bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Cryo-EM structure of the receptor-activated TRPC5

2 at 2.9 angstrom resolution

3

4

5 Jingjing Duan1,2*, Jian Li3,4*, Gui-Lan Chen5*, Bo Zeng5, Kechen Xie5, Xiaogang Peng6,

6 Wei Zhou6, Jianing Zhong4, Yixing Zhang1, Jie Xu7, Changhu Xue7, Lan Zhu8, Wei Liu8,

7 Xiao-Li Tian9, Jianbin Wang1, David E. Clapham2, Zongli Li10#, Jin Zhang1#

8

9 1School of Basic Medical Sciences, Nanchang University, Nanchang, Jiangxi, 330031,

10 China.

11 2Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA

12 3College of Pharmaceutical and Biological Engineering, Shenyang University of 13 Chemical Technology, Shenyang 110142, China

14 4Gannan Medical University，XueYuan Road 1#，Ganzhou，Jiangxi, 341000, China.

15 5Key Laboratory of Medical Electrophysiology, Ministry of Education, and Institute of

16 Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan, 646000,

17 China

18 6The Key Laboratory of Molecular Medicine, the Second Affiliated Hospital of

19 Nanchang University, Nanchang 330006, China.

20 7College of Food Science and Engineering, Ocean University of China, Qingdao 266003,

21 China. bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

22 8School of Molecular Sciences and Biodesign Center for Applied Structural Discovery,

23 Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.

24 9Human Population Genetics, Human Aging Research Institute (HARI) and School of

25 Life Science, Nanchang University, The Key Lab of Jiangxi Province for Human Aging,

26 Nanchang, 330031, China.

27 10Howard Hughes Medical Institute, Department of Biological Chemistry and Molecular

28 Pharmacology, Harvard Medical School, Boston, MA 02115, USA.

29

30 *These authors contributed equally to this work.

31 # Corresponding authors bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

32 Abstract:

33 The transient receptor potential canonical subfamily member 5 (TRPC5) is a

34 non-selective calcium-permeant cation channel. As a depolarizing channel, its function

35 is studied in the central nervous system and kidney. TRPC5 forms heteromultimers

36 with TRPC1, but also forms homomultimers. It can be activated by reducing agents

37 through reduction of the extracellular disulfide bond. Here we present the 2.9 Å

38 resolution electron cryo-microscopy (cryo-EM) structure of TRPC5. The structure of

39 TRPC5 in its apo state is partially open, which may be related to the weak activation of

40 TRPC5 in response to extracellular pH. We also report the conserved negatively charged

41 residues of the cation binding site located in the hydrophilic pocket between S2 and S3.

42 Comparison of the TRPC5 structure to previously determined structures of other TRPC

43 and TRP channels reveals differences in the extracellular pore domain and in the

44 length of the S3 helix. Together, these results shed light on the structural features that

45 contribute to the specific activation mechanism of the receptor-activated TRPC5.

46

47

48

49 bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

50 Introduction

51 The transient receptor potential canonical (TRPC) channels are Ca2+-permeant cation

52 channels that are potentiated by stimulation of G -coupled receptors or receptor

53 tyrosine kinases1,2. TRPC4 and TRPC5 share 65% sequence identity and exhibit similar

54 functional properties when expressed as independent homomers. As homomultimers, the

55 channel’s outward conductance is blocked in the 10-40 mV range3,4 by intracellular Mg2+

56 5. TRPC5 is expressed in both excitable and non-excitable cells, primarily in the brain

57 and kidney6. TRPC5 knock-out mice exhibit severe daily blood pressure fluctuations,

58 and deficits in motor coordination and innate fear behavior7-9. Although contested10,

59 TRPC5 inhibition may suppress the development of progressive kidney diseases11.

60 The cryo-EM revolution has enabled recent structural solutions for TRPC3, TRPC4, and

61 TRPC612-14. Here we present the structure of the mouse TRPC5 at pH 7.5 to an overall

62 resolution of 2.9 Å. We contrast and compare the TRPC5 structure with other TRP

63 channels to help understand the diverse functional and physiological roles of this ion

64 channel family.

65

66 Results

67 Overall structure of the mouse TRPC5 tetrameric ion channel bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

68 A mouse TRPC5 construct lacking the C-terminal 210 residues (a.a. 1-765, excluding a.a.

69 766-975) yielded more protein after purification than that of the full-length construct. The

70 baculovirus construct, consisting of a maltose binding protein (MBP) tag at the N

71 terminus, was stably purified to homogeneity in n-dodecyl β-D-maltoside (DDM) and

72 reconstituted in the amphipol poly (maleic anhydride-alt-1-decene) substituted with

73 3-(dimethylamino) propylamine (PMAL-C8; Supplementary Fig. 1). The amphipol

74 reconstituted detergent-free protein was then negatively stained and analyzed by

75 single-particle cryo-EM.

76 Single particle cryo-EM analyses of TRPC5 at an overall resolution of ~ 2.9 Å was

77 sufficient for de novo model building (Supplementary Fig. 2, Supplementary Fig. 3,

78 Supplementary Table 1). Disordered regions led to poor densities for 7 residues in the

79 S1-S2 loop, 28 residues in the distal N terminus, and 3 residues in the truncated distal C

80 terminus. Similar to other solved TRP channel structures, TRPC5 forms a four-fold

81 symmetric homotetramer (Fig. 1a, b) with dimensions of 100 Å × 100 Å × 120 Å.

82 Each of the four monomers can be divided into a compact cytosolic domain and a

83 transmembrane domain (TMD) (Fig. 1). The cytosolic domain is composed of the

84 N-terminal region with an ankyrin repeats domain (ARD) and seven α-helices (HLH);

85 the C-terminal subdomain contains a connecting helix and a coiled-coil domain. The

86 transmembrane domain (TMD) is composed of six α-helices (S1-S6), a TRP domain, and bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

87 several small helices, including a pore helix, pre-S1 elbow, pre-S1 helix and an S2-S3

88 linker helix with connecting loops (Fig. 1c, d).

89 Whole-cell patch-clamp recordings confirmed that the truncated TRPC5 construct used

90 for cryo-EM analyses retained the primary electrophysiological properties of full-length

91 TRPC5 (Fig. 2). Both the truncated and full-length constructs were activated by englerin

92 A and inhibited by ML204, a TRPC4/5 blocker (Fig. 2a, b). No significant current was

93 observed in cells transfected with empty vector (Fig. 2c).

94 The ion conduction pore

95 TRPC4 and TRPC5 are the most closely-related TRPC , with 65% amino acid

96 identity at full length, and 100% identity in S5 (a.a. 502-542), the pore helix (a.a.

97 569-580), S6 (a.a. 596-651, including the lower gate) and the selectivity filter (a.a.

98 580-584) (Supplementary Fig. 4). The architecture of the ion conduction pathway is

99 well conserved between these two channels (Fig. 3a). However, the calculated pore size

100 of TRPC5 is wider than that of TRPC4 (Fig. 3b), potentially due to the activation state of

101 the purified protein. TRPC5’s motif ‘TRAIDEPNN’ (a.a. 544-552) forms a small helix in

102 the middle of the extracellular loop connecting S5 and the pore helix (Supplementary

103 Fig. 5). This unique helix, close to the disulfide bond and on the top of the pore helix

104 (Supplementary Fig. 5), is distinct from the corresponding motif ‘ETKGLS’ of TRPC4.

105 The TRPC5 motif contains 3 more amino acids, including one more negatively-charged bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

106 residue, Asp548, and the hydrophobic residue, Ile547 (Fig. 3c, red arrow); it is thus

107 potentially more attractive to cations entering the pore.

108 Along with the pore, the filter/gate-forming residues of TRPC5 are identical to those of

109 TRPC4. Gly581 marks a restriction point of 7.2 Å between diagonally opposed residues

110 at the selectivity filter. “INQ” (Ile621, Asn625 and Gln629) defines a lower gate with a

111 4.9 Å constriction formed by the side chains of Gln629 (Supplementary Fig. 6); in

112 TRPC4, the most restricted point is 3.6 Å between opposing Asn621 residues.

113 Interestingly, the lower gate of TRPC5 appears to be partially open compared with the

114 presumed closed state of TRPC4, despite similar isolation procedures and conditions.

115 This may be related to the weak activation of TRPC5 compared to TRPC4 at pH 7.415,

116 suggesting a distinct activation mechanism for TRPC5 in response to extracellular pH.

117 Structural comparisons between TRPC5 and other TRPC channels

118 Here we compare the architectural differences between the TRPC1/4/5 and TRPC3/6/7

119 channel subfamilies (Fig. 4a). An overlay of TRPC5 with the previously solved TRPC3,

120 TRPC4, and TRPC6 channel structures shows relatively high spatial conservation of the

121 6-transmembrane bundle. Several intracellular features of TRPC channels are also

122 preserved, including the ankyrin repeats, the pre-S1 elbow in the N-terminal domain, and

123 the connecting helix running parallel to the membrane bilayer. A key feature of TRPC

124 channels is the conserved LFW motif inside the pore domain (Fig. 3c). In the TRPC5 bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

125 structure, a π–π interaction between Phe576 and Trp577 stabilizes the key pore region, as

126 is also seen in the available TRPC3/4/6 channel structures (Fig. 4b). Mutagenesis studies

127 of the LFW motif in TRPC5 result in non-functional channels16.

128 Despite the relatively high structural conservation, TRPC5’s transmembrane domain has

129 several distinct features. Unlike TRPC3/6, TRPC4/5’s extracellular domains and long

130 pore loops form intricate structures stabilized by a disulfide bond between Cys553 and

131 Cys558. As the two cysteines in the pore region are well conserved in TRPC1/4/5

132 subfamily (Fig. 3d), the disulfide bond may be important to gating. Indeed, mutation of

133 Cys553 or Cys558 to alanine activates TRPC517 (Fig. 4c). Interestingly, although TRPC4

134 and TRPC5 have similar functional properties, the loop preceding TRPC5’s disulfide

135 bond is distinct from that of TRPC4, with 3 extra residues (a.a. 546-548) in TRPC5 (Fig.

136 3c). Superimposition of the TRPC channels also reveals striking differences in the

137 arrangement of S3 (Fig. 4d); the S3 helix is longer in TRPC3 and TRPC6 and protrudes

138 into the extracellular space. In both TRPC4 and TRPC5 channels, the extracellular S3

139 region is shorter by four helical turns, limiting potential extracellular interactions.

140 Cation- and lipid-binding sites

141 A strong non-protein peak is observed in the intracellular hydrophilic pocket between

142 TRPC5’s S2 and S3 helices. Since Na+ was the only added cation in our purification

143 buffer, this peak may represent a sodium ion, but we cannot rule out another bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

144 contaminating cation, such as Ca2+. The cation is not in the putative ion conduction

145 pathway; it is coordinated by hydrogen bonding with several highly conserved residues,

146 including Glu418, Glu421, Asn436 and Asp439 (Fig. 5a). The geometry of the cation’s

147 coordination in TRPC5 resembles that of the TRPC4 structure18. Structural alignment of

148 these residues from TRPC family members reveals a well-preserved cation-binding site,

149 including Ca2+ binding sites, in the TRPM4 and TRPM2 structures19,20. Small deviations

150 are seen in TRPM4 and TRPC4; Glu421 in the S2 helix’s cytosolic end is substituted by a

151 glutamine (Fig. 5b, c). A number of negatively charged residues, including Asp633,

152 Asp636, Glu638, Asp652 and Glu653 in the TRP domain, as well as Asp424, Glu429 and

153 Asp433 on S2-S3 linker, are aligned along a potential cation entry pathway

154 (Supplementary Fig. 7). Asp652 and Glu653 in the TRP domain and negatively charged

155 residues in the cytosolic region form a cation access pathway, which should facilitate

156 cation entry to its binding site (Supplementary Fig. 7c). Further experiments are needed

157 to determine if this represents a site for internal cationic potentiation or inhibition of

158 TRPC5 activity.

159 Eight densities corresponding to lipid molecules were clearly resolved in the TRPC5

160 cryo-EM density and identified as four cholesteryl hemisuccinates (CHS, a

161 cholesterol-mimicking artificial detergent molecule; purple) and four phospholipids

162 (phosphatidic acid or ceramide-1-phosphate; yellow) (Fig. 6a). Each phospholipid is

163 embedded in the gap between the monomeric subunits. The phospholipid interacts with bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

164 the pore helix through its polar head and side chain oxygen with amino acids Phe576 and

165 Trp577 (Fig. 6b, c). Four CHS heads face down at the interface with the plasma

166 membrane, imbedding into the space among the S4, S5, S6 helices and the N-terminal

167 domain (Fig. 6b). CHS stabilizes the domain interaction through interactions with

168 Asn500 on the S4/S5 linker and with Trp315, Tyr316, and Trp322 in the TRP domain

169 (Fig. 6d). The polar heads of the identified lipids interact with positively charged regions

170 of the channel (Supplementary Fig. 8). Thus, we assume that in vivo phosphorylation or

171 dephosphorylation, and the composition of membrane lipids, regulate ion conduction by

172 altering channel topology.

173

174 Discussion

175 Here we obtained and investigated the cryo-EM structure of TRPC5 at 2.9 Å resolution.

176 The overall architecture of TRPC5 resembles that of other TRPC subfamily channels, but

177 important differences in TM bundles and pore regions may help explain many of its

178 unique functional features. Comparison of the structures of the open, or partially open,

179 TRPC5 and closed TRPC3/6, suggest that the extracellular disulfide bond is a potential

180 transducer of conformational changes that control the opening and closing of the pore

181 domain. Another unique feature is the short S3 helix that dips into the hydrophobic

182 region. Shift in this helical domain may be directly translated into the transmembrane bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

183 domains.

184 One of the unique and interesting features of TPRC4/5 channels is that they are reversibly

185 potentiated by micromolar concentrations of La3+ or Gd3+. Extensive mutagenesis studies

186 suggest that the binding site is extracellular and between Glu543 and Glu59521. Mutation

187 of glutamic acids Glu543 and Glu595/Glu598, in the extracellular mouth of the channel

188 pore, to glutamine resulted in loss of potentiation. However, Glu543 at the end of the S5

189 helix, and Glu595 at the beginning of S6, are oriented such that cation binding is not

190 possible. Since our structure was obtained in a divalent-free buffer, we can’t rule out the

191 possibility that Ca2+ binding may lead to a conformational change in this region.

192 (Supplementary Fig. 9).

193 Arg593, the arginine located in the pore loop near the proposed extracellular Gd3+

21 194 binding site, was reported to be critical for GPCR-Gq-PLC-dependent gating of TRPC5 .

195 An Arg593Ala mutation had reduced sensitivity to Gq-PLC activation, which can be

196 explained by our structure. Arg593 interacts via a salt bridge with Glu598 and forms a

197 hydrogen bond with the Val590 side chain. Breaking this electrostatic interaction may

198 lead to unfolding of this pore region. In addition, Arg593 is the only positively charged

199 residue in the corresponding positions in TRPC channels (glycine in TRPC1, glutamine

200 in TRPC4, asparagine in TRPC6, or negatively-charged aspartic acid in TRPC7). One

201 possibility that Arg593 may serve as a molecular fulcrum, allowing the efficient

202 transmission of gating force to TRPC5’s pore helix-loop. bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

203 Another unique feature of TPRC4/5 channels is their unusual current-voltage (I-V)

204 relationship, in particular the flat segment in the 10-40 mV range attributed to Mg2+ block

205 of outward current2. TRPC5’s S6 terminal Asp633 residue is close to the intracellular

206 mouth of channel pore and was proposed as the site for block of the ion conduction

207 pathway upon binding Mg2+, whereas the downstream Asp636 did not affect Mg2+

208 block22. In our structure, Asp633 is situated much closer to the channel pore than Asp636

209 (Supplementary Fig. 10), which supports it being a Mg2+ block site. However, because

210 we used a divalent-free buffer for cryo-EM, direct evidence for Mg2+-binding on Asp633

211 and restriction of the channel pore should be studied in proteins purified in

212 Mg2+-containing solution.

213 In TRPV channels, as in many other 6TM channels, the S4-S5 linker is crucial to channel

214 gating23-25. Consistent with this, a single point mutation in TRPC4/5’s S4-S5 linker,

215 Gly504Ser, fully activates it. Introduction of a second mutation (Ser623Ala) into TRPC4

216 Gly503Ser suppressed the constitutive activation and partially rescued its function26.

217 These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel

218 gating and that disruption disinhibits gating control by the sensor domain.

219 Both the TRPC5 structure in the present study and our recently published TRPC4

220 structure18 were obtained from protein samples prepared in the same divalent-free

221 solution at pH 7.5. TRPC5’s wider pore could reflect that an open state is preferred under

222 these conditions. In our patch-clamp experiments, TRPC5 always exhibited small but bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

223 consistent basal activity, while TRPC4 was inactive without drug or receptor stimulation.

224 This agrees with a previous study that TRPC5 is partially open at pH 7.4, while TRPC4 is

225 closed15. The wider pore size of TRPC5 may allow small number of cations to pass

226 through under resting conditions and suggest that the current TRPC5 structure is a

227 partially open state of the channel.

228 In summary, our structure enables new comparisons between the different subgroups of

229 the available TRPC channels. Our findings provide structural insights into conservation

230 and divergence of TRPC channels, but do not explain the mechanisms regulating

231 GPCR-Gq-PLC activation of a receptor-operated channel. Finally, the findings have

232 broad implications for understanding the structural basis underlying epitope antibody

233 selectivity, and can facilitate rational, structure-based, approaches for the design of

234 subtype-selective TRPC ligands as new therapeutic agents.

235

236 Materials and Methods

237 Protein expression and purification. A synthetic fragment encoding residues 1 to

238 765 (excluding a.a. 766-975) of mouse TRPC5 was cloned into the pEGBacMam vector.

239 The resulting protein contains a maltose binding protein (MBP) tag on its N terminus. P3

240 baculoviruses were produced in the Bac-to-Bac Baculovirus Expression System

241 (Invitrogen). HEK293S GnTI- (from ATCC) cells were infected with 10% (v/v) P3

242 baculovirus at a density of 2.0 - 3.0 × 106 cells/ml for protein expression at 37°C. After bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

243 24h, 10 mM sodium butyrate was added, and the temperature reduced to 30°C for 72h

244 before harvesting. Cells were gently disrupted and resuspended in a solution containing

245 30 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol (DTT), pH 7.5 with EDTA-free

246 protease inhibitor cocktail (Roche). Cells were solubilized for 2-3h in a solution

247 containing 1.0% (w/v) N-dodecyl-beta-D-maltopyranoside (DDM, Affymetrix), 0.1%

248 (w/v) cholesteryl hemisuccinate (CHS, Sigma), 30 mM HEPES, 150 mM NaCl, 1 mM

249 DTT, pH 7.5 with EDTA-free protease inhibitor cocktail (Roche). After 30 min, the cell

250 lysate was then centrifuged for 60 min at 100,000×g and the supernatant incubated in

251 amylose resin (New England BioLabs) at 4°C overnight. The resin was washed with 20

252 column volumes of wash buffer [25 mM HEPES, 150 mM NaCl, 0.1% (w/v) digitonin,

253 0.01% (w/v) CHS, 1 mM DTT; pH 7.5 with EDTA-free protease inhibitor cocktail

254 (Roche)]. The protein was eluted with 4 column volumes of wash buffer with 40 mM

255 maltose. The protein was then concentrated to 0.5 ml and mixed with PMAL-C8

256 (Anatrace) at 1:4 (w/w) with gentle agitation for 4h. Bio-Beads SM-2 (50 mg/ml

257 reconstitution mixture, Bio-Rad) were added to remove additional detergent; a disposable

258 poly-prep column was used to remove Bio-Beads. After incubation at 4°C overnight, the

259 protein was further purified on a Superose 6 size exclusion column in 25 mM HEPES,

260 150 mM NaCl, 1 mM DTT; pH 7.5. All purification procedures were carried out either on

261 ice or at 4 °C. The peak fractions corresponding to tetrameric TRPC5 was concentrated to

262 7.0 mg/ml and used for preparation of cryo-EM sample grids. bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

263 Electron microscopy data collection. 3.5µl of purified TRPC5 protein sample in

264 PMAL-C8 at 7.0 mg/ml was applied to glow-discharged Quantifoil R1.2/1.3 holey

265 carbon 400 mesh copper grids (Quantifoil). Grids were blotted for 7s at 100% humidity

266 and flash frozen by liquid nitrogen-cooled liquid ethane using a FEI Vitrobot Mark I

267 (FEI). The grid was then loaded onto FEI TF30 Polara electron microscope operated at

268 300 kV accelerating voltage. Image stacks were recorded on a Gatan K2 Summit direct

269 detector (Gatan) set in super-resolution counting mode using SerialEM27, with a defocus

270 range between 1.5 and 3.0 μm. The electron dose was set to 8 e-/physical pixel/s and the

271 sub-frame time to 0.2 s. A total exposure time of 8s resulted in 40 sub-frames per image

272 stack. The total electron dose was 42.3 e-/Å2 (~1.1 e-/Å2 per sub-frame).

273 Image processing and 3D reconstruction. Image stacks were gain-normalized and

274 binned by 2x to a pixel size of 1.23Å prior to drift and local movement correction using

275 motionCor228, resulted in the sums of all frames of each image stack with and without

276 dose-weighting. The dose-weighting sum were binned by 8x and subject to visual

277 inspection, images of poor quality were removed before particle picking. Particle picking

278 and subsequent bad particle elimination through 2D classification was performed using

279 Python scripts/programs29 with minor modifications in the 8x binned images. The

280 selected 2D class averages were used to build an initial model using the common lines

281 approach implemented in SPIDER30 through Maofu Liao’s Python scripts29, which was

282 applied to later 3D classification using RELION31. Contrast transfer function (CTF) bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

283 parameters were estimated using CTFFIND432 using the sum of all frames without

284 dose-weighting. Quality particle images were then boxed out from the dose-weighted

285 sum of all 40 frames and subjected to RELION 3D classification. RELION 3D

286 refinements were then performed on selected classes for the final map. The resolution of

287 this map was further improved by using the sum of sub-frames 1-14. The number of

288 particles in each dataset and other details related to data processing are summarized in

289 Table 1.

290 Model building, refinement, and validation.

291 At 2.9 Å resolution, the cryo-EM map was of sufficient quality for de novo atomic model

292 building in Coot33. Amino acid assignment was achieved based mainly on the clearly

293 defined densities for bulky residues (Phe, Trp, Tyr, and Arg) and the absence of side

294 chain densities for glycine residues. The atomic model was further visualized in COOT; a

295 few residues with side chains moving out of the density during the refinement were fixed

296 manually, followed by further refinement. The TRPC5 model was then subjected to

297 global refinement and minimization in real space using the module

298 ‘phenix.real_space_refine’ in PHENIX34. The geometries of the model were assessed

299 using Mo Probity35 in the ‘comprehensive model validation’ section of PHENIX. The

300 final model exhibited good geometry as indicated by the Ramachandran plot (preferred

301 region, 98.25%; allowed region, 1.75%; outliers, 0%). The pore radius was calculated

302 using HOLE36. bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

303 Electrophysiology

304 The full-length, truncated TRPC5 constructs or empty vector were transfected into HEK

305 293T (from ATCC) cells together with an mCherry plasmid. Cells with red fluorescence

306 were selected for whole-cell patch recordings (HEKA EPC10 USB amplifier,

307 Patchmaster 2.90 software). A 1-s ramp protocol from –100 mV to +100 mV was applied

308 at a frequency of 0.2 Hz. Signals were sampled at 10 kHz and filtered at 3 kHz. The

309 pipette solution contained (in mM): 140 CsCl, 1 MgCl2, 0.03 CaCl2, 0.05 EGTA, 10

310 HEPES, and the pH was titrated to 7.2 using CsOH. The standard bath solution contained

311 (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 D-, and the pH was

312 adjusted to 7.4 with NaOH. The recording chamber (150 µl) was perfused at a rate of ~2

313 ml/min. All recordings were performed at room temperature.

314

315 Acknowledgements

316 We thank Dr. Steve Harrison and the Cryo-EM Facility (Harvard Medical School) for use

317 of their microscopes. We thank Dr. Maofu Liao for providing the Python scripts and help in

318 image processing. J.Z. was supported by the Thousand Young Talents Program of China,

319 National Natural Science Foundation of China (Grant No. 31770795) and Jiangxi

320 Province Natural Science Foundation (Grant No. 20181ACB20014). J.L. was supported

321 by the National Natural Science Foundation of China (Grant No. 81402850). Functional bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

322 studies in this project were supported by the National Natural Science Foundation of China

323 (31300949 to B.Z. and 31300965 to G.L.C.)

324

325 Author Contributions

326 J.Z., J.D. and D.E.C. designed the project. J.Z. and J.D. designed and made constructs for

327 BacMam expression and determined the conditions used to enhance protein stability. J.Z.

328 purified the protein. Z.L. carried out detailed cryo-EM experiments, including data

329 acquisition and processing. J.L. and J.Z. built the atomic model on the basis of cryo-EM

330 maps. G.L.C., B.Z. and K.C. performed functional studies. X. P., J. Z., Y.Z., X. J., C.X.,

331 L.Z., W.L., X.L.T. and J.W. assisted with protein purification and the mutation of TRPC5

332 constructs for functional studies. J.D. and J.Z. drafted the initial manuscript. All authors

333 contributed to structure analysis/interpretation and manuscript revision. J.Z., D.E.C. and

334 Z.L. initiated the project, planned and analyzed experiments and supervised the research.

335 The authors declare no competing financial interests.

336 Data deposition: Cryo-EM electron density map of the mouse TRPC5 has been deposited

337 in the Electron Microscopy Data Bank, https://www.ebi.ac.uk/pdbe/emdb/ (accession

338 number EMD-9615), and the fitted coordinate has been deposited in the Protein Data

339 Bank, www.pdb.org (PDB ID code 6AEI). bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

340

341 Figure legends

342 Figure 1. Overall structure of mouse TRPC5.

343 a, Cryo-EM density map of mouse TRPC5 at 2.9 Å overall resolution with each monomer

344 represented in different colors (left, side view and right, top view). b, Ribbon diagrams of

345 the mouse TRPC5 model with the channel dimensions indicated. c, Ribbon diagrams

346 depicting structural details of a single subunit. d, Linear diagram depicting the major

347 structural domains of the TRPC5 monomer, color-coded to match the ribbon diagram in c.

348 Figure 2. The truncated TRPC5 construct used for cryo-EM encodes a functional

349 channel

350 Representative whole-cell patch clamp recordings and I-V relationships of (a) truncated

351 TRPC5, (b) full-length TRPC5, and (c) empty vector-transfected HEK293 cells. The time

352 course of currents measured at +80 and -80 mV and I-V relationships of the peak currents

353 in different conditions are shown. Englerin A is an activator and ML204 is an inhibitor

354 for TRPC4/5 channels.

355 Figure 3. TRPC5 ion conduction pathway compared with TRPC4 and other TRPCs

356 a, Side view of TRPC5’s pore region with chains A and C compared with TRPC4 (gray).

357 Ion conduction pathway shown as dots and mapped using HOLE with amino acid residues bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

358 labeled. b, Pore radius along the central axis. The side chains of glycine form a narrow

359 constriction at the selectivity filter. “INQ” motif forms the lower gate. c, Sequence of the

360 mouse TRPC5 aligned to TRPC4 and other TRPC subfamily members (Clustal Omega)

361 between S5 and S6 including the linker, pore helix, and pore loop. Regions corresponding

362 to different extracellular pore domains are indicated by the red arrow. The two cysteines

363 forming disulfide bonds, a conserved “LFW” motif, and the selectivity filter are

364 highlighted.

365 Figure 4. Comparison of the TRPC5 structure with other TRP channel structures

366 a, Superimposed side views of mouse TRPC5 subunit (blue) compared with other TRPC

367 family members including mouse TRPC4 (PDB: 5Z96, gray)37, human TRPC3 (PDB:

368 5ZBG, pink)12, and human TRPC6 (PDB: 5YX9, yellow)14. b, The conserved “LFW”

369 motif in the pore helix. c, Key pore loop disulfide bond between Cys553 and Cys548 in

370 TRPC5 and the corresponding pore loop-disulfide bond in TRPC4. This disulfide bond is

371 not present in TRPC3 or TRPC6 (black arrow); d, Differences in the organization of the S3

372 helix between the TRPC4/5 and TRPC3/6. The S3 helixes of TRPC3 and TRPC6 are

373 longer than those of TRPC5 and TRPC4.

374 Figure 5. The cation binding site in TRPC5

375 a, A putative Na+ (purple sphere) on the cytosolic face is in the hydrophilic pocket of the

376 S1-S4 domain, interacting with Glu418, Glu421, Asn436 and Asp439. b, Enlarged view bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

377 of the putative Na+ binding site. Comparable Ca2+ (green sphere) binding site and an

378 enlarged view of TRPM4 as “E/Q/N/D” (TRPC5: “E/E/N/D”). c, Sequence of the

379 mTRPC5 aligned to mTRPC4 and other representative TRP members (Clustal Omega).

380 The key residues are highlighted as “E/E/N/D”, which are conserved in TRPC2/3/5/6/7

381 and TRPM7 but as “E/Q/N/D” in TRPC4 and TRPM4.

382 Figure 6. Lipid coordination in TRPC5.

383 Lipid-channel interactions. a, Side and top views of ribbon diagrams of the

384 TRPC5 tetramer: four cholesterol hemisuccinate (CHS) molecules and four

385 phospholipids (potentially ceramide-1-phosphate, C1P, or phosphatidic acid, PA) are

386 shown as spheres with purple or yellow carbons, respectively. b, Side views of each CHS

387 and PA molecules per protomer. c and d, Ribbon diagram of the TRPC5 lipid-binding

388 regions. c, PA interacts with the pore helix through Trp577 and Phe576. d, CHS, shown

389 in purple, interacts with the S4/S5 linker at Asn500 and the N-terminal domains at

390 Trp315, Tyr316, and Trp322.

391

392 Supplementary Figures

393 Supplementary Figure 1. Biochemical characterization of the TRPC5 construct.

394 Size exclusion chromatography of TRPC5 proteins. Void volume (V0) and the peaks

395 corresponding to tetrameric TRPC5 and PMAL-C8 are indicated. Protein samples of the bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

396 indicated TRPC5 protein fraction were subjected to SDS-PAGE and Coomassie-blue

397 staining.

398 Supplementary Figure 2. Flow chart for TRPC5 cryo-EM data processing.

399 a, Representative image of the purified TRPC5 protein, 2D class averages of TRPC5

400 particles, side views of the 3D reconstructions from RELION 3D classification, and final

401 3D reconstructions from 3D auto-refinement. b, Fourier shell correlation (FSC) curve for

402 the 3D reconstruction (marked at overall 2.9 Å resolution). c, Local resolution estimation

403 from ResMap38 and, d, Euler distribution plot of particles used in the final

404 three-dimensional reconstruction. The length of the rod is proportional to the number of

405 particles in that view, with regions in red denoting the views containing the highest number

406 of particles.

407 Supplementary Figure 3. Cryo-EM densities of selected regions of TRPC5.

408 Density map showing the transmembrane helices (S1-S6), an (AR),

409 N-terminal helix, TRP domain, pore helix, connecting helix, coiled-coil helix, and lipids.

410 The maps were contoured at a level of 3.0 σ, carve = 2. Ankyrin repeats, AR: T144-K164,

411 N helix: P216-E234, S1: N355-Q386, S2: V402-W423, S3: F427-Y457, S4: F474-F500,

412 S5: H502-Y542, pore helix: F569-F580, S6: F596-F651, connecting helix: L707-N735 and

413 coiled-coil domain: E740-G761.

414 Supplementary Figure 4. Sequence alignment of TRPC subfamily members. bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

415 Sequence of the full-length mouse TRPC5 aligned to other TRPC subfamily members

416 (Clustal Omega); key residues indicated. Regions corresponding to the putative Na+

417 binding sites are labeled. The selectivity filter, lower gate, and two cysteines forming

418 disulfide bonds are highlighted.

419 Supplementary Figure 5. Pore loop structures of known TRP channels

420 a-h, comparison of the pore loop in TRPC5 (a, PDB accession number 6AEI) with other

421 known TRP channel, including TRPC4 (b, PDB accession number 5Z96), TRPC3 (c,

422 PDB accession number 5ZBG), TRPC6 (d, PDB accession number 5YX9), TRPM4 (e,

423 PDB accession number 6BWI), TRPM7 (f, PDB accession number 6BWD), TRPA1 (g,

424 PDB accession number 3J9P) and TRPV1 (h, PDB accession number 5IRX). i, Sequence

425 alignment of the pore loop in known TRP channels.

426 Supplementary Figure 6. Comparison of ion conducting pathways between TRPC5

427 and TRPC4.

428 Comparison of ion conduction pathways of a, TRPC5, and b, TRPC4 (PDB: 5Z96).

429 Distances between specific side chains along the pore and the key residues are labeled.

430 Supplementary Figure 7. The predicted Na+ binding sites.

431 Side and top views of electrostatic maps of predicted Na+ binding pockets in TRPC4; a,

432 Tetramer and b, Monomer. Gray dots highlight possible pathways for Na+ entry. The

433 surface is colored according to the calculated electrostatic potential, revealing the

434 tetrameric distribution of charge. Blue indicates positive potential, red negative potential, bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

435 and transparent white, neutral potential. c, A number of negatively charged residues,

436 including Asp633, Asp636, Glu638, Asp652 and Glu653 on the TRP domain, as well as

437 Asp424, Glu429 and Asp433 on S2-S3 linker, are aligned along the cation entry pathway.

438 d, The bottom view of the cytoplasmic cavity for cation.

439 Supplementary Figure 8. Lipid-channel interactions.

440 a, Side view of TRPC5 monomer. CHS (purple) and PA (yellow) molecules are shown as

441 sticks. b, Electrostatic maps of the lipids binding sites.

442 Supplementary Figure 9. Poor loop interactions

443 Key residues located in the pore loop are shown in stick representation. Arg593 forms a

444 hydrogen bond with Glu598.

445 Supplementary Figure 10. Intracellular mouth of the channel pore

446 Asp633 and Asp636 residues are shown in stick representation. Na+ bound between S2

447 and S3 are shown as purple spheres. Carbon and oxygen atoms of residues are shown in

448 green and red, respectively.

449

450 References

451 1. Clapham, D.E. TRP channels as cellular sensors. Nature 426, 517-24 (2003). 452 2. Schaefer, M. et al. Receptor-mediated regulation of the nonselective cation 453 channels TRPC4 and TRPC5. J Biol Chem 275, 17517-26 (2000). bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

454 3. Larsson, K.P. et al. Orexin-A-induced Ca2+ entry: evidence for involvement of 455 channels and protein kinase C regulation. J Biol Chem 280, 1771-81 (2005). 456 4. Strubing, C., Krapivinsky, G., Krapivinsky, L. & Clapham, D.E. TRPC1 and 457 TRPC5 form a novel cation channel in mammalian brain. Neuron 29, 645-55 458 (2001). 459 5. Obukhov, A.G. & Nowycky, M.C. TRPC5 channels undergo changes in gating 460 properties during the activation-deactivation cycle. J Cell Physiol 216, 162-71 461 (2008). 462 6. Hofmann, T., Schaefer, M., Schultz, G. & Gudermann, T. Transient receptor 463 potential channels as molecular substrates of receptor-mediated cation entry. J 464 Mol Med (Berl) 78, 14-25 (2000). 465 7. Lau, O.C. et al. TRPC5 channels participate in pressure-sensing in aortic 466 baroreceptors. Nat Commun 7, 11947 (2016). 467 8. Puram, S.V. et al. A TRPC5-regulated calcium signaling pathway controls 468 dendrite patterning in the mammalian brain. Dev 25, 2659-73 (2011). 469 9. Riccio, A. et al. Essential role for TRPC5 in amygdala function and fear-related 470 behavior. Cell 137, 761-72 (2009). 471 10. Wang, X. et al. TRPC5 Does Not Cause or Aggravate Glomerular Disease. J Am 472 Soc Nephrol 29, 409-415 (2018). 473 11. Zhou, Y. et al. A small-molecule inhibitor of TRPC5 ion channels suppresses 474 progressive kidney disease in animal models. Science 358, 1332-1336 (2017). 475 12. Fan, C., Choi, W., Sun, W., Du, J. & Lu, W. Structure of the human lipid-gated 476 cation channel TRPC3. Elife 7(2018). 477 13. Vinayagam, D. et al. Electron cryo-microscopy structure of the canonical TRPC4 478 ion channel. Elife 7(2018). 479 14. Tang, Q. et al. Structure of the receptor-activated human TRPC6 and TRPC3 ion 480 channels. Cell Res 28, 746-755 (2018). 481 15. Cui, N. et al. Involvement of TRP channels in the CO(2) chemosensitivity of 482 locus coeruleus neurons. J Neurophysiol 105, 2791-801 (2011). bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

483 16. Strubing, C., Krapivinsky, G., Krapivinsky, L. & Clapham, D.E. Formation of 484 novel TRPC channels by complex subunit interactions in embryonic brain. J Biol 485 Chem 278, 39014-9 (2003). 486 17. Xu, S.Z. et al. TRPC channel activation by extracellular . Nature 451, 487 69-72 (2008). 488 18. Duan, J. et al. Structure of the mouse TRPC4 ion channel. Nat Commun 9, 3102 489 (2018). 490 19. Autzen, H.E. et al. Structure of the human TRPM4 ion channel in a lipid nanodisc. 491 Science 359, 228-232 (2018). 492 20. Zhang, Z., Toth, B., Szollosi, A., Chen, J. & Csanady, L. Structure of a TRPM2 493 channel in complex with Ca(2+) explains unique gating regulation. Elife 7(2018). 494 21. Chen, X. et al. Molecular Determinants of the Sensitivity to Gq/11-Phospholipase 495 C-dependent Gating, Gd3+ Potentiation, and Ca2+ Permeability in the Transient 496 Receptor Potential Canonical Type 5 (TRPC5) Channel. J Biol Chem 292, 497 898-911 (2017). 498 22. Obukhov, A.G. & Nowycky, M.C. A cytosolic residue mediates Mg2+ block and 499 regulates inward current amplitude of a transient receptor potential channel. J 500 Neurosci 25, 1234-9 (2005). 501 23. Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct 502 conformations reveal activation mechanisms. Nature 504, 113-8 (2013). 503 24. Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel 504 determined by electron cryo-microscopy. Nature 504, 107-12 (2013). 505 25. Gao, Y., Cao, E., Julius, D. & Cheng, Y. TRPV1 structures in nanodiscs reveal 506 mechanisms of ligand and lipid action. Nature 534, 347-51 (2016). 507 26. Beck, A. et al. Conserved gating elements in TRPC4 and TRPC5 channels. J Biol 508 Chem 288, 19471-83 (2013). 509 27. Mastronarde, D.N. Automated electron microscope tomography using robust 510 prediction of specimen movements. J Struct Biol 152, 36-51 (2005). 511 28. Zheng, S.Q. et al. MotionCor2: anisotropic correction of beam-induced motion for 512 improved cryo-electron microscopy. Nat Methods 14, 331-332 (2017). bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

513 29. Ru, H. et al. Molecular Mechanism of V(D)J Recombination from Synaptic 514 RAG1-RAG2 Complex Structures. Cell 163, 1138-52 (2015). 515 30. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D 516 electron microscopy and related fields. J Struct Biol 116, 190-9 (1996). 517 31. Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM 518 structure determination. J Struct Biol 180, 519-30 (2012). 519 32. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation 520 from electron micrographs. J Struct Biol 192, 216-21 (2015). 521 33. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of 522 Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501 (2010). 523 34. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for 524 macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 525 213-21 (2010). 526 35. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular 527 crystallography. Acta Crystallogr D Biol Crystallogr 66, 12-21 (2010). 528 36. Smart, O.S., Neduvelil, J.G., Wang, X., Wallace, B.A. & Sansom, M.S. HOLE: a 529 program for the analysis of the pore dimensions of ion channel structural models. 530 J Mol Graph 14, 354-60, 376 (1996). 531 37. Duan, J. et al. Structure of full-length human TRPM4. Proc Natl Acad Sci U S A 532 (2018). 533 38. Kucukelbir, A., Sigworth, F.J. & Tagare, H.D. Quantifying the local resolution of 534 cryo-EM density maps. Nat Methods 11, 63-5 (2014).

535 bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 1.

a

90 °

b

90 °

Disulfide bond helix helix Pore

d S1 S2 S3 S4 S5 S6 c S6

Pore

Pre- S1 helix helix

S2 S4 TRP domain S2-S3 linker S5 S6 S1 S3 Pre-S1 elbow Pre-S1 elbow H4 H5 H6 H7

TRP domain H3 H2 HLH HLH N-terminal H1

AR4 AR4 Coiled-coil

domain AR3 AR3 ARD Coiled- ARD coil

AR2 AR2 domain domain

AR1 AR1

bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 2.

a b c

TRPC5 truncated TRPC5 full-length Empty vector Englerin A 100 nM Englerin A 100 nM 2

2 ML204 50 M 2 ML204 50 M 1 4 nA Englerin A 4 nA Englerin A 1.0 nA 0 Englerin A 100 nM 2 0 2 ML204 ML204 0 0.5 Basal Basal Englerin A -2 -100 100 -100 100 Basal

mV mV

Current(nA) Current(nA) Current(nA) -2 -2 -2 -100 100 -1 mV -4 -4 -0.5 -4 -6 -6 -1.0 -4 -2 0 1 2 3 0 1 2 3 4 0 1 2 3 Time (min) Time (min) Time (min) bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 3.

a TRPC5 TRPC4 b

N584 N580

G581 G577 Selectivity filter Central cavity

I621 I617 Lower gate

N625 N621 Q629 Q625

Disulfide bond Selectivity filter c LFW motif

S5 linker Pore helix Pore loop and linker S6 bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 4.

d a b, c b, c d

180 °

b c d

C553

C558 S2 S3 bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5. a

E418

D439

E421 Na+ N436

b

E828

D868

Q831 2+ Ca N865

E418 E421 N436 D439 c c

S2 S3 S2-S3 linker 445 444 868 932 798 486

S2 S2-S3 linker S3 bioRxiv preprint doi: https://doi.org/10.1101/467969; this version posted November 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 6.

a

90 °

c S5 PA Pore helix b W577

F576 S6

d

S5 CHS

S4 N500

W322 TRP domain

W315

Y316