[Analysis of the Divalent Cation Blocking in Ion Channels by Crystal Structure and Molecular Dynamics Simulations].

Neural activity generates essential responses, such as thinking, memory formation, and muscle contraction. It is controlled by the well-coordinated activity of various cation-selective channels of the cell membrane. The divalent cation block plays an essential role in various tetrameric ion channels. For example, N-methyl-D-aspartic acid receptors, which are tetrameric ion channels involved in memory formation, are inhibited by magnesium ions. Divalent cations are thought to bind in the ion pathway of the ion channel and as a consequence block the channel current, however, direct observation of such a block has not been reported yet. As a consequence, the behavior of these blocking divalent cations remains poorly understood. NavAb, a similar tetrameric sodium channel cloned from Arcobacter butzleri, is one of the most structurally analyzed tetrameric channels that is not inhibited by divalent cations. In this study, we elucidated the molecular mechanism of the divalent cation block by reproducing the divalent cation block in NavAb. The X-ray crystal structure of divalent-cation-block mutants show electron density in the ion transmission pathway of the divalent cation blocked mutants, indicating that the mutations increasing the hydrophilicity of the inner vestibule of the pore domain enable a divalent cation to stack into the ion pathway. In molecular dynamics simulations, the stacked calcium ion repels the sodium ions near the channel lumen's entrance at the selective filter's bottom. These results suggest the primary process of the divalent cation block mechanism in tetrameric cation channels and suggest a process of functional acquisition in ion channel evolution.

Voltage sensors of a Na+ channel dissociate from the pore domain and form inter-channel dimers in the resting state.

Understanding voltage-gated sodium (Nav) channels is significant since they generate action potential. Nav channels consist of a pore domain (PD) and a voltage sensor domain (VSD). All resolved Nav structures in different gating states have VSDs that tightly interact with PDs; however, it is unclear whether VSDs attach to PDs during gating under physiological conditions. Here, we reconstituted three different voltage-dependent NavAb, which is cloned from Arcobacter butzleri, into a lipid membrane and observed their structural dynamics by high-speed atomic force microscopy on a sub-second timescale in the steady state. Surprisingly, VSDs dissociated from PDs in the mutant in the resting state and further dimerized to form cross-links between channels. This dimerization would occur at a realistic channel density, offering a potential explanation for the facilitation of positive cooperativity of channel activity in the rising phase of the action potential.

The structural basis of divalent cation block in a tetrameric prokaryotic sodium channel.

Divalent cation block is observed in various tetrameric ion channels. For blocking, a divalent cation is thought to bind in the ion pathway of the channel, but such block has not yet been directly observed. So, the behaviour of these blocking divalent cations remains still uncertain. Here, we elucidated the mechanism of the divalent cation block by reproducing the blocking effect into NavAb, a well-studied tetrameric sodium channel. Our crystal structures of NavAb mutants show that the mutations increasing the hydrophilicity of the inner vestibule of the pore domain enable a divalent cation to stack on the ion pathway. Furthermore, non-equilibrium molecular dynamics simulation showed that the stacking calcium ion repel sodium ion at the bottom of the selectivity filter. These results suggest the primary process of the divalent cation block mechanism in tetrameric cation channels.

Recognition Mechanism of a Novel Gabapentinoid Drug, Mirogabalin, for Recombinant Human α2δ1, a Voltage-Gated Calcium Channel Subunit.

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2δ1. Here, to reveal the mirogabalin recognition mechanisms of α2δ1, we present structures of recombinant human α2δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α2δ1 to those of the α2δ2, α2δ3, and α2δ4 isoforms, of which α2δ3 and α2δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α2δ1 ligand recognition.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations.

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.

The insights into calcium ion selectivity provided by ancestral prokaryotic ion channels.

Prokaryotic channels play an important role in the structural biology of ion channels. At the end of the 20th century, the first structure of a prokaryotic ion channel was revealed. Subsequently, the reporting of structures of various prokaryotic ion channels have provided fundamental insights into the structure of ion channels of higher organisms. Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling. Similar to other proteins, Cavs were predicted to have a prokaryotic counterpart; however, it has taken more than 20 years for one to be identified. The homotetrameric channel obtained from Meiothermus ruber generates the calcium ion specific current, so it is named as CavMr. Its selectivity filter contains a smaller number of negatively charged residues than mutant Cavs generated from other prokaryotic channels. CavMr belonged to a different cluster of phylogenetic trees than canonical prokaryotic cation channels. The glycine residue of the CavMr selectivity filter is a determinant for calcium selectivity. This glycine residue is conserved among eukaryotic Cavs, suggesting that there is a universal mechanism for calcium selectivity. A family of homotetrameric channels has also been identified from eukaryotic unicellular algae, and the investigation of these channels can help to understand the mechanism for ion selection that is conserved from prokaryotes to eukaryotes.

Development of Novel AKR1C3 Inhibitors as New Potential Treatment for Castration-Resistant Prostate Cancer.

Aldo-keto reductase (AKR) 1C3 catalyzes the synthesis of active androgens that promote the progression of prostate cancer. AKR1C3 also contributes to androgen-independent cell proliferation and survival through the metabolism of prostaglandins and reactive aldehydes. Because of its elevation in castration-resistant prostate cancer (CRPC) tissues, AKR1C3 is a promising therapeutic target for CRPC. In this study, we found a novel potent AKR1C3 inhibitor, N-(4-fluorophenyl)-8-hydroxy-2-imino-2H-chromene-3-carboxamide (2d), and synthesized its derivatives with IC50 values of 25-56 nM and >220-fold selectivity over other AKRs (1C1, 1C2, and 1C4). The structural factors for the inhibitory potency were elucidated by crystallographic study of AKR1C3 complexes with 2j and 2l. The inhibitors suppressed proliferation of prostate cancer 22Rv1 and PC3 cells through both androgen-dependent and androgen-independent mechanisms. Additionally, 2j and 2l prevented prostate tumor growth in a xenograft mouse model. Furthermore, the inhibitors significantly augmented apoptotic cell death induced by anti-CRPC drugs (abiraterone or enzalutamide).

Crystal structure of a human plasma membrane phospholipid flippase.

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p-Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1-CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

A native prokaryotic voltage-dependent calcium channel with a novel selectivity filter sequence.

Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na+ channels (Navs). It is known that prokaryotic Navs can obtain Ca2+ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca2+ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca2+ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs. These findings provide new insight into the Ca2+ selectivity mechanism that is conserved from prokaryotes to eukaryotes.

Electrical signals in the brain and muscles allow animals ­ including humans ­ to think, make memories and move around. Cells generate these signals by enabling charged particles known as ions to pass through the physical barrier that surrounds all cells, the cell membrane, at certain times and in certain locations. The ions pass through pores made by various channel proteins, which generally have so-called "selectivity filters" that only allow particular types of ions to fit through. For example, the selectivity filters of a family of channels in mammals known as the Cavs only allow calcium ions to pass through. Another family of ion channels in mammals are similar in structure to the Cavs but their selectivity filters only allow sodium ions to pass through instead of calcium ions. Ion channels are found in all living cells including in bacteria. It is thought that the Cavs and sodium-selective channels may have both evolved from Cav-like channels in an ancient lifeform that was the common ancestor of modern bacteria and animals. Previous studies in bacteria found that modifying the selectivity filters of some sodium-selective channels known as BacNavs allowed calcium ions to pass through the mutant channels instead of sodium ions. However, no Cav channels had been identified in bacteria so far, representing a missing link in the evolutionary history of ion channels. Shimomura et al. have now found a Cav-like channel in a bacterium known as Meiothermus ruber. Like all proteins, ion channels are made from amino acids and comparing the selectivity filter of the M. ruber Cav with those of mammalian Cavs and the calcium-selective BacNav mutants from previous studies revealed one amino acid that plays a particularly important role. This amino acid is a glycine that helps select which ions may pass through the pore and is also present in the selectivity filters of many Cavs in mammals. Together these findings suggest that the Cav channel from M. ruber is similar to the mammal Cav channels and may more closely resemble the Cav-like channels thought to have existed in the common ancestor of bacteria and animals. Since other channel proteins from bacteria are useful genetic tools for studies in human and other animal cells, the Cav channel from M. ruber has the potential to be used to stimulate calcium signaling in experiments.

A single K+-binding site in the crystal structure of the gastric proton pump.

The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 Å of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of the H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfills the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+ recognition is resolved and supported by molecular dynamics simulations, establishing how the H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold-one of the highest cation gradients known in mammalian tissue-across the membrane.

Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression.

The effects of autonomic innervation of tumors on tumor growth remain unclear. Here we developed a series of genetic techniques to manipulate autonomic innervation in a tumor- and fiber-type-specific manner in mice with human breast cancer xenografts and in rats with chemically induced breast tumors. Breast cancer growth and progression were accelerated following stimulation of sympathetic nerves in tumors, but were reduced following stimulation of parasympathetic nerves. Tumor-specific sympathetic denervation suppressed tumor growth and downregulated the expression of immune checkpoint molecules (programed death-1 (PD-1), programed death ligand-1 (PD-L1), and FOXP3) to a greater extent than with pharmacological α- or ß-adrenergic receptor blockers. Genetically induced simulation of parasympathetic innervation of tumors decreased PD-1 and PD-L1 expression. In humans, a retrospective analysis of breast cancer specimens from 29 patients revealed that increased sympathetic and decreased parasympathetic nerve density in tumors were associated with poor clinical outcomes and correlated with higher expression of immune checkpoint molecules. These findings suggest that autonomic innervation of tumors regulates breast cancer progression.

Morphologic determinant of tight junctions revealed by claudin-3 structures.

Tight junction is a cell adhesion apparatus functioning as barrier and/or channel in the paracellular spaces of epithelia. Claudin is the major component of tight junction and polymerizes to form tight junction strands with various morphologies that may correlate with their functions. Here we present the crystal structure of mammalian claudin-3 at 3.6 Å resolution. The third transmembrane helix of claudin-3 is clearly bent compared with that of other subtypes. Structural analysis of additional two mutants with a single mutation representing other subtypes in the third helix indicates that this helix takes a bent or straight structure depending on the residue. The presence or absence of the helix bending changes the positions of residues related to claudin-claudin interactions and affects the morphology and adhesiveness of the tight junction strands. These results evoke a model for tight junction strand formation with different morphologies - straight or curvy strands - observed in native epithelia.

Crystal structures of the gastric proton pump.

The gastric proton pump-the H+, K+-ATPase-is a P-type ATPase responsible for acidifying the gastric juice down to pH 1. This corresponds to a million-fold proton gradient across the membrane of the parietal cell, the steepest known cation gradient of any mammalian tissue. The H+, K+-ATPase is an important target for drugs that treat gastric acid-related diseases. Here we present crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8 Å resolution. The drugs have partially overlapping but clearly distinct binding modes in the middle of a conduit running from the gastric lumen to the cation-binding site. The crystal structures suggest that the tight configuration at the cation-binding site lowers the pK a value of Glu820 sufficiently to enable the release of a proton even into the pH 1 environment of the stomach.

Enhancement of the thermostability of mouse claudin-3 on complex formation with the carboxyl-terminal region of Clostridium perfringens enterotoxin improves crystal quality.

Tight junctions regulate substance permeation through intercellular spaces as a physical barrier or a paracellular pathway, and play an important role in maintaining the internal environment. Claudins, which are tetraspan-transmembrane proteins, are pivotal components of tight junctions. In mammals 27 claudin subtypes have been identified, each of which interacts with specific subtypes. Although the crystal structures of several subtypes have been determined, the molecular mechanisms underlying subtype specificity remain unclear. Here, mouse claudin-3 (mCldn3) was crystallized in complex with the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) for the structural analysis of an additional claudin subtype. mCldn3 alone was difficult to crystallize, but complex formation with C-CPE enhanced the thermostability of mCldn3 and facilitated its crystallization. The introduction of an S313A mutation into C-CPE further improved its thermostability, and the resolution limits of the diffraction data sets improved from 8 Šfor the wild-type complex to 4.7 Šfor the S313A mutant complex.

Optimized expression and purification of NavAb provide the structural insight into the voltage dependence.

Voltage-gated sodium channels are crucial for electro-signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high-resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well-established standard of prokaryotic voltage-gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high-resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild-type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage-gated sodium channels.

Molecular determinants of prokaryotic voltage-gated sodium channels for recognition of local anesthetics.

Local anesthetics (LAs) inhibit mammalian voltage-gated Na(+) channels (Navs) and are thus clinically important. LAs also inhibit prokaryotic Navs (BacNavs), which have a simpler structure than mammalian Navs. To elucidate the detailed mechanisms of LA inhibition to BacNavs, we used NavBh, a BacNav from Bacillus halodurans, to analyze the interactions of several LAs and quaternary ammoniums (QAs). Based on the chemical similarity of QA with the tertiary-alkylamine (TAA) group of LAs, QAs were used to determine the residues required for the recognition of TAA by NavBh. We confirmed that two residues, Thr220 and Phe227, are important for LA binding; a methyl group of Thr220 is important for recognizing both QAs and LAs, whereas Phe227 is involved in holding blockers at the binding site. In addition, we found that NavBh holds blockers in a closed state, consistent with the large inner cavity observed in the crystal structures of BacNavs. These findings reveal the inhibition mechanism of LAs in NavBh, where the methyl group of Thr220 provides the main receptor site for the TAA group and the bulky phenyl group of Phe227 holds the blockers inside the large inner cavity. These two residues correspond to the two LA recognition residues in mammalian Navs, which suggests the relevance of the LA recognition between BacNavs and mammalian Navs.

Control of Spontaneous Ca2+ Transients Is Critical for Neuronal Maturation in the Developing Neocortex.

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation.

Tight junctions. Structural insight into tight junction disassembly by Clostridium perfringens enterotoxin.

The C-terminal region of Clostridium perfringens enterotoxin (C-CPE) can bind to specific claudins, resulting in the disintegration of tight junctions (TJs) and an increase in the paracellular permeability across epithelial cell sheets. Here we present the structure of mammalian claudin-19 in complex with C-CPE at 3.7 Å resolution. The structure shows that C-CPE forms extensive hydrophobic and hydrophilic interactions with the two extracellular segments of claudin-19. The claudin-19/C-CPE complex shows no density of a short extracellular helix that is critical for claudins to assemble into TJ strands. The helix displacement may thus underlie C-CPE-mediated disassembly of TJs.

Two alternative conformations of a voltage-gated sodium channel.

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.