-sensitive cation channel TRPC3

The TRPC channels are crucially involved in store-operated calcium entry and calcium homeostasis, and they are thus implicated in human diseases such as neurodegenerative disease, cardiac hypertrophy, and spinocerebellar ataxia. We present structure of the full-length human TRPC3, a lipid-gated TRPC member, in a lipid-occupied, closed state at 3.3 Angstrom. TRPC3 has an acorn-like shape with four elbow-like membrane reentrant helices prior to the first transmembrane helix. The TRP helix is perpendicular to, and thus disengaged from, the pore-lining S6, suggesting a different gating mechanism. The third transmembrane helix S3 is remarkably long, resulting in a windmill-like transmembrane domain, and constituting an extracellular domain that may serve as a sensor of external stimuli. We identified two lipid binding sites, one being sandwiched between the pre-S1 elbow and the S4-S5 linker, and the other being close to the ion-conducting pore, where the conserved LWF motif of the TRPC family is located.


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The cytosolic free Ca 2+ concentration is strictly regulated because calcium is crucial to 39 most cellular processes, from transcription control, to neurotransmitter release, to hormone 40 molecule synthesis (Berridge et al., 2003;Kumar and Thompson, 2011;Sudhof, 2012). A major 41 mechanism regulating calcium homeostasis is store-operated calcium entry (SOCE), which is 42 triggered by the depletion of calcium stored in the endoplasmic reticulum (ER) (Ong et al., 2016; 43 Smyth et al., 2010). This process activates store-operated channels (SOCs) in the plasma 44 membrane, resulting in the influx of calcium that refills the calcium stores of the ER for further 45 cellular stimulation (Prakriya and Lewis, 2015). A key component of SOCE has been identified 46 as the TRPC channels, which are calcium-permeable, nonselective cation channels belonging to 47 the TRP superfamily (Liu et al., 2003;Zhu et al., 1998;Zhu et al., 1996). 48 Among the seven members in TRPC family, TRPC3, TRPC6, and TRPC7 are the closest 49 homologues, and they are unique in being activated by the lipid secondary messenger 50 diacylglycerol (DAG), a degradation product of the signaling lipid phosphatidylinositol 51 4,5-bisphosphate (PIP2) (Itsuki et al., 2012;Tang et al., 2001). However, the molecular 52 mechanism of such activation remains elusive due to a lack of knowledge of the lipid binding 53 sites. TPRC3, TRPC6, and TRPC7 share several functional domains, including N-terminal 54 ankyrin repeats (AR), a transmembrane domain (TMD) with six transmembrane helixes (S1-S6), 55 and a C-terminal coiled-coil domain (CTD). They also exhibit an unusually long S3 helix, but 56 the function of the S3 helix is poorly understood (Vazquez et al., 2004). 57 TRPC3 is abundantly expressed in the cerebellum, cerebrum, and smooth muscles, and it 58 plays essential role in the regulation of neurogenesis and extracellular/intracellular calcium 59 signaling (Gonzalez-Cobos and Trebak, 2010; Li et al., 1999). Dysfunction of TRPC3 has been 60 linked to neurodegenerative disease, cardiac hypertrophy, and ovarian adenocarcinoma (Becker  Overall architecture 70 Our three-dimensional reconstruction of hTRPC3 was of sufficient quality to allow de novo 71 modeling of almost the entire protein ( Fig. S 1, 2), with the exception of the first 21 N-terminal 72 residues; the region connecting the TRP helix and the C-terminal domain (residues 688-757); the 73 loop connecting the linker domain LD6 and LD7 (residues 281-291); and the last 30 C-terminal 74 residues. Characterized by a distinctive one head-two tails shape, we identified two lipid-like 75 densities, one sandwiched between the pre-S1 elbow and the S4-S5 linker, and the other wedged 76 between the P loop and S6 of the adjacent subunit. Notably, we modeled two lipid molecules at 77 these two sites, nevertheless, we were not able to determine the identity of the lipids at current 78 resolution. Interestingly, the TRP helix is perpendicular to the S6, and the density of the hinge 79 region is poorly defined, even though both the TRP helix and S6 exhibit excellent densities 80 (Figure 1a, b).

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The overall structure of TRPC3 resembles an acorn, and it has a solely alpha-helical 82 composition (Figure 1a-d). While TRPC3 shares a similar architecture of the TMD with other 83 TRPCs, the third transmembrane helix, S3, is nearly twice as long as the S3 in any other 84 DAG-insensitive TRPC channels (Fig. S 3). It elongates into the extracellular space and connects 85 to the S4 through a remarkably long loop, where a glycosylation site is observed (Figure 1c, d).

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The extended structure of S3 gives rise to a windmill-like TMD, distinctive to voltage-gated

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Transmembrane domain and lipid-binding sites 99 The TMD of TRPC3 shares topology similar to that of other TRP channels and voltage-gated 100 ion channels, consisting of the S1-S4 domain and the pore domain arranged in a 101 domain-swapped manner (Figure 1e, 3a). Nevertheless, the distinct activation mechanism of 102 TRC3, TRPC6, and TRPC7 by DAG implies unique features of their TMD. Indeed, comparison 103 of the relative arrangement of the S1-S4 domains with the pore domain shows remarkable 104 differences between TRPC3 and TRPA1 or TRPM4, yet overall agreement with TRPV1 ( Fig. S 105 4). Detailed inspection of the TMD in TRPC3 reveals two unique features: a large elbow-like 106 pre-S1 domain harboring a lipid-binding site (lipid 1), and unusually long S3 helix forming an 107 extracellular domain (ECD), along with the S1-S2 linker and S3-S4 linker (Figure 3a, b).

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The pre-S1 elbow, embedded in the lipid bilayer, consists of two half transmembrane 109 helices (half TM1 and half TM2). The half TM1 connects to LD9, which is the last alpha helix in 110 the LD; the half TM2 connects to the pre-S1 helix, a short alpha helix prior to S1 running 111 horizontally along the intracellular face of the membrane (Figure 2a, 3c, 3d). This unique 112 configuration pulls the intracellular half of S1 away from the pore center, resulting in a 113 hydrophobic pocket behind the pre-S1 elbow and surrounded by half TM1 and S1 (Figure 3c).

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Moreover, the outward movement of S1 opens a window between itself and S5 from the adjacent 115 subunit, exposing the intracellular half of S4 and the S4-S5 linker, which are key regions for TRP 116 channel gating, to the lipid environment ( Figure 3d). Indeed, we observed a lipid-shaped density (lipid 1) in this pocket (Figure 3c, d). The head 118 group of lipid 1 is well defined in the density map, forming several hydrogen bonds and polar 119 interactions with residues in the LD9, the pre-S1 elbow, half TM1, and the S4-S5 linker, while the 120 two hydrocarbon tails are in contact with S1, S4, the pre-S1 elbow, and half TM1 (Figure 3e, f). A 121 similar pre-S1 elbow structure with lipid-like density has been observed in the Drosophila  We also identified a second lipid-like density (lipid 2) in the lateral fenestration of the pore 127 domain, wedged between the P loop and S6 of adjacent subunit and forming both hydrophobic and 128 hydrophilic interactions (Figure 3g, h). Moreover, lipid 2 is in close contact with the LFW motif on 129 the P loop, which is highly conserved throughout the TRPC family and is crucial to channel 130 function ( Fig. S 3). Replacing this motif by three alanine residues in TRPC5 and TRPC6 resulted 131 in a nonfunctional channel (Strubing et al., 2003). Therefore, the lipid 2 binding site likely 132 represents another important modulation site. In addition to interaction with lipid 2, the LFW motif 133 forms multiple hydrophobic interactions within the pore domain and therefore plays an important 134 role in maintaining the proper structure of the pore domain ( Figure 3i).

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A second unique feature of TRPC3 is the remarkably long S3, stretching out into the 136 extracellular side and supporting the formation of the ECD (Figure 3a). Within the ECD we observed a cavity-like feature (Figure 3j), with S3 and the S3-S4 linker as a -back wall and roof‖, 138 and the S1-S2 linker forming the entrance. This cavity is located right above the lipid bilayer, 139 and its interior is filled with both charged and hydrophobic residues ( Figure 3j). Moreover, a 140 tyrosine residue (Y589) in the loop connecting the S5 and the P loop plugs into the cavity ( Figure   141 3i). We speculate that the cavity may serve as a binding site for small molecules and that binding   unknown, it clearly contributes to the channel assembly through three major interfaces. The first 220 interface is contributed by the vertical CTD pole helices of the four subunits winding into a 221 tetrameric coiled-coil assembly (Figure 6b, c). This is a common feature employed to specify 222 subunit assembly and assembly specificity within the voltage-gated ion channel superfamily 223 (Figure 6b, c). The second interface is formed by the horizontal CTD rib helix penetrating through 224 the tunnel composed of ARD and LD from neighboring subunits, thus tethering them together 225 (Figure 6c, d). Notably, the rib helix is rich in positively charged residues, forming multiple 226 interactions with the charged residues in the LD. The third interface is located between LD and 227 LD/pre-S1 elbow of the adjacent subunit (Figure 6e). All these interactions knit the tetramer 228 together.

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The TRPC3 structure displays a unique acorn-like architecture. Distinct to the TRPM, TRPV or 231 TRPA channels whose TRP helix and S6 form a continuous alpha helical structure, the TRP helix 232 in TRPC3 is disengaged from the S6, which aligns with the unique gating mechanism of TRPC, 233 perhaps linked to the lipid activation or voltage independence. The remarkably long S3 endows 234 TRPC3 a windmill-like TMD and frames the ECD in which a cavity may act as a binding site for 235 small molecules, suggesting a role for the ECD in sensing extracellular stimuli. We identified 236 two lipid binding sites, one buried in a pocket surrounded by the pre-S1 elbow, S1, and the S4-S5 237 linker, and the other inserted into the lateral fenestration of the pore domain. Our structure 238 provides a framework for understanding the complex gating mechanism of TRPC3.  molecule is buried inside the pocket formed by pre-S1 elbow, S1, and the S4-S5 linker. Two      relative organization of the S1-S4 domain with the pore domain in TRPC3 is similar to that in 304 TRPV1, but the S1-S4 domain in TRPC3 exhibits a clockwise rotation relative to TRPA1 or 305 TRPM4. The purified TRPC3 protein sample (2.5 μL) at a concentration of 5 mg/mL was applied onto a 335 glow-discharged Quantifoil holey carbon grid (gold, 1.2/1.3 μm size/hole space, 300 mesh). The 336 gird was blotted for 1.5 s at 100% humidity by using a Vitrobot Mark III, and then was plunged  filtered to 10 Å) was applied to the two half maps.

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Model building 366 The model of TRPC3 was built in Coot using the TMD domain of TRPM4 structure (PDB 5wp6) 367 as a guide (Emsley et al., 2010). De novo building was mainly guided by bulky residues and 368 secondary structure prediction (Fig. S 3). The TRPC3 structure chiefly consists of α helices, 369 which greatly assisted register assignment. In the initial de novo-built model, the order and 370 length of the secondary structure features, as well as the positions of bulky residues within each 371 secondary structure feature are in good agreement with the prediction (Fig. S 3) conditions. All the authors contributed in cryo-EM data collection and processing, structure 392 analysis, and preparation of the manuscript. CTD "rib" helix Pre-S1 "elbow" Pre-S1 "elbow" Pre-S1 "elbow" TRP helix TRP helix Pre-S1 "elbow" Pre-S1 helix Pre-S1 helix Pre-S1 helix   Pre-S1 "elbow" Pre-S1 "elbow" Pre-S1 helix S1 S1-S2 linker S2