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Cryo-EM structure of the heptameric calcium homeostasis modulator 1 channel

2022; Elsevier BV; Volume: 298; Issue: 5 Linguagem: Inglês

10.1016/j.jbc.2022.101838

1083-351X

Yue Ren, Yang Li, Yaojie Wang, Tianlei Wen, Xuhang Lu, Shenghai Chang, Xing Zhang, Yuequan Shen, Xue Yang,

Advanced Memory and Neural Computing

Calcium homeostasis modulator 1 (CALHM1) is a voltage- and Ca2+-gated ATP channel that plays an important role in neuronal signaling. However, as the previously reported CALHM structures are all in the ATP-conducting state, the gating mechanism of ATP permeation is still elusive. Here, we report cryo-EM reconstructions of two Danio rerio CALHM1 heptamers with ordered or flexible long C-terminal helices at resolutions of 3.2 Å and 2.9 Å, respectively, and one D. rerio CALHM1 octamer with flexible long C-terminal helices at a resolution of 3.5 Å. Structural analysis shows that the heptameric CALHM1s are in an ATP-nonconducting state with a central pore diameter of approximately 6.6 Å. Compared with those inside the octameric CALHM1, the N-helix inside the heptameric CALHM1 is in the "down" position to avoid steric clashing with the adjacent TM1 helix. Molecular dynamics simulations show that as the N-helix moves from the "down" position to the "up" position, the pore size of ATP molecule permeation increases significantly. Our results provide important information for elucidating the mechanism of ATP molecule permeation in the CALHM1 channel. Calcium homeostasis modulator 1 (CALHM1) is a voltage- and Ca2+-gated ATP channel that plays an important role in neuronal signaling. However, as the previously reported CALHM structures are all in the ATP-conducting state, the gating mechanism of ATP permeation is still elusive. Here, we report cryo-EM reconstructions of two Danio rerio CALHM1 heptamers with ordered or flexible long C-terminal helices at resolutions of 3.2 Å and 2.9 Å, respectively, and one D. rerio CALHM1 octamer with flexible long C-terminal helices at a resolution of 3.5 Å. Structural analysis shows that the heptameric CALHM1s are in an ATP-nonconducting state with a central pore diameter of approximately 6.6 Å. Compared with those inside the octameric CALHM1, the N-helix inside the heptameric CALHM1 is in the "down" position to avoid steric clashing with the adjacent TM1 helix. Molecular dynamics simulations show that as the N-helix moves from the "down" position to the "up" position, the pore size of ATP molecule permeation increases significantly. Our results provide important information for elucidating the mechanism of ATP molecule permeation in the CALHM1 channel. Adenosine triphosphate (ATP) release channels play an important role in various cellular signaling events (1Abbracchio M.P. Burnstock G. Verkhratsky A. Zimmermann H. Purinergic signalling in the nervous system: An overview.Trends Neurosci. 2009; 32: 19-29Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar). Consequently, their malfunctions are associated with many pathophysiological processes, including neurological disorders, inflammation, and cancer progression (2Ma Z. Taruno A. Ohmoto M. Jyotaki M. Lim J.C. Miyazaki H. Niisato N. Marunaka Y. Lee R.J. Hoff H. Payne R. Demuro A. Parker I. Mitchell C.H. Henao-Mejia J. et al.CALHM3 is essential for rapid ion channel-mediated purinergic neurotransmission of GPCR-mediated tastes.Neuron. 2018; 98: 547-561Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To date, extensive research has identified five family proteins as human ATP release channels, although some of them need to be further verified. These five family proteins are connexins, pannexins, calcium homeostasis modulators (CALHMs), volume-regulated anion channels, and maxi-anion channels (2Ma Z. Taruno A. Ohmoto M. Jyotaki M. Lim J.C. Miyazaki H. Niisato N. Marunaka Y. Lee R.J. Hoff H. Payne R. Demuro A. Parker I. Mitchell C.H. Henao-Mejia J. et al.CALHM3 is essential for rapid ion channel-mediated purinergic neurotransmission of GPCR-mediated tastes.Neuron. 2018; 98: 547-561Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Convergent evolution analysis showed that they all have four transmembrane helices (TMs) in the monomer that form an oligomer with a central pore (3Siebert A.P. Ma Z. Grevet J.D. Demuro A. Parker I. Foskett J.K. Structural and functional similarities of calcium homeostasis modulator 1 (CALHM1) ion channel with connexins, pannexins, and innexins.J. Biol. Chem. 2013; 288: 6140-6153Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These oligomers are usually called hemichannels which release ATP to mediate the subsequent purinergic signal transduction of neighboring cells (4Contreras J.E. Saez J.C. Bukauskas F.F. Bennett M.V. Gating and regulation of connexin 43 (Cx43) hemichannels.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11388-11393Crossref PubMed Scopus (339) Google Scholar). Hemichannels formed by some isoforms of connexin and CALHM (such as connexin 26, connexin 43, connexin 50, CALHM2, CALHM4, etc.) can dock with each other and further form gap junction channels that allow direct exchange of small metabolites between cells (5Goldberg G.S. Lampe P.D. Nicholson B.J. Selective transfer of endogenous metabolites through gap junctions composed of different connexins.Nat. Cell Biol. 1999; 1: 457-459Crossref PubMed Scopus (264) Google Scholar, 6Kumar N.M. Gilula N.B. The gap junction communication channel.Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1622) Google Scholar, 7Taruno A. ATP release channels.Int. J. Mol. Sci. 2018; 19: 808Crossref PubMed Scopus (92) Google Scholar). CALHM1 is a large-pore nonselective ion channel gated by voltage and extracellular Ca2+ concentration (8Ma Z. Tanis J.E. Taruno A. Foskett J.K. Calcium homeostasis modulator (CALHM) ion channels.Pflugers Arch. 2016; 468: 395-403Crossref PubMed Scopus (51) Google Scholar). In addition, CALHM1 is a voltage-gated ATP release channel that mediates purinergic neurotransmission of sweet, bitter, and umami tastes from type II taste bud cells to the taste nerve (9Taruno A. Vingtdeux V. Ohmoto M. Ma Z. Dvoryanchikov G. Li A. Adrien L. Zhao H. Leung S. Abernethy M. Koppel J. Davies P. Civan M.M. Chaudhari N. Matsumoto I. et al.CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes.Nature. 2013; 495: 223-226Crossref PubMed Scopus (309) Google Scholar, 10Romanov R.A. Lasher R.S. High B. Savidge L.E. Lawson A. Rogachevskaja O.A. Zhao H. Rogachevsky V.V. Bystrova M.F. Churbanov G.D. Adameyko I. Harkany T. Yang R. Kidd G.J. Marambaud P. et al.Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex.Sci. Signal. 2018; 11eaao1815Crossref PubMed Scopus (48) Google Scholar). Compared with the slow activation kinetics of CALHM1 homo-oligomerized channels, CALHM1/CALHM3 can form hetero-oligomerized ion channels with rapid voltage-gated activation kinetics that are closer to physiological states in vivo (2Ma Z. Taruno A. Ohmoto M. Jyotaki M. Lim J.C. Miyazaki H. Niisato N. Marunaka Y. Lee R.J. Hoff H. Payne R. Demuro A. Parker I. Mitchell C.H. Henao-Mejia J. et al.CALHM3 is essential for rapid ion channel-mediated purinergic neurotransmission of GPCR-mediated tastes.Neuron. 2018; 98: 547-561Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Structural studies of CALHM family proteins have recently made great progress (11Foskett J.K. Structures of CALHM channels revealed.Nat. Struct. Mol. Biol. 2020; 27: 227-228Crossref PubMed Scopus (4) Google Scholar). Different CALHM isoforms form diverse oligomeric assemblies, ranging from 8 monomers to 13 monomers (12Demura K. Kusakizako T. Shihoya W. Hiraizumi M. Nomura K. Shimada H. Yamashita K. Nishizawa T. Taruno A. Nureki O. Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies.Sci. Adv. 2020; 6eaba8105Crossref PubMed Scopus (13) Google Scholar, 13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar, 14Syrjanen J.L. Michalski K. Chou T.H. Grant T. Rao S. Simorowski N. Tucker S.J. Grigorieff N. Furukawa H. Structure and assembly of calcium homeostasis modulator proteins.Nat. Struct. Mol. Biol. 2020; 27: 150-159Crossref PubMed Scopus (32) Google Scholar). The CALHM1 structures of vertebrate species have been reported to be an octamer and form a hemichannel (12Demura K. Kusakizako T. Shihoya W. Hiraizumi M. Nomura K. Shimada H. Yamashita K. Nishizawa T. Taruno A. Nureki O. Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies.Sci. Adv. 2020; 6eaba8105Crossref PubMed Scopus (13) Google Scholar, 13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar, 14Syrjanen J.L. Michalski K. Chou T.H. Grant T. Rao S. Simorowski N. Tucker S.J. Grigorieff N. Furukawa H. Structure and assembly of calcium homeostasis modulator proteins.Nat. Struct. Mol. Biol. 2020; 27: 150-159Crossref PubMed Scopus (32) Google Scholar), while the Caenorhabditis elegans CLHM1 can assemble as a nonamer, decamer, or undecamer and form two conformations of hemichannels and gap junctions (12Demura K. Kusakizako T. Shihoya W. Hiraizumi M. Nomura K. Shimada H. Yamashita K. Nishizawa T. Taruno A. Nureki O. Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies.Sci. Adv. 2020; 6eaba8105Crossref PubMed Scopus (13) Google Scholar, 13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar). Recent research on the CALHM1–CALHM2 chimera structure suggested that interactions between the long C-terminal helices (LCHs) and the TM4-LCH linker may determine the oligomeric state of the CALHM channels (14Syrjanen J.L. Michalski K. Chou T.H. Grant T. Rao S. Simorowski N. Tucker S.J. Grigorieff N. Furukawa H. Structure and assembly of calcium homeostasis modulator proteins.Nat. Struct. Mol. Biol. 2020; 27: 150-159Crossref PubMed Scopus (32) Google Scholar). Ren et al. reported a Danio rerio CALHM1 octamer with ordered long C-terminal helices (octamer+LCH) and proposed that the extracellular loop 1 region within the dimer interface may contribute to oligomeric assembly (13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar). In this research, we reported cryo-EM reconstructions of two D. rerio CALHM1 heptamers with ordered long C-terminal helices (heptamer+LCH) or flexible long C-terminal helices (heptamer-noLCH) and one D. rerio CALHM1 octamer with flexible long C-terminal helices (octamer-noLCH). The comparison between different CALHM1 oligomers from the same species suggests that the CALHM1 channel may have multiple conformational states. The structures of the drCALHM1 channels were determined by cryo-EM. drCALHM1 channels were purified in the presence or absence of Ca2+ and concentrated for cryo-EM sample preparation. All datasets were collected using a Titan Krios electron microscope operated at 300-kV accelerating voltage with a K2 Summit direct electron-counting detector. Details on the data collection and data processes are shown in Figs. S1–S4. The heptamer+LCH structure was reconstructed from the dataset in the presence of Ca2+. The heptamer-noLCH and octamer-noLCH structures were reconstructed from datasets in the absence of Ca2+. The final 3D reconstructions of heptamer+LCH, heptamer-noLCH, and octamer-noLCH were determined at overall resolutions of 3.2 Å, 2.9 Å, and 3.5 Å, respectively (Figs. S1 and S2). The cryo-EM maps are sufficient to resolve most amino acid side chains (Figs. S3 and S4). Due to the quality of density, the N-helix of heptamer+LCH, heptamer-noLCH, or octamer-noLCH structures only builds an alanine model (Figs. S3 and S4). The overall structure of heptameric drCALHM1 is shaped like a barrel and can be divided into three parts: the extracellular loop, transmembrane domain, and cytoplasmic domain (Fig. 1). From the top view, its extracellular region forms a ring with a diameter of approximately 98 Å, and seven small helices in the middle of the ring can be clearly observed (Fig. 1, A and D). The side view shows that the height of the drCALHM1 heptamer is approximately 92 Å. There are multiple lipid molecules that exist inside a protomer and within the interface of two protomers (Fig. 1, B and E). From the cytosolic view, it can be observed that the cytoplasmic domain mainly consists of one long helix. Seven LCHs intercross each other (Fig. 1, C and F), resulting in a ring diameter of approximately 100 Å. The overall drCALHM1 heptamer is quite similar to that of a previously reported octamer (13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar) except for the different oligomerization states. As shown in Figure 1, the heptamer has smaller pores than the octamer, and we further calculated the exact pore size with the N-helix having all alanines in the current model (Fig. 2, A and B). The narrowest diameter of the heptamer+LCH channel is approximately 14 Å (Fig. 2C). If the putative side chain was built for the N-helix, the pore diameter was reduced to 6.6 Å, which blocked ATP permeation through the pore but allowed ion flux. To further validate this result, we took advantage of molecular dynamics (MD) simulation methodology. Coarse-grained simulations of heptamer+LCH in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids were carried out. We added hydrated Na+, hydrated K+, and Cl− ions on either side of the POPC bilayer to the Martini, version 2.0, forcefield (15Monticelli L. Kandasamy S.K. Periole X. Larson R.G. Tieleman D.P. Marrink S.-J. The MARTINI coarse-grained force field: Extension to proteins.J. Chem. Theor. Comput. 2008; 4: 819-834Crossref PubMed Scopus (1693) Google Scholar). Ca2+ ions were added on the extracellular side. During replicates of 10-μs coarse-grained MD simulations, we observed that multiple Na+ cations, K+ cations, and Cl− anions crossed the membrane through the heptamer+LCH channel. In contrast, Ca2+ ions were still on the extracellular side of the POPC bilayer after simulations and were unable to pass through the heptameric channel (Fig. 2D). These simulation results indicated that the heptamer+LCH channel favors the permeation of monovalent ions (Na+, K+, and Cl−) but repulses Ca2+ ions. Moreover, its pore size is too narrow for bulky ATP molecules. We also calculated the electrostatic surface potential distribution of the heptamer+LCH channel and found that the positive potential at the center of the pores of the heptamer is stronger than that of the octamer (Fig. S5), which may be due to the change in the oligomerization state to make the heptameric channel more compact. The strong positive potential distribution inside pores may indicate the voltage-dependent characteristics of ion flux through the CALHM1 channel, as shown by electrophysiological studies (16Tanis J.E. Ma Z. Foskett J.K. The NH2 terminus regulates voltage-dependent gating of CALHM ion channels.Am. J. Physiol. Cell Physiol. 2017; 313: C173-C186Crossref PubMed Scopus (12) Google Scholar). To address the underlying mechanism of the formation of two different oligomers from the same CALHM1 protomer, we first compared the heptamer and octamer by superimposing one protomer of each assembly (Fig. 3A). The entire heptamer forms a smaller circle than the octamer. The protomer is composed of four TMs (TM1, TM2 plus TM2b, TM3 plus TM3b, and TM4), one extracellular helix, and one LCH. The protomers from the heptamer and octamer are extremely similar except for the position of the N-helix. Compared with the N-helix of the octamer, the N-helix of the heptamer moves toward the cytosolic direction by approximately 6 Å (Fig. 3B) (defined as the "down" position in the heptamer and the "up" position in the octamer hereafter). In the heptamer, the N-helix is aligned parallel to and interacts with TM1 (Fig. 3C). The side chain of Leu28 has hydrophobic interactions with Ala10. Moreover, the N-helix from one protomer has multiple interactions with TM1 from the neighboring protomer. One hydrogen bond was formed between the side chain of Gln32 and the main chain carboxyl oxygen atom of Ala9. Additionally, the side chain of Ile25 has hydrophobic interactions with Ala16. We anticipated more interactions between the N-helix and TM1 if the side chain of the N-helix could be built. We then superimposed the dimers from the heptamer and the octamer. These two dimers are similar except for two N-helices (Fig. 3D). Similar to the protomer comparison result, two N-helices in the dimer adapted from the heptamer obviously move down. After superimposing one protomer together, the neighboring protomer from the heptamer occupies a closer position to the pore than that from the octamer (Fig. 3E), resulting in the N-helix from the octamer having a steric clash with TM1 from the heptamer (as shown by the red arrow). Therefore, the N-helix is mandatory in the "down" position in the heptamer. In order to check whether drCALHM1 maintains a functional channel in human cells, we transfected DNA encoding GFP-tagged full-length drCALHM1 into HEK293T cells and conducted electrophysiological studies. A large voltage-dependent outward current was observed using the whole-cell configuration (Fig. S6), indicating that the expression of drCALHM1 in HEK293T cells forms a channel similar to the previously reported Homo sapiens CALHM1 channel (17Ma Z. Siebert A.P. Cheung K.H. Lee R.J. Johnson B. Cohen A.S. Vingtdeux V. Marambaud P. Foskett J.K. Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: E1963-E1971Crossref PubMed Scopus (98) Google Scholar), although the oligomeric states of drCALHM1 are unknown. To test the possibility that the movement of the N-helix can be involved in the pore size of CALHM1, we carried out MD simulations. With the help of unbiased supervised MD and biased targeted MD simulations for heptamer+LCH, we accelerated the movement of the N-helix in one protomer of the heptamer from the "down" position to the "up" position on a time scale accessible to MD simulations. During the supervised MD simulations, we monitored the root mean square deviation (RMSD) of the N-helix of protomer 1 (P1) in the current structure compared with that of P1 in the octamer to represent the movement of the N-helix. To monitor the pore size, we calculated the minimum distance of any atom between two N-helices from P1 and P4 protomers (denoted as P1−P4 hereafter), from P2 and P5 protomers (P2−P5), from P3 and P6 protomers (P3−P6), and from P4 and P7 protomers (P4−P7) (Fig. S7A). There was obvious deformation of the 7-fold symmetry structure in the N-helix region caused by the movement of the N-helix in P1 during the supervised MD simulations. Then, we calculated the potential of mean force for the RMSD of the N-helix versus. the P4−P7 distance (Fig. 4A). The potential of mean force map shows the transition process of the heptamer and clearly depicts three different conformational states (state 1, state 2, and state 3) of the heptamer starting from the initial state (state 0) (Fig. 4B). In initial state 0, the RMSD of the N-helix is approximately 6.66 Å, and the P4–P7 distance is approximately 9.57 Å. In states 1, 2, and 3 transformed from state 0, the RMSD of the N-helix is 5.63, 4.80, and 4.70 Å, and the P4−P7 distances are approximately 9.79, 11.89, and 13.26 Å, respectively. The conformational state of the N-helix moves closer to that of P1 in the octamer. Meanwhile, the pore size gradually increased from 9.79 Å to 13.26 Å. Furthermore, the targeted MD also observed that the pore size increased due to the movement of the N-helix (Fig. S7B). Conventional MD simulations were performed as controls, and the position of the N-helix and the pore size of the heptamer remained similar to those in the initial state throughout the simulation time (Fig. S7C). Additionally, we performed conventional MD simulations to show the time-dependent RMSD values for the Cα atoms of the heptamer+LCH or octamer+LCH structures relative to conformations obtained from the cryo-EM structures (Fig. S7, D and E). The RMSDs exhibited small variations during the atomic simulations, implying that the two structures were conformationally stable. Since the heptamer+LCH structure was determined using the dataset in the presence of Ca2+, we tried to understand whether Ca2+ can induce different oligomerization states of drCALHM1. Reprocessing the dataset in the absence of Ca2+, in addition to the previously reported octamer+LCH structure (13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar), we were able to identify two new conformation states of the drCALHM1 channel, in which the LCH is disordered (Fig. S2). We named them heptamer-noLCH (Fig. 5, A–F) and octamer-noLCH (Fig. 5, G–L). These results suggest that Ca2+ is unlikely to be the cause of the different oligomerization states of the drCALHM1 channel. The overall structures of heptamer-noLCH versus heptamer+LCH and octamer-noLCH versus octamer+LCH are highly similar except for LCHs (Fig. S8, A and B). Moreover, the pore diameters remained similar in these structures with or without LCHs (Fig. S8, C and D), indicating that the LCH did not regulate the pore size. We also compared the buried surface area of the protomer in different conformational states. With the LCH, the buried surface areas are 948.6 Å2 and 959.6 Å2 for the monomer in the heptamer+LCH structure and octamer+LCH structure, respectively. However, without the LCH, the buried surface areas are 627.6 Å2 and 603.3 Å2 for the protomer in the heptamer-noLCH structure and octamer-noLCH structure, respectively. These results suggest that the LCH may serve as a scaffold to stabilize oligomerized CALHM1 channels. In this study, we reported a heptameric drCALHM1 channel with an ATP nonconducting conformation. Compared with the octameric drCALHM1 channel (13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar), the heptameric drCALHM1 channel undergoes two major changes. One is a much smaller channel ring, leading to a smaller pore and thus possibly blocking ATP permeation. Since the narrowest pore diameter is approximately 6.6 Å, it is likely that the ions can pass through the pores of the heptamer. Indeed, MD results showed that Na+, K+, and Cl− can pass through the pore. However, Ca2+ was precluded from the pore, which may be caused by the positively charged environment inside the pore. Nevertheless, such narrow pores will unlikely allow ATP molecule permeation. The other difference is that the N-helix moves toward the cytosolic direction by approximately 6 Å to avoid the steric clash with neighboring TM1 helices, suggesting that the change in the oligomer state may correlate with the movement of the N-helix. Supervised MD simulations and targeted MD simulations indeed showed that the movement of the N-helix to the "up" position led to the deformation of the 7-fold symmetric structure and significantly increased pore size. We also determined two conformational states of heptameric and octameric drCALHM1 channels with flexible LCHs. Although the current structures of heptamer-noLCH and octamer-noLCH were reconstructed from the Ca2+-free dataset, these two conformational states can be observed from the dataset in the presence of Ca2+. Moreover, it has been noted that the final models of heptamer-noLCH and octamer-noLCH do not include LCHs due to the intrinsic flexibility in these specific conformational states instead of protein degradation. The purified protein sample did not show obvious degraded bands from the SDS-PAGE gel. Therefore, it is reasonable to hypothesize that for the same type of oligomer, the drCALHM1 channel may exhibit equilibrium between two conformational states (ordered versus flexible LCHs). Together with previously published results, we proposed a molecular mechanism of ATP permeation through the CALHM1 channel (Fig. S9). In the resting state, the CALHM1 channel may form a heptamer and equilibrium between ordered LCHs and flexible LCHs. Upon sensing stimulus, the N-helix inside the pore moves up toward the extracellular direction, driving conformational changes and oligomeric rearrangement to facilitate ATP molecule permeation. Compared with the published ATP permeation mechanism of another ATP release channel, pannexin 1 (18Ruan Z. Orozco I.J. Du J. Lu W. Structures of human pannexin 1 reveal ion pathways and mechanism of gating.Nature. 2020; 584: 646-651Crossref PubMed Scopus (59) Google Scholar), our proposed mechanism of ATP permeation has a common point: both use the C-terminal tail to regulate ATP permeation. The major difference is that the C-terminal tail has to be cleaved during ATP permeation in pannexin 1, while the C-terminal part in CALHM1 may switch to the disordered region for further assembly into higher-order oligomers to permeate ATP molecules. To date, several CALHM subtype structures have been reported to form various oligomers (12Demura K. Kusakizako T. Shihoya W. Hiraizumi M. Nomura K. Shimada H. Yamashita K. Nishizawa T. Taruno A. Nureki O. Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies.Sci. Adv. 2020; 6eaba8105Crossref PubMed Scopus (13) Google Scholar, 13Ren Y. Wen T. Xi Z. Li S. Lu J. Zhang X. Yang X. Shen Y. Cryo-EM structure of the calcium homeostasis modulator 1 channel.Sci. Adv. 2020; 6eaba8161Crossref Scopus (6) Google Scholar, 14Syrjanen J.L. Michalski K. Chou T.H. Grant T. Rao S. Simorowski N. Tucker S.J. Grigorieff N. Furukawa H. Structure and assembly of calcium homeostasis modulator proteins.Nat. Struct. Mol. Biol. 2020; 27: 150-159Crossref PubMed Scopus (32) Google Scholar, 19Choi W. Clemente N. Sun W. Du J. Lu W. The structures and gating mechanism of human calcium homeostasis modulator 2.Nature. 2019; 576: 163-167Crossref PubMed Scopus (28) Google Scholar, 20Drozdzyk K. Sawicka M. Bahamonde-Santos M.I. Jonas Z. Deneka D. Albrecht C. Dutzler R. Cryo-EM structures and functional properties of CALHM channels of the human placenta.Elife. 2020; 9e55853Crossref PubMed Scopus (12) Google Scholar). Consequently, the "protein gate" or "lipid gate" model has been proposed to explain ATP permeation (14Syrjanen J.L. Michalski K. Chou T.H. Grant T. Rao S. Simorowski N. Tucker S.J. Grigorieff N. Furukawa H. Structure and assembly of calcium homeostasis modulator proteins.Nat. Struct. Mol. Biol. 2020; 27: 150-159Crossref PubMed Scopus (32) Google Scholar, 19Choi W. Clemente N. Sun W. Du J. Lu W. The structures and gating mechanism of human calcium homeostasis modulator 2.Nature. 2019; 576: 163-167Crossref PubMed Scopus (28) Google Scholar, 20Drozdzyk K. Sawicka M. Bahamonde-Santos M.I. Jonas Z. Deneka D. Albrecht C. Dutzler R. Cryo-EM structures and functional properties of CALHM channels of the human placenta.Elife. 2020; 9e55853Crossref PubMed Scopus (12) Google Scholar). Our results are unfavorable for these two models. We did not observe any TM1 movement when compared with the closed and open states of drCALHM1 channels. Additionally, MD simulations did not support the argument that lipids enter the pore. During the 10-μs coarse-grained MD simulations, the lipids assembled into clearly defined upper and lower leaflets around both proteins but could not be accommodated within both channel pores of proteins (Fig. S10). Although the molecular mechanism of ATP permeation in CALHM channels requires further investigation, the feature of dynamic assembly in CALHM channels is certainly quite unique. The correlation between dynamic assembly and biological function remains to be ascertained in the future. Full-length D. rerio CALHM1 (UniProtKB number: E7F2J4, synthesized by Genewiz Inc) was ligated into the EcoRI-NotI restriction sites of the pEG-BacMam vector, with a tobacco etch virus protease cleavage site and enhanced green fluorescent protein at its N-terminus. The BacMam viruses were generated and amplified following the standard Bac-to-Mam Baculovirus expression system (Invitrogen) in Spodoptera frugiperda (Sf9) cells. The bacmid produced by DH10Bac cells was transfected into Sf9 cells using X-tremeGENE HP DNA Transfection Reagent (Roche) and then cultured at 27 °C. HEK293S GnTi- cells were cultured in Freestyle 293 expression medium (Thermo Fisher Scientific) at 37 °C with 5% CO2. When the cell density reached 2 × 106 cells per mL, the cells are transfected with the second-generation virus (P2). After transfection, the cells were incubated at 37 °C for 24 h, and then sodium butyrate was added to a final concentration of 10 mM to facilitate protein expression. The cells were cultured at 30 °C for another 60 h before harvest. The cells were collected by centrifugation at 800g, resuspended in lysis buffer consisting of 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM PMSF, and then lysed through sonication for 15 min. The membrane pellets were collected by ultracentrifugation at 180,000g for 1 h and then homogenized in the buffer consisting of 20 mM Tris-HCl pH 7.5, 200 mM NaCl, and 1 × protease inhibitor cocktail (Roche), and solubilized in 1% (w/v) n-dodecyl-β-D-maltoside (DDM, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma–Aldrich) at 4 °C for 3 h. Insolub