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An allosteric mechanism of inactivation in the calcium-dependent chloride channel BEST1
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Publication date (Print):
05 November 2018
Journal:
The Journal of General Physiology
Publisher:
Rockefeller University Press
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Abstract
BEST1 is a chloride channel that is activated by calcium. Vaisey and Long demonstrate that ionic currents through BEST1 inactivate by an allosteric mechanism in which the binding of a C-terminal peptide to a surface-exposed receptor controls a physically distant inactivation gate within the pore.
Abstract
Bestrophin proteins are calcium (Ca 2+)-activated chloride channels. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Whole-cell recordings show that ionic currents through BEST1 run down over time, but it is unclear whether this behavior is intrinsic to the channel or the result of cellular factors. Here, using planar lipid bilayer recordings of purified BEST1, we show that current rundown is an inherent property of the channel that can now be characterized as inactivation. Inactivation depends on the cytosolic concentration of Ca 2+, such that higher concentrations stimulate inactivation. We identify a C-terminal inactivation peptide that is necessary for inactivation and dynamically interacts with a receptor site on the channel. Alterations of the peptide or its receptor dramatically reduce inactivation. Unlike inactivation peptides of voltage-gated channels that bind within the ion pore, the receptor for the inactivation peptide is on the cytosolic surface of the channel and separated from the pore. Biochemical, structural, and electrophysiological analyses indicate that binding of the peptide to its receptor promotes inactivation, whereas dissociation prevents it. Using additional mutational studies we find that the “neck” constriction of the pore, which we have previously shown to act as the Ca 2+-dependent activation gate, also functions as the inactivation gate. Our results indicate that unlike a ball-and-chain inactivation mechanism involving physical occlusion of the pore, inactivation in BEST1 occurs through an allosteric mechanism wherein binding of a peptide to a surface-exposed receptor controls a structurally distant gate.
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Cryo-EM structures of the TMEM16A calcium-activated chloride channel
Shangyu Dang, Shengjie Feng, Jason Tien … (2017)
Calcium-activated chloride channels (CaCCs) encoded by TMEM16A 1–3 control neuronal signaling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system 4–7 . To understand how CaCCs mediate and control anion permeation to fulfill these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues is essential. TMEM16A forms a dimer with two pores 8,9 . Thus far, CaCC structural analyses relied on homology modeling of a fungal homolog nhTMEM16 that functions primarily as a lipid scramblase 10–12 and subnanometer-resolution electron cryo-microscopy (cryo-EM) 12 . Here, we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol (LMNG) as determined by single-particle cryo-EM. These structures reveal the ion permeation pore and represent different functional states. The LMNG structure has one Ca2+ ion resolved within each monomer with a constricted pore, likely corresponding to a closed state because CaCC with single Ca2+ occupancy requires membrane depolarization to open 13 . The nanodisc structure has two Ca2+ ions per monomer and its pore in a closed conformation likely reflects channel rundown, a gradual loss of channel activity following prolonged CaCC activation in 1 mM Ca2+. Structure-based mutagenesis and electrophysiological studies identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these data clarify the basis of CaCC anion conduction. Our screen of deletion constructs of the mouse TMEM16A splice variant 1 that contains exon ‘a’ but not exons ‘b-d’ identified a C-terminally truncated mutant with high expression and stability (Extended Data Fig. 1a) and channel function (Extended Data Fig. 1b). Its increased Ca2+ sensitivity is reminiscent of gating changes observed in other TMEM16A truncation mutants 14,15 . Reducing negatively charged lipids via poly-L-lysine (PLL) caused desensitization by reducing Ca2+ sensitivity without altering the plateau current (Extended Data Fig. 1c). This truncated TMEM16A including amino acids 1–903 was purified in detergent followed by reconstitution into lipid nanodiscs (Extended Data Fig. 1d) or kept solubilized in LMNG (Extended Data Fig. 1h) for further analyses. N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) assay 16,17 revealed a similar melting temperature (Tm) for Ca2+-free TMEM16A while stepwise increase of Ca2+ concentration caused Tm to gradually increase for TMEM16A in nanodisc but not for LMNG-solubilized TMEM16A (Extended Data Fig. 1e, i); this stabilizing effect of Ca2+ reached plateau at ~1 μM Ca2+ (Extended Data Fig. 1e), as did channel activation (Extended Data Fig. 1b). The CPM assay thus revealed that TMEM16A in nanodiscs or LMNG differ in their Ca2+ binding characteristics. Negative stain electron microscopy (EM) of LMNG-solubilized TMEM16A and dodecylmaltoside (DDM):cholesterol hemisuccinate (CHS)-solubilized TMEM16A reconstituted into lipid nanodiscs revealed homogeneous preparations suitable for high resolution structural studies (Extended Data Fig. 1f, g, j, k). Using single-particle cryo-EM we determined the structures of TMEM16A to better than 4 Å nominal resolutions (Extended Data Fig. 2, 3, 4b). Local resolution analysis showed that the core transmembrane domains have the highest (better than 4 Å) resolution and the extracellular and cytoplasmic domains have the poorest (Extended Data Fig. 2f, 3f), suggesting conformational flexibility of these regions. The resolution of the LMNG-solubilized TMEM16A reconstruction is anisotropic because images of particles oriented along certain axes were lacking (Extended Data Fig. 3c, h, i). Because the missing views are within a small angular range, there is no distortion regarding the definition of the transmembrane helices with respect to the membrane. The anisotropy is considerably reduced by adding particles from a dataset in which a monoclonal fragment of antigen binding (Fab) isolated from a human naïve Fab phage library 18,19 is bound to TMEM16A (Extended Data Fig. 3b, h, i). The final density maps allowed reliable assignment of most of the side chains in all ten transmembrane segments and enabled building de novo atomic models of the transmembrane segments of dimeric TMEM16A in both nanodisc and LMNG (Fig. 1a, b; Extended Data Fig. 4a, c, d, 5a–f). Cross-validation between the atomic model and the density maps of the transmembrane domains suggested that the average resolutions are around 4 Å (Extended Data Fig. 2g, 3g). The nanodisc-reconstituted TMEM16A is structurally similar to the subnanometer-resolution structure of digitonin-solubilized TMEM16A 12 but missing part of the C-terminal cytoplasmic domain owing to the truncation construct used in our study (Extended Data Fig. 5g–i). To locate the pore, we used the HOLE program 20 to delineate potential water-filled profiles in these structures. By testing whether anion selectivity could be altered by mutations of residues lining the potential pore within each monomer or the potential pore at the dimer interface that is associated with lipids, we identified the pore surrounded by transmembrane helices TM3–8 (Fig. 1c–e and Extended Data Fig. 6d). This pore is not exposed to the lipid bilayer as proposed 21–24 . Similar to a previous study 12 , our cryo-EM studies revealed a pore too narrow for the passage of permeant anions. It remains possible that some open conformation of the pore is partly lined by lipids. A comparison of nanodisc-reconstituted and LMNG-solubilized TMEM16A density maps (Fig. 2a, b) revealed differences primarily in the positions and orientations of TM3, TM4, and TM6 (Fig. 2c–f; Extended Data Fig. 7a–f). These pore-lining helices in the TMEM16A channel are also displaced noticeably from the positions of the equivalent helices of the nhTMEM16A lipid scramblase 10 . We found some helical distortions in the nanodisc-reconstituted TMEM16A starting from G640 and extending towards the lower part of TM6 (Extended Data Fig. 4a, 7a, b). In the LMNG reconstruction, the lower part of the TM6 could not be resolved beyond G640 (Extended Data Fig. 4a, 7d, e). These observations are in agreement with mutagenesis studies implicating G640 as a flexible hinge that affects channel gating 13 . Additionally, the two structures differ in the orientation of TM3 and in the arrangement of the TM5-TM6 loop that is near the pore entrance and harbors K599 and R617 important for anion selectivity 25 , and the TM9-TM10 loop that harbors R784 important for anion selectivity 25 and packs against the TM5-TM6 loop (Extended Data Fig. 7g–l). We observed densities consistent with the presence of ordered lipids near the dimer interface but not near the pore of both nanodisc-reconstituted and LMNG-solubilized TMEM16A (Fig. 1a; Extended Data Fig. 7b, c, e, f). And the number of ordered lipids observed in the nanodisc structure is greater than that in the LMNG structure. It is possible that lipids may stabilize TMEM16A in the more native environment of nanodiscs. These two structures showed a surprising difference near the putative Ca2+ binding sites even though both structures were determined in the presence of 1 mM calcium. Consistent with previous studies 10,26,27 , the acidic residues E650, E698, E701, E730 and D734 group together to form two Ca2+ binding sites in nanodisc-reconstructed TMEM16A (Fig. 2g, h). Comparisons of the maps and models suggest that these residues are well organized in nanodisc-reconstructed TMEM16A, but less so in LMNG-solubilized TMEM16A (Fig. 2h, i). The difference maps between the experimental map and the map from the refined structure that do not contain Ca2+ ions showed a clear omit density that we assigned as a bound Ca2+ ion, for LMNG-solubilized TMEM16 at σ = 13 and for nanodisc-reconstituted TMEM16A at σ = 8. The location of this bound Ca2+ ion matches well with one of the two bound Ca2+ ions in the crystal structure of nhTMEM16A 10 . In the nanodisc structure, there is another omit density (σ = 10) nearby that may correspond to the second bound Ca2+ ion (Fig. 2h). In contrast, in the LMNG structure, no other significant omit density was found nearby, suggesting there is only one Ca2+ ion bound (Fig. 2h). Notably, E650 on the lower half of TM6 is not resolved in LMNG-solubilized TMEM16A (Fig. 2h; Extended Data Fig. 4a, 7d). Our mutagenesis studies showing that alanine substitution of E650 raises the Ca2+ concentration for half maximal activation (EC50) and reduces the Hill coefficient from two to one 13,26 indicate that this residue in TM6 is critical for Ca2+ binding. It is therefore understandable that the more flexible TM6 harboring E650 results in partial Ca2+ occupancy of LMNG-solubilized TMEM16A (Fig. 2h, i), raising the possibility that some of the conformational differences between the two structures could reflect different channel conformations. It appears that the native-like lipid environment of nanodiscs allowed stabilization of TM6 so that two Ca2+ ions can be fully coordinated by their binding sites (Fig. 2g, h). Based on our structures (see Extended Data Fig. 8 for structural determination details), mutagenesis of residues lining the pore revealed that a subset of these residues affected anion selectivity (Fig. 3a–c). Besides R511 on TM3 and K599 on the TM5-TM6 loop important for anion selectivity 25 , and K584 on TM5 that partially accounts for the selectivity for anions over cations 28 (Fig. 3b), we tested alanine substitutions of 24 other pore-lining residues including N542 and D550 on TM4, N587 and V595 on TM5, Q705 and F712 on TM7, and S635 on TM6 (Fig. 3a, b and Extended Data Fig. 6d). The permeability ratios determined by replacing external Cl− with I− or SCN− were reduced by 40% by S635A but increased by 40–130% by the other six mutations (Fig. 3d–f). Whereas the side chains of Q705 and F712 do not appear to be pointing into the pore, it remains an open question whether they may reorient and face the pore as the channel opens. These ten pore-lining residues identified thus far for their roles in anion selectivity (Fig. 3c) may reflect an extended selectivity filter, or multiple pore regions for permeant ion interactions. We noticed narrow constrictions of the pore of LMNG-solubilized TMEM16A (Fig. 4a–c), likely representing a stable closed conformation. To test whether residues facing the pore affect channel gating possibly by altering the relative stability of the open versus closed states, we examined alanine substitutions of 21 residues including N542 and I546 on TM4, Y589 and I592 on TM5, and F708 on TM7. These five alanine mutations increased the apparent Ca2+ sensitivity whereas alanine substitution of V595 on TM5 and L639 on TM6 decreased the apparent Ca2+ sensitivity (Fig. 4d–f and Extended Data Fig. 9c). In contrast to the pore-lining residues important for anion selectivity that are spread over more than 25 Å along the length of the pore (Fig. 3c), these seven residues affecting the Ca2+ dependence of channel activation are all located within ~10 Å from the pore constrictions (Fig. 4c), indicating that these residues at the constricted pore influence the stability of the channel conformation important for gating. TMEM16A-CaCC likely have multiple open states 2,13 and closed states 13 (Fig. 5a). With the same anion concentration on both sides of the membrane, occupancy of the first Ca2+ binding site allows the channel to open when the membrane potential is depolarized to positive values 13 , whereas CaCC with double Ca2+ occupancy activates in a voltage independent manner 13 . The currents conducted by the C-terminally truncated TMEM16A also displayed voltage dependence at 30 nM Ca2+ but not 1 mM Ca2+ (Fig. 5b). Given the single Ca2+ occupancy of TMEM16A in LMNG, this structure is in a closed conformation probably because of the lack of depolarization. Moreover, we found that the K584Q mutation altered the ion selectivity in low but not high Ca2+ (Extended Data Fig. 9a, b), suggestive of multiple open states with differences in the permeation pathway 2 – a finding of potential relevance to the different mutagenesis results 8,9,28 regarding this pore-lining residue (Fig. 3a–c). The nanodisc-reconstituted TMEM16A structure likely corresponds to a closed conformation due to channel rundown following prolonged activation in 1 mM Ca2+ (Fig. 5c–e) 12 . Intrigued by the involvement of the hydrophobic V595 in anion selectivity, we tested multiple substitutions and found bulky basic residues to be less effective than alanine in enhancing the relative permeability of large anions (Extended Data Fig. 6a–d). These studies underscore the importance of structure-based functional analyses. In this study, we identify for the first time pore-lining residues of CaCC based on structural analyses of TMEM16A as well as mutagenesis of these highly conserved pore-lining residues (Extended Data Fig. 10). Unlike previous efforts searching among positively charged residues for those involved in anion permeation 12,25,28 , we tested residues facing water-filled potential pore structures to delineate the permeation pathway based on the involvement of pore-lining residues in anion selectivity. For ion permeation and channel gating, we classify pore-lining residues into two groups: one group is distributed along the entire pore and is important for anion selectivity, and the other group is clustered together and is important for gating. For the first group, we validated the ability of ten pore-lining residues to interact with permeant ions by showing that mutations of these residues alter anion selectivity in this study and in two previous studies 25,28 . Visualization of the pore-lining residues and functional assessments of their role in anion selectivity thus firmly establishes their pore assignment. For the second group, we demonstrate a role for seven residues facing the constricted pore in channel gating, indicating that channel opening likely involves displacement of these residues that affect the relative stability of the open and closed states. Our study provides an essential framework to guide further studies of gating and anion permeation of calcium-activated chloride channels. METHODS Protein expression and purification Truncated mouse TMEM16A was C-terminally fused to a 3C consensus sequence, a Strep-tag II peptide, and a GFP moiety and expressed in HEK293 GnTi− cells using the BacMam system as described previously 29,30 . Protein purification and sample processing were carried out at 4°C. For nanodisc-reconstituted preparations, approximately 10 g of cell pellet (from about 1 L culture) was lysed by stirring for 40 min in 200 ml hypotonic buffer containing 50 mM TrisNO3 (pH 9.0) supplemented with 0.1 mg/ml DNase, 1× complete protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl (PMSF). The membrane fraction was collected by centrifugation at 30,000g for 30 min, and then homogenized with a Dounce homogenizer in extraction buffer containing 50 mM TrisNO3 (pH 9.0), 150 mM KNO3, and 10 mM CaCl2 supplemented with 0.1 mg/ml DNase, 1× complete protease inhibitor cocktail, and 1 mM PMSF. Protein was extracted in 200 ml extraction buffer plus 0.5% n-dodecyl-b-d-maltopyranoside (DDM) and 0.1% cholesteryl hemisuccinate (CHS) with gentle stirring for 2 h. The insoluble fraction was removed by centrifugation at 30,000g for 30 min. The recombinant protein was affinity purified with an anti-GFP nanobody immobilized on CNBr-activated sepharose resin (GE Healthcare) in wash buffer containing 10 mM TrisNO3 (pH 9.0), 150 mM KNO3, 1 mM CaCl2, 0.05% DDM, and 0.01% CHS supplemented with a 0.1 mg/ml lipid mixture containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoylsn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) at a ratio of 3:1:1. The purified protein was recovered by incubation with 1.5 CV wash buffer containing 0.5 mM dithiothreitol (DTT) and 50 mg 3C protease overnight. To reconstitute the protein in nanodiscs, the lipid was prepared as described previously in buffer containing 10 mM TrisNO3 and 150 mM KNO3. After purification, the protein sample was mixed with MSP2N2 and soy PC (Avanti) at a molar ratio of TMEM16A monomer: MSP2N2: soy PC = 1:4:100. The mixture was allowed to equilibrate for 1 h and Bio-beads SM2 (Bio-Rad) were added to the mixture three times within 24 h to gradually remove detergents from the system. Afterwards, the sample was filtered through a 0.45 μm filter, and reconstituted protein was separated on a Superdex-200 column in column buffer containing 10 mM TrisNO3 (pH 9.0), 150 mM KNO3, and 1 mM CaCl2. The peak fraction was collected and concentrated to 0.6–0.7 mg/ml using a 100 kDa MWCO Amicon Ultra filter device (Millipore). The lipid compositions used for protein purification and nanodisc reconstitutions are chosen based on previous studies 30,31 . The same purification procedures were used to prepare LMNG solubilized preparations except 5 mM LMNG was in the extraction buffer and 0.02 mM LMNG was used in the wash buffer instead of DDM and CHS. After digestion by 3C, the protein was collected and immediately separated on a Superdex-200 column in column buffer containing 10 mM TrisNO3 (pH 9.0), 150 mM KNO3 and 1 mM CaCl2. The peak fractions were collected and concentrated to 1 mg/ml using 100-kDa Amicon Ultra filter device. N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl] maleimide (CPM) assay of TMEM16A stability as function of calcium concentration To assess the stability of TMEM16A in the presence of a range of Ca2+ concentrations via CPM assay 16 , we dialyzed TMEM16A in nanodiscs or LMNG in solutions containing buffered calcium made (at pH7.5 buffered with HEPES) by combining Ca(OH)2 and EGTA to a ratio predicted by CaBuf softward (KU Leuven). Calcium concentrations of the solutions containing dialyzed TMEM16A protein preparations were subsequently measured directly against calcium standards, using a Fluo-8 fluorescent assay in a BioTek Synergy H4 plate reader. MSP proteins have no cysteines and empty nanodiscs generate no CPM fluorescence signals 17 , so the CPM signal measured via a fluoromax fluorometer (excitation at 387 nm, emission at 463 nm) provides a readout of cysteine exposure upon unfolding of TMEM16A at temperature ranging from 25°C to 75°C. Isolation of monoclonal Fab (fragment antigen binding) for TMEM16A To obtain Fabs to be used as fiducial markers for cryo-EM studies, TMEM16A was reconstituted into nanodiscs to provide a native-like lipid bilayer environment for presentation of the calcium-activated chloride channel to the recombinant Fab library. Once reconstituted, TMEM16A in nanodiscs was used for in-solution selections. A subtractive (nanodisc alone) and competitive panning strategy was used to find selective binders for TMEM16A, from our fully human naïve Fab phage library 18,19 . The selections yielded 23 clones with a signal 4 times higher than the background in an ELISA assay. Out of these 23 clones, 3 clones showed a very specific signal for TMEM16A and almost no signal for empty nanodiscs. Clone 2F11 was then expressed and purified, and complex formation with TMEM16A was tested with size exclusion chromatography. Fab(2F11) and TMEM16A indeed formed a complex which was also demonstrated by two-dimensional class averages of electron microscopy negative stain images (Extended Data Fig. 3b). Electrophysiology HEK293 cells were maintained at 37°C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin, and were passaged upon reaching confluency (every 2–4 days) by digestion with 0.05% Trypsin-EDTA. 24 hours before recording, cells were transiently transfected with TMEM16A constructs using Lipofectamine 2000 (Invitrogen). On the morning before recording, cells were re-plated onto poly-L-lysine coated coverslips using trypsin and allowed to settle for at least 1 hour. For patch clamp electrophysiology, coverslips were transferred into a recording bath solution containing 140 mM NaCl, 10 mM HEPES, and 5 mM EGTA, with pH adjusted to 7.2 with NaOH and osmolarity adjusted to 305–315 mOsm with mannitol. All recordings were made at room temperature. For whole cell recordings, intracellular solutions contained 140 mM NaCl, 10 mM HEPES, 1 mM CaCl2 and 2 mM MgCl2 with pH adjusted to 7.2 with NaOH and osmolarity adjusted to 310 mOsm with mannitol. For anion selectivity measurements, recordings from HEK293 cells transiently expressing wild-type or mutant TMEM16A under bi-ionic conditions using whole-cell patch-clamp and subjected to one-second ramp protocols between −80 and +80 mV. Extracellular solutions are exchanged between voltage ramp commands and contain 140 mM NaCl, 140 mM NaI, or 140 mM NaSCN. Permeability ratios for iodide/chloride ions (2.70 ± 0.09, N = 28 for WT; 3.73 ± 0.12, N = 11 for N542A; 5.32 ± 0.25, N = 9 for D550A; 4.85 ± 0.16, N = 7 for N587A; 4.00 ± 0.16, N = 9 for V595A; 1.82 ± 0.08, N = 7 for S635A; 4.65 ± 0.06, N = 5 for Q705A; 4.45 ± 0.10, N = 6 for F712A), or thiocyanate/chloride ions (5.47 ± 0.21, N = 28 for WT; 8.99 ± 0.24, N = 8 for N542A; 13.33 ± 0.66, N = 9 for D550A; 9.10 ± 0.29, N = 7 for N587A; 9.96 ± 0.30, N = 9 for V595A; 3.24 ± 0.26, N = 7 for S635A; 13.36 ± 0.32, N = 5 for Q705A;10.78 ± 0.34, N = 6 for F712A), are calculated by fitting the GHK equation to currents recorded using voltage ramps (Extended Data Fig. 6d). For Fig. 3d, the reversal potential (at 0 nA) is −46 mV for WT and −57 mV for N587A in external SCN−, −27 mV for WT and −40 mV for N587A in external I−, −2 mV for WT and 0 mV for N587A in external Cl−. For inside out patch recording, intracellular solutions consisted of normal bath solution supplemented with 2 mM MgCl2. Intracellular solutions with varying calcium concentrations were generated by buffering calcium ions with EGTA at pH 7.2 and were made by combining Ca(OH)2 and EGTA to a ratio predicted by CaBuf software (KU Leuven) and then adding 140 mM NaCl or NMDG-Cl and 10 mM HEPES to the desired volume. Calcium concentrations were subsequently measured directly against calcium standards using a Fluo-8 or Fluo-8FF fluorescent assay in a BioTek Synergy H4 plate reader. To assess calcium sensitivity of channel activation via recordings from inside-out patches excised from HEK293 cells transiently expressing wild-type or mutant TMEM16A, the membrane potential was held at +60 mV, and patches were exposed to intracellular solutions containing 140 mM NaCl and increasing concentrations of free Ca2+. Solutions (a)–(f) in Fig. 4d contained 150 nM, 300 nM, 400 nM, 600 nM, 5.5 μM, and 1 mM free Ca2+, respectively. Ca2+-dependent currents from wild-type (WT) and mutant TMEM16A channels were normalized to their maximum values and fit to the Hill equation. The EC50 values in nM (796 ± 66, N = 10 for WT; 295 ± 24, N = 10 for N542A; 148 ± 31, N = 6 for I546A; 192 ± 13, N = 6 for Y589A; 252 ± 17, N = 6 for I592A; 2036 ± 52, N = 7 for V595A; 2008 ± 63, N = 7 for L639A; 205 ± 30, N = 8 for F708A) were compared using one-way ANOVA followed by the Bonferroni post-hoc test for significance (Extended Data Fig. 9c). External or internal solutions using alternate anions were made by replacing NaCl with the indicated sodium salt at equimolar concentrations. Patch pipettes were pulled from 1.5/0.86 (OD/ID) glass and polished to 2–2.5 MΩ resistance (inside-out patch) or 3–5 MΩ (whole cell patch). Perfusion exchange was performed using a VM-8 perfusion apparatus with Octaflow software (ALA Scientific). Data were collected at 10 kHz sampling rate and low pass filtered online at 1 kHz. Recordings were made using an Axon Instruments Multiclamp 700 with Digidata 1440 and were collected into pClamp10 software. All patch clamp seals were allowed to reach at least 3 GΩ resistance before patch rupture, but typical seal resistance usually exceeded 10 GΩ. All recordings were made using a 1 M KCl agar bridge to prevent baseline fluctuation at the reference electrode. For Data analysis, all offline data analysis for patch clamp recording was performed using Graphpad Prism 6, Clampfit 10, and Microsoft Excel. Permeability ratios were determined from bi-ionic conditions using a reduced form of the Goldman-Hodgkin-Katz voltage equation: E REV = R T z F Ln P X [ X - ] o P Cl [ Cl - ] i where P X represents relative permeability of ion species “X” and F, R and T have their usual thermodynamic meanings. Concentration-dependence curves for Ca2+ were generated by fitting data to an equation of the form: I I MAX = 1 1 + ( K D [ Ca 2 + ] ) n H Where I/I MAX denotes current normalized to the maximum amplitude in the highest [Ca2+] tested, KD denotes the dissociation constant for that ion and nH denotes the Hill coefficient. Statistical analysis for data acquired using patch clamp employed one-way ANOVA followed by Bonferroni post-hoc tests for statistical significance. p 0.99 and p = 0.73 for “Inst” and “Overall”, respectively). Mean ± SEM are shown in b and c. d, Size-exclusion chromatography of TMEM16A reconstituted into lipid nanodiscs with MSP2N2. The peak fractions corresponding to nanodisc-reconstituted TMEM16A (16A) and free MSP2N2 are indicated. The 16A peak fraction was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). TMEM16A and MSP2N2 (MSP) monomers are approximately 105 kDa and 46 kDa, respectively. The faint band at 210 kDa may correspond to incompletely disassociated TMEM16A dimers. e, CPM analysis 16,17 of nanodisc-reconstituted TMEM16A in 0, 71 nM, 293 nM, 782 nM, 4120 nM or 1 mM Ca2+. f, Raw micrographs of nanodisc-reconstituted TMEM16A examined by negative-stain EM. g, 2D class averages of particles from negative-stain EM of TMEM16A reconstituted into nanodiscs h, Size-exclusion chromatography of TMEM16A solubilized in LMNG. The peak fraction was examined by SDS-PAGE. i, CPM analysis of LMNG-solubilized TMEM16A in 0, 83 nM, 333 nM, 1122 nM, 5290 nM or 1 mM Ca2+. j, Raw micrographs of LMNG-solubilized TMEM16A examined by negative-stain EM. Both micrographs, (f) and (j), showed mono-dispersed and homogeneous particles. k, 2D class averages of particles from negative-stain EM of TMEM16A solubilized in LMNG. Extended Data Figure 2 Cryo-EM analysis of TMEM16A reconstituted in nanodiscs a, A representative cryo-EM micrograph of nanodisc-reconstituted TMEM16A. Green circles indicate individual particles. b, Representative 2D class averages from boxed particles with 256-pixel box size (261.12 Å). c, Eular angle distribution of all particles included in the final 3D reconstruction. The size of the spheres is proportional to the number of particles seen from that specific orientation. d, FSC curves of two independently refined maps before (blue) and after (red) post-processing in RELION. Curves with resolution corresponding to FSC = 0.143 are shown. e, Planar slices through the unsharpened EM density map at different levels along the channel symmetry axis. f, Local resolution of TMEM16A as estimated by RELION and shown with pseudo-color representation of resolution. g, Cross-validation using FSC curves of the density map calculated from the refined model versus half map 1 (work), versus half map 2 (free), and versus summed map. h, Directional FSC from different Fourier cones. Each curve indicates a different direction. i, Calculated resolution from different views. The directions are indicated as x, y, and z in the 3D resolution map. The highest and lowest resolutions are labeled with red and blue circles respectively. The green circle shows global average resolution. Extended Data Figure 3 Cryo-EM analysis of TMEM16A solubilized in LMNG a, A representative cryo-EM micrograph of LMNG-solubilized TMEM16A. Green circles indicate individual particles. b, Representative 2D class averages from boxed particles with a 256 pixels box size (261.12 Å) and TMEM16A in complex with Fabs (bottom row, with the two right panels showing particles after subtraction of densities for Fabs). c, Eular angle distribution of all particles included in the final 3D reconstruction. The size of the spheres is proportional to the number of particles visualized from that specific orientation. d, FSC curves of two independently refined maps before (blue) and after (red) post-processing in RELION. Curves with resolution corresponding to FSC = 0.143 are shown. e, Planar slices through the unsharpened EM density map at different levels along the channel symmetry axis. f, Local resolution of TMEM16A as estimated by RELION and shown with pseudo-color representation of resolution. g, Cross-validation using FSC curves of the density map calculated from the refined model versus half map 1 (work), versus half map 2 (free), and versus summed map. h, Directional FSC (dFSC) from different Fourier cones. Each curve indicates a different direction. dFSC for TMEM16A alone in LMNG in grey (average in yellow); dFSC for combination of TMEM16A alone and with Fabs bound in LMNG in purple (average in red). i, Calculated resolution from different views (grey for combination of TMEM16A alone and with Fabs bound, yellow for TMEM16A alone). The directions are indicated as x, y, and z in the 3D resolution map. The highest and lowest resolutions are labeled with red and blue circles respectively. The green circle shows global average resolution. Extended Data Figure 4 Cryo-EM densities of the ten transmembrane helices of TMEM16A, summary of cryo-EM data collection and processing, and summaries of sidechain assignments a, Representative cryo-EM densities of the ten transmembrane helices (TM1-TM10) of nanodisc-reconstituted TMEM16A (right) or LMNG-solubilized TMEM16A (left) are superimposed on the corresponding atomic model. The EM densities are shown in blue meshes for nanodisc-reconstituted TMEM16A, or green meshes for LMNG-solubilized TMEM16A, and the model is show as sticks and colored according to atom type (C: light grey; N: blue; O: red; S: yellow). b, Summary of cryo-EM data collection and model refinement. c, Summary of sidechain assignment of TMEM16A in nanodiscs. d, Summary of sidechain assignment of TMEM16A in LMNG. Extended Data Figure 5 Atomic models of TMEM16A in two conformations a–c, Ribbon diagrams of TMEM16A reconstituted in nanodiscs (in green and yellow) with lipids (in red), overlayed with EM density map (sharpened, in light grey). Two Ca2+ ions (orange spheres) are present in each monomer. d–f, Ribbon diagrams of TMEM16A solubilized in LMNG (in blue) with lipids (in red), overlayed with EM density map (sharpened, in light grey). One Ca2+ ion (orange sphere) is present in each monomer g–i, EM densities of nanodisc-reconstituted TMEM16A (unsharpened, in green and yellow) overlayed with digitonin-solubilized TMEM16A 12 (in grey). Extended Data Figure 6 Anion selectivity depends on residues lining the pore surrounded by TM3–8 but not TM10 residues at the dimer interface a, Bi-ionic conditions for assessing the effect of the V595L mutation on permeability ratios. b, Effects of different substitutions of V595 on the permeability ratio PI-/PCl- (2.70 ± 0.09, N = 28 for WT; 4.00 ± 0.16, N = 9 for V595A; 3.49 ± 0.21, N = 6 for V595K; 3.81 ± 0.07, N = 7 for V595L; 3.48 ± 0.14, N = 7 for V595R). c, Effects of different substitutions of V595 on the permeability ratio PSCN-/PCl- (5.47 ± 0.21, N = 28 for WT; 9.96 ± 0.30, N = 9 for V595A; 6.64 ± 0.58, N = 6 for V595K; 7.86 ± 0.37, N = 7 for V595L; 6.30 ± 0.37, N = 7 for V595R). d, Permeability ratios determined in bi-ionic conditions for TMEM16A mutants. The exact n values (independent experimental samples from individually recorded HEK293 cells) are given for every experiment. The P-values are generated after a Dunnett’s posthoc test following one-way ANOVA. For these multiplicity adjusted P-values, values smaller than 0.0001 cannot be estimated precisely; Prism’s documentation suggests this approach is the most rigorous and conservative way to generate a P-value from a multiple comparison test 53 . Extended Data Figure 7 Comparisons of extracellular loops and lipids in nanodisc-resonstituted and LMNG-solubilized TMEM16A. a–c, Lipids (in red) in the nanodisc-reconstituted TMEM16A (in green and yellow, overlayed with EM density map in light grey) (b, c), with helical distortions of TM6 near G640 (a). d–f, Lipids (in red) in LMNG-solubilized TMEM16A (in blue, overlayed with EM density map in light grey) (e, f), with the lower half of TM6 beyond G640 disordered and hence absent from the reconstruction (d). g–i, Extracellular domains of nanodisc-reconstituted TMEM16A (unsharpened, in green and yellow) overlayed with those of LMNG-solubilized TMEM16A (unsharpened, in blue). j–l, Extracellular TM5-TM6 and TM9-TM10 loops in ribbon diagrams for nanodisc-reconstituted TMEM16A (in green and yellow) overlayed with those of LMNG-solubilized TMEM16A (in blue). Extended Data Figure 8 Data processing of TMEM16A in nanodisc (a) or LMNG (b). a, Data processing of nanodisc-reconstituted TMEM16A. Particle picking was performed with Gautomatch with templates from 2D classes from the LMNG dataset and generated 927,414 particles in total. All particles were extracted and binned 4 (pixel size is 4.08 Å) and then 2D classified. 341,875 particles from good 2D classes were used in 3D refinement with an initial model from the LMNG structure low pass filtered to 60 Å, giving rise to a 5.5 Å map. The 5.5 Å map was then low pass filtered to 10 Å as the initial model for 3D classification without applied symmetry for all particles with a 1.02 Å pixel size. Of the seven classes, two classes (15.92% and 11.15% of the 927,414 particles) gave maps with improved resolution (4.7 Å and 5.1 Å respectively) after 3D auto-refinement with C2 symmetry. These two classes were combined together yielding a total of 251,851 particles, and another 3D auto-refinement was run to generate the unmasked map at a resolution of 4.6 Å. The map was then masked to get the final map at resolution of 3.8 Å. b, Data processing of LMNG-solubilized TMEM16A. Approximately 4000 particles were manually picked and classified by 2D classification in SAMUEL to generate the templates for automatic particle picking with samautopick.py. 533,545 particles were identified after manually inspection. The crystal structure of nhTMEM16A 10 (PDB: 4WIS) was converted to mrc with e2pdb2mrc.py and low pass filtered to 60 Å as the initial model. 44 of 200 2D classes were used for 3D auto-refinement with C2 symmetry. Since 3D classification failed for further separation, the reported resolution of the final map was 3.8 Å. To reduce anisotropy due to underrepresentation of side views, this dataset was merged with another dataset for Fabs bound to TMEM16A in LMNG. Starting with 338,705 particles from automatic particle picking, 4 of 40 2D classes (132,444 particles) were used for 3D auto-refinement. Then the Fab density for each particle was subtracted. The 132,444 subtracted particles without Fab density were combined with the 342,875 particles from all 5 classes of TMEM16A in LMNG dataset with resolution of 3.8 Å and processed for 3D auto-refinement to generate the unmasked map with resolution of 3.9 Å. This map was then masked to get the final map at resolution of 3.4 Å. Pixel sizes are shown in parenthesis for each class. Extended Data Figure 9 Multiple open and closed states of TMEM16A calcium-activated chloride channel (CaCC) and involvement of pore-lining residues in channel gating. a, Reduction from 150 mM NaCl to 15 mM NaCl in the intracellular solution containing 1 μM or 1 mM Ca2+ caused identical shift of reversal potential of wildtype TMEM16A but not K584Q mutant channels in excised inside-out patch held at +80 mV and subjected to a ramp to −80 mV. Repeated independently 8 times for WT with similar results, and 5 times for K584Q with similar results. b, The K584Q mutation altered the permeability ratio PNa+/PCl- at 1 μM but not 1 mM Ca2+. N = 8 for WT, N = 5 for K584Q. c, Calcium sensitivity of channel activation of wildtype and mutant TMEM16A channels (number of independent experiments and P values are given in this table). Permeability ratios for mutants were compared to those of wild-type (WT) using one-way ANOVA followed by the Bonferroni post-hoc test for significance; **** designates p < 0.0001; *** designates p < 0.001; ** designates p < 0.005; data are presented as mean ± SEM. Extended Data Figure 10 Sequence alignment of TMEM16A homologues. a, Sequences of TMEM16A homologues were analyzed by Clustal omega. Conserved residues are highlighted. Transmembrane helices are indicated above the sequences. The residues that were shown in this study (D550, N587, S635, Q705 and F712) and previous studies (R511, K584 and K599) to be crucial for selectivity are marked in orange and blue respectively. The residues (I546, Y589, I592, L639 and F708) that were shown in this study to be critical for gating are marked in green. The residues (N542 and V595) that contributed to both selectivity and gating property is marked in purple. The residues (E650, E698, E701, E730 and D734) important for Ca2+ binding are marked in red. b, Sequence alignment of mouse TMEM16A and nhTMEM16. Conserved residues are highlighted. Transmembrane helices of TMEM16A are indicated above the sequence.
- Record: found
- Abstract: found
- Article: not found
Hydrophobic gating in ion channels.
Biological ion channels are nanoscale transmembrane pores. When water and ions are enclosed within the narrow confines of a sub-nanometer hydrophobic pore, they exhibit behavior not evident from macroscopic descriptions. At this nanoscopic level, the unfavorable interaction between the lining of a hydrophobic pore and water may lead to stochastic liquid-vapor transitions. These transient vapor states are "dewetted", i.e. effectively devoid of water molecules within all or part of the pore, thus leading to an energetic barrier to ion conduction. This process, termed "hydrophobic gating", was first observed in molecular dynamics simulations of model nanopores, where the principles underlying hydrophobic gating (i.e., changes in diameter, polarity, or transmembrane voltage) have now been extensively validated. Computational, structural, and functional studies now indicate that biological ion channels may also exploit hydrophobic gating to regulate ion flow within their pores. Here we review the evidence for this process and propose that this unusual behavior of water represents an increasingly important element in understanding the relationship between ion channel structure and function.
- Record: found
- Abstract: found
- Article: not found
Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line
C Milstein, J. V. Kilmartin, B Wright (1982)
Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.
Author and article information
Journal
Journal ID (nlm-ta): J Gen Physiol
Journal ID (iso-abbrev): J. Gen. Physiol
Journal ID (publisher-id): jgp
Journal ID (hwp): jgp
Title:
The Journal of General Physiology
Publisher:
Rockefeller University Press
ISSN
(Print):
0022-1295
ISSN
(Electronic):
1540-7748
Publication date
(Print):
05
November
2018
Volume: 150
Issue: 11
Pages: 1484-1497
Affiliations
Author notes
Correspondence to Stephen B. Long:
longs@
123456mskcc.org
Author information
George Vaisey
http://orcid.org/0000-0002-8359-1314
Stephen B. Long
http://orcid.org/0000-0002-8144-1398
Article
Publisher ID: 201812190DOI: 10.1085/jgp.201812190
PMC ID: 6219684
PubMed ID: 30237227
SO-VID: 93e55424-c758-47a2-b58d-b6556d0f08d2
Copyright © © 2018 Vaisey and Long
License:
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
History
Date
received
: 27
July
2018
Date
accepted
: 30
August
2018
Funding
Funded by: Boehringer Ingelheim Fonds, DOI https://doi.org/10.13039/501100001645;
Funded by: National Institutes of Health, DOI https://doi.org/10.13039/100000002;
Award ID: R01 GM110396
Award ID: P30 CA008748
Categories
Subject:
Research Articles
Subject:
Research Article
Subject:
508
Subject:
500
Subject:
501
ScienceOpen disciplines: Anatomy & Physiology
Data availability:
ScienceOpen disciplines: Anatomy & Physiology