14.1. Introduction

Calcium ions play important roles in many physiological processes, including neurotransmitter release, excitation-contraction coupling, cell motility, and gene expression [1]. Cellular calcium levels are precisely tuned by various channels and transporters. Transient receptor potential (TRP) channels, which are generally nonselective cation channels, conduct Ca²⁺ in response to disparate activators, including sensory stimuli such as temperature, touch, and pungent chemicals [2]. Members 5 and 6 of vanilloid subfamily (TRPV5 and TRPV6, previously named ECaC1 and ECaC2/CaT1, respectively) are uniquely Ca²⁺-selective (P_Ca/P_Na > 100) [3,4] TRP channels, both of which were identified in 1999 by expression cloning strategies utilizing cDNA libraries from rabbit kidney [5] and rat duodenum [6], respectively. While TRPV5 expression is mainly restricted to the kidney, TRPV6 is expressed in various tissues including the stomach, small intestine, prostate, esophagus, colon, and placenta. Genetic knockout of TRPV5 or TRPV6 in mice suggests the importance of these channels for Ca²⁺ homeostasis. TRPV5 knockout mice showed defects in renal Ca²⁺ reabsorption and reduced bone thickness [7], and the knockout of TRPV6 resulted in defective intestinal calcium absorption, decreased bone mineral density, reduced fertility, and hypocalcemia when challenged with a low Ca²⁺ diet [8]. Further support for the role of these channels in Ca²⁺ absorption and homeostasis stems from the robust regulation of their expression by the calciotropic hormone vitamin D [9–12] (see Chapter 13).

TRPV6 has been shown to be aberrantly expressed in numerous cancer types, including carcinomas of the colon, prostate, breast, and thyroid [13–18]. The correlation between TRPV6 expression and tumor malignancy and its potential contribution to cancer cell survival has highlighted TRPV6 as a target for cancer diagnosis and treatment [15,17,19,20]. Indeed, a selective inhibitor of TRPV6 activity derived from northern short-tailed shrew venom [21] has entered phase I clinical trials in patients with advanced solid tumors of tissues known to express TRPV6, including the pancreas and ovary [22].

Structurally, TRPV5 and TRPV6 share ∼75% sequence identity with each other and are ∼25% identical to the founding member of the TRPV subfamily TRPV1. The transmembrane (TM) domain has the same topology as tetrameric K⁺ channels [23], with six TM helices (S1–S6) and a pore-forming re-entrant loop between the S5 and S6. Importantly, this loop contains a conserved aspartate residue that is critical for the calcium permeability of TRPV5 and TRPV6 [24,25], suggesting that this residue at least in part comprises the selectivity filter. Flanking the TM domain are relatively large intracellular N- and C-termini. The N-terminus, which includes six ankyrin repeats [26], is critical for proper channel assembly and function [27,28], while the C-terminus contains domains involved in Ca²⁺/calmodulin-dependent inactivation [29–31] (see Chapter 13).

To help understand the functional mechanisms of TRPV5/6 and potentially inform rational drug design, we sought to obtain a high-resolution structure of an intact channel. Until several years ago, the only viable method of obtaining such a structure was x-ray crystallography. However, producing well-diffracting crystals can be a notoriously difficult and resource-consuming process because membrane proteins typically have low expression and purification yields, poor stability in detergent, and inherent flexibility [32]. However, structural biologists are now able to circumvent this major bottleneck, owing to recent advances in single-particle cryo-electron microscopy (cryo-EM), which have facilitated the determination of membrane protein structures at near-atomic resolutions without prior crystallization [33]. These advances have had a particularly profound effect on the TRP channel field, as atomic-level cryo-EM structures have been determined for TRPV1 [34–36]; TRPV2 [37,38]; and ankyrin subfamily member [39]. The ability to computationally select specific conformational states from a heterogeneous cryo-EM sample can be especially powerful when studying mechanisms of gating, as exemplified by studies of TRPV1 in various ligand-induced conformations [34–36]. Cryo-EM will surely continue to be exploited with great effect to elucidate structures of TRP channels and other membrane proteins.

As yet, there are several benefits that may make obtaining an x-ray structure desirable over cryo-EM. First, crystallographers can use true statistical approaches such as the Free R value [40] to evaluate the accuracy of atomic models against experimental data, while analogous methods in cryo-EM [41–43] are still relatively nascent. Second, the resolution of a cryo-EM map usually varies widely across a single reconstruction, with more flexible regions, typically in the periphery, being less resolved or completely absent. For example, in TRPV1, while the TM domain is well resolved, the first two ankyrin repeats at the distal N-terminus are missing from the electron density maps [34–36], presumably due to their flexibility (Figure 14.1a). In x-ray structures, the resolution obtained from the diffraction data is more representative of the structure as a whole, and peripheral or flexible regions may be stabilized by crystal contacts and thus adequately resolved (Figure 14.1b). Third and perhaps most importantly, the position of anomalous scatterers, such as selenium atoms in selenomethionine-labeled protein, sulfur atoms in native cysteine or methionine side chains, or heavy atoms bound to the protein, can be accurately identified with little ambiguity. Anomalous scattering can therefore be utilized to robustly aid or validate sequence registry (Figure 14.1c), which is especially important for low-resolution structures and/or regions with poor electron density. Using anomalous scattering to identify bound ions is particularly useful for studying ion channel structures, as ion binding at specific locations is vital for understanding permeation and ion channel block. For TRPV6, we used these techniques to identify binding sites for the permeant cations Ca²⁺ and Ba²⁺, as well as the channel blocker Gd³⁺ (Figure 14.1d through g). Methods to unambiguously identify specific atoms or small labels in cryo-EM electron density maps have yet to be developed. We were motivated by each of these factors as we attempted to crystallize TRPV5/6 in the midst of the cryo-EM “resolution revolution.”

Figure 14.1

Crystallographic analysis of TRPV6. (a) Cartoon model of TRPV1 (yellow, PDB ID: 3J5P) with cryo-EM density (blue surface, EMD-5778) superimposed. Note the peripheral regions of the model, consisting of the first two ankyrin repeats, do not have corresponding (more...)

In 2016, we reported the crystal structure of intact rat TRPV6 at 3.25 Å resolution [44]. To our knowledge, this represented the first crystal structure of a TRP channel and the second crystal structure of a naturally occurring Ca²⁺-selective channel, after the structure of the calcium release-activated calcium (CRAC) channel Orai reported in 2012 [45]. A detailed description of the TRPV6 structure can be found elsewhere [44]. In this chapter we will focus on the multiyear journey taken to determine the structure, in which >150 constructs were purified and subjected to crystallization screening, and thousands of crystals were tested for diffraction. We will summarize the methods used to screen constructs and precrystallization conditions, express and purify protein, grow and optimize crystals, and collect and analyze diffraction data. Finally, we will briefly compare the structural bases of Ca²⁺-selective permeation in TRPV6 and Orai.

14.2. Precrystallization Screening of Protein Expression and Biochemical Behavior Using FSEC

Obtaining milligram quantities of pure, monodisperse protein is a general prerequisite for protein crystallography studies. As large-scale membrane protein expression and purification can be very costly and time consuming, methods to efficiently and rapidly screen genetic constructs and buffer compositions at smaller scales are necessary to optimize protein expression levels and biochemical behavior. For this project, we employed fluorescence-detection size exclusion chromatography (FSEC) [46], where the target protein is expressed as a fusion with green fluorescent protein (GFP) (or other fluorescent proteins) and crude lysate containing just nanogram quantities of the fusion protein is loaded onto a gel filtration column. The resulting chromatogram provides a quick assessment of the identity (elution time), biochemical behavior (number, sharpness, and symmetry of chromatographic peaks), and expression level (peak amplitude) of the target protein. FSEC experiments can routinely be conducted using adherent Sf9 or HEK cells in a six-well plate (Figure 14.2a) to inform larger scale expression, which can be carried out in polycarbonate Erlenmeyer flasks (Figure 14.2b). A concurrent advantage of expressing the target protein as a GFP fusion, even at the large scale, is that expression levels can be monitored in real time by epifluorescence microscopy (Figure 14.2c).

Figure 14.2

TRPV6 expression and FSEC screening.

Initially, we used FSEC to screen C-terminal GFP fusions of ∼20 TRPV5/6 orthologues for crystallographic studies. To facilitate affinity purification and proteolytic removal of the GFP tag, a strep tag (WSHPQFEK) or 8x His tag was added to the C-terminus and thrombin site cleavage site (LVPRGS) was inserted between the target protein and GFP. While a majority of orthologues showed poor chromatographic behavior, rat TRPV6 (rTRPV6), which displayed a single sharp, dominant FSEC peak, was selected as a promising candidate for further study. As our initial efforts to crystallize wildtype rTRPV6 protein were unsuccessful, we generated truncation mutations and screened them by FSEC. A small-scale FSEC screen of C-terminal truncations is shown in Figure 14.2d. Several of these truncations improved the expression level of rTRPV6. In parallel, we used FSEC to screen a panel of detergents used for the extraction (solubilization) and purification of the target protein (Figure 14.2e). We also used FSEC to optimize parameters for protein expression, including incubation time (Figure 14.2f) and the concentration of histone deacetylase inhibitor sodium butyrate (NaBu) added to improve the expression level (Figure 14.2g). Exploiting FSEC at the small-scale to inform large-scale purifications was critical to maximizing the efficiency of the crystallization trials.

14.3. Large-Scale Purification and Crystallization

Large-scale purifications (800 mL–6 L) were conducted by the baculovirus infection of Sf9 cells or baculovirus-mediated transduction of HEK293 cells [47]. We found that the BacMam system provided ∼2x greater yields (∼1 mg/L of cells depending on the construct) than Bac-to-Bac (∼0.5 mg/L). A flow chart of the process of going from a sequenced plasmid containing the desired target construct to a purified protein ready for crystallization trials, which takes approximately 2–3 weeks, is shown in Figure 14.3a. Throughout the project, we kept the same purification protocol overall but varied the expressed protein construct and the detergent (and lipid) used for solubilization.

Figure 14.3

Purification of TRPV6.

For large-scale expression, suspension-adapted HEK 293S cells lacking N-acetyl-glucosaminyltransferase I were grown in Freestyle 293 media (Life Technologies) supplemented with 2% FBS at 37°C in the presence of 5% CO₂. The culture was transduced with P2 baculovirus once cells reached a density of 2.5–3.5 x 10⁶ per mL. After 8–12 h, 10 mM NaBu was added and the temperature was changed to 30°C. Cells were harvested 48–72 h posttransduction and resuspended in a buffer containing 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM β-mercaptoethanol (βME), 0.8 μM aprotinin, 2 μg/mL leupeptin, 2 mM pepstatin A, and 1 mM phenylmethysulfonyl fluoride (PMSF). The cells were disrupted using a Misonix Sonicator (12 x 15 s, power level 7), and the resulting homogenate was clarified by spinning in a Sorvall centrifuge at 7500 rpm for 15 min. Crude membranes were collected by ultracentrifugation for 1 h in a Beckman Ti45 rotor at 40,000 rpm. The membranes were mechanically homogenized and subsequently solubilized for 2–4 h in a buffer containing 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM βME, 20 mM n-dodecyl-β-d-maltopyranoside (DDM), 0.8 μM aprotinin, 2 μg/mL leupeptin, 2 mM pepstatin A, and 1 mM PMSF. After insoluble material was removed by ultracentrifugation, streptavidin-linked resin was added to the supernatant and rotated for 4–16 h. Resin was washed with 10 column volumes of wash buffer containing 150 mM NaCl, 20 mM Tris pH 8.0, 1 mM βME, and 1 mM DDM, and the protein was eluted using wash buffer supplemented with 2.5 mM d-desthiobiotin. The eluted fusion protein was concentrated to ∼1.0 mg/mL and digested with thrombin at a mass ratio of 1:100 (thrombin:protein) for 1.5 h at 22°C. The digested protein was concentrated and injected into a Superose 6 column equilibrated in a buffer composed of 150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM βME, and 0.5 mM DDM. Ten millimolar tris(2-carboxyethyl)phosphine (TCEP) was added to fractions with elution time corresponding to the tetrameric channel, and protein was concentrated to 2.5–3.0 mg/mL using a 100 kDa MWCO concentrator. All purification steps were conducted at 4°C. Typical purifications yielded ∼1 mg of purified protein per liter of transduced cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 14.3b) and FSEC (Figure 14.3c) analysis revealed a final product with high chemical and conformational homogeneity.

We produced TRPV6 protein labeled with the unnatural amino acid selenomethionine for the subsequent detection of the selenium atoms to aid sequence registry during model building. In regions of the protein devoid of methionines in the natural sequence, we introduced methionine substitutions. Protocols to express selenomethionine-labeled protein in HEK cells were adapted from literature [48]. Specifically, 6–8 h after transduction with P2 baculovirus, cells were pelleted and resuspended in DMEM (Life Technologies) supplemented with 10% FBS and lacking l-methionine. After incubating methionine-depleted cells for 6 h at 37°C, 60 mg of l-selenomethionine was added per liter of cells. Thirty-six to forty-eight hours after transduction, cells were harvested and protein was purified using the same protocol as described earlier, except for the addition of 4 mM l-methionine to all purification buffers, excluding the final gel filtration buffer. This procedure yielded ∼0.4 mg of selenomethionine-labeled protein per liter of transduced cells.

Purified protein was routinely subjected to high-throughput screening (∼100 nL protein per drop) of crystallization facilitated by liquid dispensing robotics and automated imaging systems. Initial tests identified the best range of protein concentrations (2–3 mg/mL) and crystallization temperatures (20°C–22°C) that were maintained in a majority of subsequent crystallization screening experiments. Primary “hits” from the screening experiments were imaged for UV fluorescence (Figure 14.4a, d, and g) and scaled up (1–2 μL protein per drop) in sitting drop or hanging drop vapor diffusion trays. Overall, we found that hanging drop vapor diffusion resulted in the largest and most reproducible crystals (Figure 14.4a, d, and g). Crystallization conditions were optimized for pH, precipitant concentration, salt concentration, salt type, and protein to mother liquor ratio. To obtain structures in the presence of various cations, protein was incubated with Ca²⁺, Ba²⁺, or Gd³⁺ for ∼1 h prior to crystallization. Selenomethionine-labeled protein crystallized in similar conditions to native protein but yielded significantly smaller crystals. We also manually screened for additives (salts, volatiles, other small molecules, or detergents) to improve crystal size or morphology by briefly incubating the protein with the additive prior to crystallization. In addition, a variety of postcrystallization treatments to improve diffraction quality were attempted, including dehydration, chemical cross-linking of free amines, and screening various cryoprotectants [49].

Figure 14.4

Crystallization of TRPV6 mutants in various space groups. (a, d, and g) Initial crystals from high-throughput screen imaged using white (top left) or UV (bottom left) light and manually optimized crystals (right) in C2 (a), C222₁ (d), or P42₁2 (g) space (more...)

14.4. Collection and Processing of Diffraction Data

X-ray diffraction data were collected at NSLS X29, APS NECAT 24 ID-C/E, and ALS 5.0.1/5.0.2 synchrotron beamlines. Prior to data collection, crystals were harvested in nylon loops matching the crystal size, cryoprotected by incubating in cryoprotectant solution (usually the mother liquor used for crystal growth with added glycerol, ethylene glycol, or low molecular weight polyethylene glycol) and plunged and stored in liquid nitrogen. Crystals were transferred to ALS-style or universal pucks for shipping and to aid remote-controlled robotics for crystal positioning and data collection at the beamline.

Crystals were initially screened for diffraction quality by visually examining diffraction patterns after exposure to x-rays at several angles and locations along the crystal. Those with satisfactory diffraction properties were selected for the collection of a complete data set. The angular coverage necessary to collect a complete diffraction data set depends on the symmetry of the crystal lattice. Thus, the strategy employed for collecting full data sets was chosen based on the crystal form. For example, for crystals in the P42₁2 space group, the crystal must be rotated at least 90° during data collection (with 1–3 frames collected per degree, depending on the detector used and crystal mosaicity) to collect a complete data set with the maximum number of unique diffraction spots. To minimize radiation decay during data collection, crystals significantly larger than the x-ray beam cross section were translated along a vector orthogonal to the beam.

To crystallographically identify cations such as Ca²⁺, Ba²⁺, and Gd³⁺ (Figure 14.1e through g), the x-ray beam was tuned to specific wavelengths at the synchrotron to maximize the anomalous scattering of the cation. The wavelengths chosen were 1.75 Å for Ca²⁺ and Ba²⁺ and 1.56 Å for Gd³⁺. To detect anomalous scattering, precise differences between each diffraction spot and its centrosymmetrically related partner (Friedel pair) must be calculated. As such, care must be taken to minimize radiation damage in the time between collections of Friedel pairs. We thus employed the “inverse beam” data collection approach, in which diffraction from small (∼10°) wedges related centrosymmetrically (separated by 180°) are collected consecutively. The same approach was used to identify selenium atoms in crystals with selenomethionine-labeled protein, as well as sulfur atoms in crystals with native protein.

The experimentally collected spot intensities in diffraction data were indexed, integrated, scaled, and merged together using processing programs such as XDS [50], HKL2000 [51], and IMOSFLM [52]. The scaled intensities were converted into the structure factor amplitudes by the CTRUNCATE program in CCP4 suite [53]. While the x-ray detector provides the structure factor amplitudes, it cannot collect data to provide the corresponding phase information, which is necessary to convert the structure factors into electron density. Thus, to obtain the structure, crystallographers must solve the “phase problem” in one of several ways [54]. When homologous (∼25% identity or greater) structures of the protein or its domain(s) are available, the phases can be obtained by the method of molecular replacement (MR). In MR, the homologous structure(s), or “search model(s),” are rotated and translated in a crystal lattice according to various algorithms [55] and structure factors are back calculated and compared to experimental data. If the MR program can find a search model orientation that adequately matches the experimental diffraction, electron density maps can be calculated from the obtained phases. The resulting model is then refined against the map to improve the phases and the accuracy of the structure.

We used the Phaser [56] program to obtain phases and an initial structural solution by MR with the structure of mouse TRPV6 ankyrin domain (PDB code 2RFA; consisting of residues 44–225) as a search model. The resulting structural model was iteratively refined in REFMAC [57] or PHENIX [58] and built in COOT [59]. The TM domain was built using the TRPV1 cryo-EM reconstruction (PDB code: 3J5P) [35] as a guide. The structural model improvement was monitored by the gradual decrease in R-factors. At the final stages, the model quality was assessed by low values of the R-factors and good stereochemistry (low values for Ramachandran outliers, bond angle, and bond length deviations).

14.5. Protein Engineering to Improve Crystal Packing

Obtaining well-diffracting crystals of membrane proteins requires the optimization of many parameters, including the target genetic construct, expression system, detergents/lipids used for protein solubilization, buffer composition, crystallization method (vapor diffusion, microbatch, lipid crystalline phase, etc.), crystallization condition, and postcrystallization treatments. Perhaps the most important determinant for the crystallization of membrane proteins is the composition of its amino acid sequence; genetic engineering of the crystallizing construct has been critical for making various classes of membrane proteins amenable to crystallization. For example, the tethering of fusion partners such as T4 lysozyme has aided the crystallization of many different G-protein coupled receptors (GPCRs) [60], while the screening of mutants to improve thermostability has been employed for various targets including ionotropic glutamate receptor [61], serotonin transporter [62,63], and GPCRs [64–66].

Initial efforts to improve crystallization of TRPV6 by construct engineering included N- and C-terminal truncations, deletions of hypervariable regions such as the extracellular loop connecting TM segments S1 and S2, mutations of exposed cysteines to prevent nonspecific aggregation, mutations of a conserved N-linked glycosylation site [67], and mutations of high entropy residues such as lysine and glutamic acid [68]. After the optimization of purification conditions and crystallization of these constructs as described earlier, the diffracting power of the crystals was limited to approximately 6 Å resolution.

While the data at this resolution were not useful for building an accurate structural model of TRPV6, we were able to obtain MR solutions (see preceding text) that accurately placed the protein’s ankyrin domains in a crystal lattice, thus providing information about secondary structure elements or residues involved in crystal lattice contacts. Based on this information, we generated mutants (substitutions, deletions, and insertion of fusion partners) aimed at strengthening crystal contacts or favoring new ones. Each of these mutants was tested for expression and biochemical behavior by FSEC prior to purification and crystallization trials. While a majority of the >100 mutants informed by this MR solution-based strategy either had no appreciable effect or completely ablated crystallization, several constructs resulted in new crystal forms that have different crystal packing contacts (Figure 14.4b, c, e, f, h, and i). The construct used to build the final structural model contained three substitutions in the ankyrin domain (I62Y, L92N, and M96Q), in addition to a single substitution in the TM domain and a C-terminal truncation. Of these three ankyrin domain mutations, single substitution I62Y completely favored crystallization in the P42₁2 (Figure 14.4g through i) space group over C222₁ (Figure 14.4d through f), despite identical purification protocols and similar crystallization conditions. The best crystals in the P42₁2 space group diffracted to a much higher resolution limit (∼3.3 Å) than in C222₁ (∼4.0 Å), allowing us to build a significantly more accurate and complete structural model. Overall, these results are a striking example of how subtle changes in the protein construct can have enormous effects on the success of a membrane protein crystallization project.

14.6. Comparison of TRPV6 and Orai Structures

Rat TRPV6 [44] and Drosophila Orai [45] are the only eukaryotic Ca²⁺-selective ion channels with crystal structures available. The recently published 4.2 Å cryo-EM structure of the Ca_v1.1 complex [69] and crystal structures of engineered Ca²⁺-selective prokaryotic channels [70] are beyond the scope of this discussion. Orai proteins serve as the pore-forming subunits of the CRAC channel, which is activated by interaction with the intracellular calcium sensor, the stromal interaction molecule (STIM) [71,72]. TRPV6 and Orai share similar biophysical properties, including high calcium selectivity, permeability to monovalent cations in the absence of external divalents, channel block by trivalent cations, strong inward rectification, and Ca²⁺/calmodulin-dependent inactivation [72,73]. Due to these similarities, in 2001 TRPV6 was proposed to also compose the pore of the CRAC channel [4]. However, distinct features of TRPV6 and CRAC channels, including higher Cs⁺ permeability in TRPV6, activation of CRAC channel but not TRPV6 by ionomycin-induced Ca²⁺ store depletion, and voltage-dependent Mg²⁺ block of TRPV6, but not CRAC channels, subsequently rebuffed this idea [74].

While TRPV6 and Orai differ completely in sequence, fold, and oligomeric state (Figure 14.5a and e), close comparison of their structures reveals similarities that underlie resemblances in their biophysical properties. The most striking similarities lie in the extracellular vestibule and selectivity filters. Both TRPV6 and Orai have highly electronegative extracellular vestibules that may serve to attract divalent metal cations (normally Ca²⁺ and Mg²⁺) to the pore (Figure 14.5b and f). In TRPV6, acidic residues from the extracellular pore loops connecting the pore helix and selectivity filter to S5 and S6 contribute to the electronegativity (Figure 14.5c and d), while in Orai, aspartates in the M1–M2 loop are involved (Figure 14.5g and h). Interestingly, in co-crystals of TRPV6 with Ba²⁺ or Gd³⁺, we observed peaks in the anomalous difference Fourier maps in the vicinity of acidic side chains in the extracellular vestibule (Figure 14.1f and g), indicating that these residues might constitute “recruitment sites” for divalent and trivalent cations.

Figure 14.5

Structural comparison of rat TRPV6 and Drosophila Orai. (a and e) Membrane topology diagram of TRPV6 (a) and Orai (e). TRPV6 assembles as a tetramer with each subunit containing six TM helices (S1–S6) and a re-entrant pore loop between S5 and (more...)

Early mutagenesis studies of TRPV6 [25,75] and Orai [76–78] highlighted a single aspartate or glutamate residue (D541 in rat TRPV6, E178 in Drosophila Orai, D542 in human TRPV6, and E106 in human Orai1) as determinants of their permeation properties, and it was proposed for both channels that high Ca²⁺ selectivity is conferred by the coordination of Ca²⁺ by these side chains. The crystal structures reveal that these residues reside at analogous locations; their side chains protrude toward the central pore axis to produce constrictions at the pore mouth. Further, co-crystallization or soaking experiments showed that these residues comprise binding sites for Ca²⁺, Ba²⁺, and Gd³⁺ in both Orai and TRPV6. Interatomic distances between the cation and the carboxylate oxygen suggest that in both cases, the acidic side chain directly coordinates the (at least) partially dehydrated cation. Notably, TRPV6 contains two additional Ca²⁺-binding sites along the permeation pathway (Figure 14.5d), while the aforementioned E178 site seems to be the only cation-binding site in Orai. On the other hand, basic residues in the lower region of the Orai pore appear to form a binding site for anion(s) that plug this pore (Figure 14.5h). Thus, apart from the binding of Ca²⁺ and other cations at acidic residues in the pore mouth, TRPV6 and Orai have distinct permeation mechanisms. Additional structures of these channels in activated states (TRPV6 requires PI(4,5)P₂ for activation [31] while Orai is opened by STIM [71,72]) will be required to obtain a more complete structural understanding of how these channels contribute to calcium entry in non-excitable cells.

Acknowledgments

We thank E.C. Twomey for comments on the manuscript. This work was supported by the NIH grants R01 NS083660 (A.I.S) and T32 GM008281 (K.S.), by the Pew Scholar Award in Biomedical Sciences, the Schaefer Research Scholar Award, the Klingenstein Fellowship Award in the Neurosciences, and the Irma T. Hirschl Career Scientist Award (A.I.S.).

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Kei Saotome

Department of Neuroscience

Howard Hughes Medical Institute

Dorris Neuroscience Center

The Scripps Research Institute

La Jolla, California

and

Department of Integrative Structural and Computational Biology

The Scripps Research Institute

La Jolla, California

Appu K. Singh

Department of Biochemistry and Molecular Biophysics

Columbia University

New York, New York

Alexander I. Sobolevsky

Department of Biochemistry and Molecular Biophysics

Columbia University

New York, New York