Convergence of IRBIT,phosphatidylinositol (4,5) bisphosphate,and WNK/SPAK kinases in regulation of the Na+-HCO3? cotransporters family

Fluid and HCO₃⁻ secretion is a vital function of secretory epithelia, involving basolateral HCO₃⁻ entry through the Na⁺-HCO₃⁻ cotransporter (NBC) NBCe1-B, and luminal HCO₃⁻ exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl⁻/HCO₃⁻ exchangers. HCO₃⁻ secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP₂). Activation of the transporters by IRBIT and PIP₂ is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO₃⁻ transporters and thus in the regulation of epithelial fluid and HCO₃⁻ secretion.Fluid and HCO₃⁻ secretion is a vital function of secretory epithelia that involves basolateral HCO₃⁻ entry through the Na⁺-HCO₃⁻ cotransporter (NBC) NBCe1-B and luminal HCO₃⁻ exit mediated by the concerted activity of cystic fibrosis transmembrane conductance regulator (CFTR) and members of the solute carrier family 26 (SLC26) transporter family (1). HCO₃⁻ secretion is osmotically active owing to an influx of Na⁺-2HCO₃⁻ (2, 3) and the exchange of Cl⁻/2HCO₃⁻ by Slc26a6 (4, 5), resulting in net osmolyte secretion in the form of HCO₃⁻. HCO₃⁻ secretion is a highly regulated activity, with several signaling pathways converging to regulate key transporters activity to tune the secretion (1); however, very little is known about the molecular mechanisms that regulate fluid and HCO₃⁻ secretion, particularly the regulation of NBCe1-B.NBCe1-B was originally designated pancreatic NBC 1 (6) and was later renamed NBCe1-B as a member of the electrogenic NBCe1 subfamily of the Na⁺-coupled bicarbonate transporter (NCBT) superfamily (2, 3). NBCe1-B is is expressed in the basolateral membrane of most secretory epithelia, including the pancreas, salivary glands, airway, and intestines (1). The NCBTs encompass 13 transmembrane domains with varying cytoplasmic N and C termini among the isoforms (3). Most members of the NCBT superfamily have a unique N terminus (the first 85 residues in NBCe1-B) (7). This domain has been shown to function as an autoinhibitory domain (AID) in NBCe1-B (8–10). Very little is known about the regulation of NBCe1-B or other members of the superfamily beyond that NBCe1-B may be modestly activated by cAMP (11, 12), although inhibition of NBCe1-B by cAMP was subsequently reported by the same group (13). NBCe1-B appears to be constitutively phosphorylated by protein kinase A in Thr49 (11); however, its role in activation of the transporter is not clear, given that both the T49A and T49D mutations were found to prevent activation by cAMP (11). Activation of NBCe1-B and NBCe1-C by intracellular Ca²⁺ through an unknown mechanism was reported recently (5). NBCe1-A is activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) (14), but direct activation of NBCe1-B and NBCe1-C by PIP₂ has not been examined. The site of interaction of PIP₂ in regulating the activity of any NCBT family member remains to be determined.NBCe1-B (9, 10, 15, 16) and NBCe1-C (17) are potently activated by the inositol 1,4,5-triphosphate (IP₃) receptors binding protein released with IP₃ (IRBIT) and are inhibited by the with no lysine kinase (WNK) and Ste20-related proline alanine rich kinase (SPAK) (15). IRBIT also regulates CFTR (15, 16) and sodium-hydrogen exchanger 3 (NHE3) (18). IRBIT activates NBCe1-B by recruiting protein phosphatase 1 (PP1), reversing inhibition by the WNK/SPAK pathway through dephosphorylation of NBCe1-B (15) and relief of inhibition by the AID (9, 10, 15, 16). The WNKs function as scaffolds to recruit SPAK to NBCe1-B, which in turn phosphorylates the transporter at unknown sites. Similarly, the site of IRBIT–AID interaction is unknown. Deletion of the first 16 residues of NBCe1-B prevents activation by IRBIT (9), although the effect of this truncation on IRBIT binding is unclear. On the other hand, an in vitro binding assay revealed binding of IRBIT to NBCe1-B(1-62), but not to NBCe1-B(1-37) (10). This finding suggests that the IRBIT binding site may be located within NBCe1-B(37-62); however, the possible binding of IRBIT to NBCe1-B(37-62) or a site within has not yet been examined.The extent to which these pathways regulate other members of the NBC family is unknown, although IRBIT may activate an unspecified member of the NBCn1 subfamily (2). The NBCn1 subfamily was established with the discovery of NBC3, later renamed NBCn1-A. NBCn1-A is a widely expressed electroneutral NBC (3, 7) that mediates HCO₃⁻ salvage in secretory epithelia (19, 20). Given that sequence analysis has shown significant conservation of the N termini of NBCe1-B and NBCn1-A, we deemed it useful to compare the regulation of these NBCs by IRBIT/PP1, PIP₂, and SPAK to evaluate the generality of this regulation. In the present studies, we also investigated whether these multiple regulatory pathways converge on the same domain to regulate the transporters.We have identified a positively charged domain within NBCe1-B(37-65) that is conserved in NBCn1-A and most members of the NCBT superfamily and is required for interaction and activation of the transporters by IRBIT. The same domain mediates regulation of NBCe1-B and NBCn1-A by PIP₂. Importantly, activation of the transporters by IRBIT and PIP₂ is nonadditive but complementary. Phosphorylation of Ser65 within this domain mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 within NBCe1-B(37-65) is required for regulation by the activator IRBIT and the inhibitor SPAK. Moreover, a sequence search using the conserved module identified a similar module in Slc26a6 and CFTR that is required for regulation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in the AID of the NBCs and thus in the regulation of epithelial fluid and HCO₃⁻ secretion.     

Intracellular Cl? as a signaling ion that potently regulates Na+/HCO3? transporters

Cl⁻ is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl⁻ (Cl⁻_in) as a signaling ion has not been previously evaluated. Here we report that Cl⁻_in functions as a regulator of cellular Na⁺ and HCO₃⁻ concentrations and transepithelial transport through modulating the activity of several electrogenic Na⁺-HCO₃⁻ transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl⁻_in concentrations highlighting the role of GXXXP motifs in Cl⁻ sensing. Regulation of the ubiquitous Na⁺-HCO3⁻ co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl⁻_in interacting at a low affinity GXXXP-containing site. IP₃ receptor binding protein released with IP₃ (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl⁻_in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl⁻_in interacting site. NBCe1-A is unaffected by Cl⁻_in between 5 and 140 mM. However, deletion of NBCe1-A residues 29–41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl⁻_in. These findings reveal a cellular Cl⁻_in sensing mechanism that plays an important role in the regulation of Na⁺ and HCO₃⁻ transport, with critical implications for the role of Cl⁻ in cellular ion homeostasis and epithelial fluid and electrolyte secretion.Cl⁻ and HCO₃⁻ are the two major intracellular anions in mammalian cells. Specific transporters, channels, and the membrane potential tightly regulate their extracellular and intracellular concentrations. In turn, Cl⁻ and HCO₃⁻ regulate the concentration of other ions, including Na⁺, K⁺, and SO₄²⁻, either directly or indirectly. Known ubiquitous Cl⁻- and HCO₃⁻-coupled transporters include the NaCl cotransporters NCCs, the KCl cotransporters KCCs, the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (1), the SLC26 Cl⁻/HCO₃⁻ exchangers and channels (2, 3), and members of the SLC4 exchangers and cotransporters family (4). The intracellular Cl⁻ (Cl⁻_in) concentration is also regulated by the ClCs (5) and Anoctamines Cl⁻ channels (6). Cl⁻ plays a role in a wide variety of cellular transport functions, including regulation of the membrane potential (6), cell volume (2), systemic and cellular acid–base balance (4), and transepithelial fluid and electrolyte secretion (7). In addition, Cl⁻ was reported to regulate transient receptor potential (TRP) channels (8), receptors assembly and function (9–11), activation of Neutrophil β2 Integrins (12), and the cell cycle (13).Like Cl⁻, HCO₃⁻ also has many important physiological roles, being the principal biological pH buffer (7) and an activator of the soluble adenylyl cyclase (14). In epithelia, HCO₃⁻ has a key role in tissue/cell viability. Among other fundamental roles, HCO₃⁻ drives Cl⁻ absorption and fluid secretion, stimulates mucin secretion, and controls solubilization of secreted macromolecules (7). Epithelial HCO₃⁻ secretion is fueled by the cellular Na⁺ gradient, which provides the driving force for HCO₃⁻ entry across the basolateral membrane mediated by the Na⁺-HCO₃⁻ cotransport NBCe1-B. HCO₃⁻ then exits the luminal membrane by the coordinated and coupled functions of the Cl⁻ channel cystic fibrosis transmembrane conductance regulator (CFTR) and the electrogenic Cl⁻/HCO₃⁻ exchanger slc26a6 (3, 7, 15). In the kidney, NBCe1-A mediates basolateral HCO₃⁻ extrusion (16, 17). In secretory ducts the basolateral NBCe1-B–mediated HCO₃⁻ influx is coupled to apical HCO₃⁻ secretion and Cl⁻ absorption via CFTR and slc26a6 (7). Cl⁻_in is reduced along the ducts as luminal Cl⁻ is reduced and luminal HCO₃⁻ is increased (18, 19).Members of the SLC4 superfamily of HCO₃⁻ cotransporters are key transporters involved in cellular HCO₃⁻ and Cl⁻ homeostasis (4, 7). The family consists of several subfamilies, including the electrogenic NBCe1 and NBCe2, electroneutral NBCn1 and NBCn2, Cl⁻-coupled anion exchangers AEs, and the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger NDCBE (4). In a wide variety of tissues, NBCe1-B mediates the electrogenic transport of 1Na⁺ and 2HCO₃⁻ ions (likely Na⁺-CO₃²⁻) (4, 17) and functions as the main epithelial HCO₃⁻ entry mechanism in the basolateral membrane of polarized cells (7). Cell-specific electrogenic NBC transporters include NBCe1-A, which is expressed in the basolateral membrane of the renal proximal tubule (4, 17), and NBCe2-C, which is found in the choroid plexus (20). IRBIT, which regulates NBCe1-B (21–23) and NBCn1-A (24), binds to the IP₃ binding domain of the IP₃ receptors (IP₃Rs) (25). IRBIT is released from the IP₃Rs upon an increase in cellular IP₃ (26), becoming available for regulation of NBCe1-B (21–23), NBCn1-A (24), CFTR (22, 23), and slc26a6 (27), thereby coordinating the activation of these transporters and epithelial fluid and HCO₃⁻ secretion (27).Cl⁻_in has not been previously considered as a signaling ion. Rather, Cl⁻ has heretofore mainly been viewed as an ion that is transported by various channels, coupled to Na⁺, K⁺, or HCO₃⁻ transport, and that plays a role regulating the plasma membrane potential (1, 7). Importantly, several studies have provided clues that Cl⁻_in may have regulatory and perhaps signaling functions. High Cl⁻_in was reported to inhibit the activity of the epithelial Na⁺ channel ENaC (28), the permeability of CFTR to HCO₃⁻ (29), TRPM7 activity (8), and perhaps the activity of the Na⁺/H⁺ exchanger NHE1 (30). Muscarinic stimulation of salivary gland acinar cells resulted in reduction in Cl⁻_in that is required for Na⁺ influx by the Na⁺/H⁺ exchanger and the Na⁺/K⁺/2Cl⁻ cotransporter (31).In the present study we asked whether Cl⁻_in functions as a signaling ion that modulates cellular Na⁺ and HCO₃⁻ concentrations through regulation of electrogenic NBC transporters. Our data indicate that Cl⁻_in regulates the function of NBCe1-B, NBCe2-C, and NBCe1-A via a cryptic Cl⁻ sites. In the basal state NBCe1-B is inhibited by Cl⁻_in interacting with a low affinity Cl⁻ site, whereas NBCe1-A is resistant to Cl⁻_in. By contrast, IRBIT-activated NBCe1-B is inhibited by low and high concentrations of Cl⁻_in due to interaction with high and low affinity Cl⁻_in motifs that depend on GXXXP motifs. Mutation of the G and P or of His in GXHXP in the autoinhibitory domain of NBCe1-B eliminated inhibition by low Cl⁻_in, whereas sparing inhibition of NBCe1-B by high Cl⁻_in. Mutation of a second GXXXP motif was required to eliminate inhibition by low affinity Cl⁻_in site. NBCe2-C is inhibited by a single high affinity GXXXP motif-dependent Cl⁻_in interacting site. Remarkably, deletion of the first 48 residues of NBCe1-A or of residues 29–41 uncovered inhibition of NBCe1-A by Cl⁻_in that was mediated by a GXXXP motif-dependent site homologous with the second GXXXP motif of NBCe1-B. These findings reveal a Cl⁻_in sensing mechanism that modulates the activity of NBCe1-B, NBCe2-C, and NBCe1-A. In transporting epithelia, such as the pancreatic and salivary ducts and the choroid plexus, we predict that a reduction in Cl⁻_in will dramatically increase transepithelial HCO₃⁻ transport and fluid secretion.     

Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).     

From the Cover: Water access points and hydration pathways in CLC H+/Cl? transporters

CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl⁻ has been established, the pathway of H⁺ translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H⁺-translocation. An extended (0.4 µs) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, site-specific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-Å hydrophobic region between the two known H⁺ transport sites [E148 (Glu_ex) and E203 (Glu_in)]. Our finding that the formation of these water wires requires the presence of Cl⁻ explains the previously mystifying fact that Cl⁻ occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H⁺ transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H⁺ and Cl⁻ ions in CLC transporters.The chloride channel (CLC) family (1, 2) includes both passive Cl⁻ channels and secondary active H⁺-coupled Cl⁻ transporters (3–8). The latter, also known as H⁺/Cl⁻ exchangers, drive uphill movement of H⁺ by coupling the process to downhill movement of Cl⁻ or vice versa, thereby exchanging the two types of ions across the membrane at fixed stoichiometry (9). ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3–8, 12–17). Detailed structural and functional studies of ClC-ec1 (9, 11, 18–27) have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl⁻ permeation pathway and its lining residues (10, 18, 25) and established the role of E148, also known as Glu_ex, as the extracellular gate for the Cl⁻ pathway (9, 11).Although much less is known about the H⁺ translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H⁺ transport (9, 13, 14, 20, 22, 27, 28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H⁺ transport (Fig. 1A): E148 (Glu_ex), which acts as the main extracellular H⁺ binding site (9, 11, 27), and E203 (Glu_in), which plays a similar role on the cytoplasmic side (20, 22, 28). Neutralization of either glutamate eliminates H⁺ translocation by ClC-ec1 (9, 28). However, the discovery of these H⁺ binding sites also raised a mechanistic puzzle (3, 23): How do protons translocate between the two sites, which are separated by a ∼15-Å-long, largely hydrophobic region within the lumen of the protein?Open in a separate windowFig. 1.Cl⁻ and H⁺ permeation pathways in ClC-ec1. (A) View of the ClC-ec1 structure in a lipid bilayer (the simulation system used here), with the identical subunits shown in yellow and orange. The presumed Cl⁻/H⁺ permeation pathways are indicated by green and red lines, respectively. The dashed segment of the red line denotes the pathway investigated in this study. (B) Close-up of the central hydrophobic region, with the residues forming this region shown as orange sticks and labeled. Also shown are key glutamate residues (E202, E203, and E148) as well as the Cl⁻ at the central anion binding site. (C) Hydration of the central hydrophobic region during the 0.4-µs equilibrium simulation, measured as the number of water molecules in this region for each subunit.Since the report of its first crystal structure, a large number of computational studies have aimed at investigating various molecular details related to the CLC H⁺ transport mechanism (27, 29–34). One model emerging from these studies proposes that water molecules may connect the two H⁺ sites (Glu_ex and Glu_in) and, thereby, facilitate H⁺ transport (29, 30, 34). This idea was initially proposed by Kuang and coworkers (29) on the basis of a hole-searching algorithm applied to static crystal structures of ClC-ec1. In their proposed pathway, water molecules are suggested to form two half-wires that are then connected by the hydroxyl group of Y445 to form a complete path for H⁺ transfer. However, it is known from experiments on the Y445F mutant that this hydroxyl is not required for H⁺ transport (20). Wang and Voth (30) proposed another pathway by combining an improved search algorithm for buried water with short molecular dynamics (MD) simulations, thereby taking into account the dynamic nature of the protein. Their pathway did not rely on Y445 but required reorientation of the side chain of E203 to connect the two H⁺ sites. In another study, these investigators further carried out semiempirical free energy calculations to investigate the Cl⁻/H⁺ coupling mechanism (33).Although the idea of water-mediated H⁺ transport is intriguing and could be key to understanding H⁺ transport in ClC-ec1, several questions relevant to a water wire mechanism remain unanswered: Can the hydrophobic region between the two H⁺ sites actually be hydrated under equilibrium conditions? What is the access/entry point or points for water from the bulk into the hydrophobic region, which is buried inside the protein, approximately at the midpoint of the membrane? Is it possible to observe the spontaneous formation of water wires through MD simulations? If so, how much do the simulated wire structures differ from the ones proposed by the prior studies based on search algorithms? How could the protein affect the dynamics and/or the thermodynamics of water wires?In the current study, we have addressed these questions through a combined computational and experimental approach. An extended 0.4-µs MD simulation of a membrane-embedded model of wild-type (WT) ClC-ec1 reveals that the central hydrophobic region can indeed be hydrated by water molecules mainly from the cytoplasmic bulk phase through pathways near the dimer interface via a portal lined by residues E202, E203, and A404. Water wires connecting the two H⁺ sites form spontaneously and repeatedly during the equilibrium simulation. Formation of wires requires a side-chain conformational change of I109 and the occupancy of the central Cl⁻ binding site, S_cen. These simulation results make two strong and testable predictions: that mutations at A404 and I109 will reduce ClC-ec1 activity and that the reduction in activity occurs via effects on the H⁺ branch of the transport mechanism. Our experimental tests and additional simulations performed on one mutant form of the protein fully support these predictions.     

Fenton chemistry at aqueous interfaces

In a fundamental process throughout nature, reduced iron unleashes the oxidative power of hydrogen peroxide into reactive intermediates. However, notwithstanding much work, the mechanism by which Fe²⁺ catalyzes H₂O₂ oxidations and the identity of the participating intermediates remain controversial. Here we report the prompt formation of O=Fe^IVCl₃⁻ and chloride-bridged di-iron O=Fe^IV·Cl·Fe^IICl₄⁻ and O=Fe^IV·Cl·Fe^IIICl₅⁻ ferryl species, in addition to Fe^IIICl₄⁻, on the surface of aqueous FeCl₂ microjets exposed to gaseous H₂O₂ or O₃ beams for <50 μs. The unambiguous identification of such species in situ via online electrospray mass spectrometry let us investigate their individual dependences on Fe²⁺, H₂O₂, O₃, and H⁺ concentrations, and their responses to tert-butanol (an ·OH scavenger) and DMSO (an O-atom acceptor) cosolutes. We found that (i) mass spectra are not affected by excess tert-butanol, i.e., the detected species are primary products whose formation does not involve ·OH radicals, and (ii) the di-iron ferryls, but not O=Fe^IVCl₃⁻, can be fully quenched by DMSO under present conditions. We infer that interfacial Fe(H₂O)_n²⁺ ions react with H₂O₂ and O₃ >10³ times faster than Fe(H₂O)₆²⁺ in bulk water via a process that favors inner-sphere two-electron O-atom over outer-sphere one-electron transfers. The higher reactivity of di-iron ferryls vs. O=Fe^IVCl₃⁻ as O-atom donors implicates the electronic coupling of mixed-valence iron centers in the weakening of the Fe^IV–O bond in poly-iron ferryl species.High-valent Fe^IV=O (ferryl) species participate in a wide range of key chemical and biological oxidations (1–4). Such species, along with ·OH radicals, have long been deemed putative intermediates in the oxidation of Fe^II by H₂O₂ (Fenton’s reaction) (5, 6), O₃, or HOCl (7, 8). The widespread availability of Fe^II and peroxides in vivo (9–12), in natural waters and soils (13), and in the atmosphere (14–18) makes Fenton chemistry and Fe^IV=O groups ubiquitous features in diverse systems (19). A lingering issue regarding Fenton’s reaction is how the relative yields of ferryls vs. ·OH radicals depend on the medium. For example, by assuming unitary ·OH radical yields, some estimates suggest that Fenton’s reaction might account for ∼30% of the ·OH radical production in fog droplets (20). Conversely, if Fenton’s reaction mostly led to Fe^IV=O species, atmospheric chemistry models predict that their steady-state concentrations would be ∼10⁴ times larger than [·OH], thereby drastically affecting the rates and course of oxidative chemistry in such media (20). Fe^IV=O centers are responsible for the versatility of the family of cytochrome P450 enzymes in catalyzing the oxidative degradation of a vast range of xenobiotics in vivo (21–28), and the selective functionalization of saturated hydrocarbons (29). The bactericidal action of antibiotics has been linked to their ability to induce Fenton chemistry in vivo (9, 30–34). Oxidative damage from exogenous Fenton chemistry likely is responsible for acute and chronic pathologies of the respiratory tract (35–38).Despite its obvious importance, the mechanism of Fenton’s reaction is not fully understood. What is at stake is how the coordination sphere of Fe²⁺ (39–46) under specific conditions affects the competition between the one-electron transfer producing ·OH radicals (the Haber–Weiss mechanism) (47), reaction R1, and the two-electron oxidation via O-atom transfer (the Bray–Gorin mechanism) into Fe^IVO²⁺, reaction R2 (6, 23, 26, 27, 45, 48–51):Ozone reacts with Fe²⁺ via analogous pathways leading to (formally) the same intermediates, reactions R3a, R3b, and R4 (8, 49, 52, 53):At present, experimental evidence about these reactions is indirect, being largely based on the analysis of reaction products in bulk water in conjunction with various assumptions. Given the complex speciation of aqueous Fe²⁺/Fe³⁺ solutions, which includes diverse poly-iron species both as reagents and products, it is not surprising that classical studies based on the identification of reaction intermediates and products via UV-absorption spectra and the use of specific scavengers have fallen short of fully unraveling the mechanism of Fenton’s reaction. Herein we address these issues, focusing particularly on the critically important interfacial Fenton chemistry that takes place at boundaries between aqueous and hydrophobic media, such as those present in atmospheric clouds (16), living tissues, biomembranes, bio-microenvironments (38, 54, 55), and nanoparticles (56, 57).We exploited the high sensitivity, surface selectivity, and unambiguous identification capabilities of a newly developed instrument based on online electrospray mass spectrometry (ES-MS) (58–62) to identify the primary products of reactions R1–R4 on aqueous FeCl₂ microjets exposed to gaseous H₂O₂ and O₃ beams under ambient conditions [in N₂(g) at 1 atm at 293 ± 2 K]. Our experiments are conducted by intersecting the continuously refreshed, uncontaminated surfaces of free-flowing aqueous microjets with reactive gas beams for τ ∼10–50 μs, immediately followed (within 100 μs; see below) by in situ detection of primary interfacial anionic products and intermediates via ES-MS (Methods, SI Text, and Figs. S1 and S2). We have previously demonstrated that online mass spectrometric sampling of liquid microjets under ambient conditions is a surface-sensitive technique (58, 62–67).     

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Proteins containing C2 domains are the sensors for Ca²⁺ and PI(4,5)P₂ in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca²⁺ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P₂ and IP₃ at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P₂/PI(3,4,5)P₃, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P₂. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca²⁺-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1–4). Many of these C2 domains have been demonstrated to function in a Ca²⁺-dependent membrane-binding manner and hence act as cellular Ca²⁺ sensors. Calcium ions bind in a cup-shaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca²⁺ ions are incomplete (5–7). This incomplete coordination sphere can be occupied by neutral and anionic (7–9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P₂ simultaneously (10). This revealed an additional lipid-binding site located in the polybasic region formed by β3–β4 strands that preferentially binds to PI(4,5)P₂ (11–15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10, 16–19). Given the importance of PI(4,5)P₂ for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20–23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca²⁺ and PI(4,5)P₂ combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16, 20, 22, 24–28). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5, 26, 27, 29, 30). However, the impossibility of obtaining crystal structures of these domains in complex with Ca²⁺ and phosphoinositides has hindered the understanding of the molecular mechanism driving the PI(4,5)P₂–C2 domain interaction. Here, we sought to unravel the molecular mechanism of Ca²⁺ and PI(4,5)P₂ binding to the C2A domain of rabphilin 3A by X-ray crystallography. A combination of site-directed mutagenesis together with isothermal titration calorimetry (ITC), fluorescence resonance of energy transfer (FRET), and aggregation experiments has enabled us to propose a molecular mechanism of Ca²⁺/PI(4,5)P₂-dependent membrane interaction through two different motifs that could bend the membrane and accelerate the vesicle fusion process. A comparative analysis revealed the structural basis for the different phosphoinositide affinities of C2A and -B domains. Furthermore, the C2A domain of synaptotagmin 1 lacks one of the key residues responsible for the PI(4,5)P₂ interaction, confirming it is a non-PI(4,5)P₂ responder.     

Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Ca²⁺ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30–45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO₃⁻, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca²⁺ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.Mammalian spermatozoa, unlike spermatozoa of many other animals, are not able to fertilize on leaving the male body. They must undergo physiological changes, collectively called “capacitation,” to become fertilization-competent. Under normal conditions, sperm capacitation takes place within the female tract, but it can also occur in chemically defined media, as first demonstrated by Toyoda et al. (1) in the mouse. Although compositions of media necessary for successful in vitro capacitation vary between species, most are basically modified Tyrode’s and Krebs–Ringer’s solutions containing HCO₃⁻ and Ca²⁺, supplemented with energy metabolites and a cholesterol acceptor such as serum albumin. Capacitation enables spermatozoa to undergo the acrosome reaction and to exhibit vigorous motility called hyperactivation (2, 3). Both the acrosome reaction and hyperactivation are believed to be essential for successful sperm penetration into oocytes (4). Molecular changes associated with capacitation include an increase in intracellular pH (pH_i) (5), an increase in intracellular Ca²⁺ concentration [Ca²⁺]_i (6), activation of a cAMP/PKA pathway (7, 8), hyperpolarization of the sperm plasma membrane potential (9–11), loss of membrane cholesterol (12, 13) and modifications of other membrane lipids (14), and an increase in protein tyrosine phosphorylation (8, 15). How these events interact with each other to render spermatozoa capable of initiating the acrosome reaction and hyperactivation is not well understood. Recent studies using gene knock-out mice revealed that both cAMP- (14–17) and Ca²⁺-regulated signaling pathways (16–18) are intricately involved in these processes.Involvement of Ca²⁺ in the sperm acrosome reaction and in hyperactivation has been known for a long time (4). Ca²⁺ ionophore, which transports extracellular Ca²⁺ into cells or releases Ca²⁺ from intracellular stores (19), induces increased respiration (20), motility (21), and the acrosome reaction (22) in mammalian spermatozoa. Several studies have shown that Ca²⁺ ionophore A23187 increases intracellular Ca²⁺ concentration excessively, rendering spermatozoa immotile (23–26). However, immobilized spermatozoa are not dead, as demonstrated by Suarez et al. (25), who found that spermatozoa could regain motility when high concentrations of BSA were added to the medium. The high affinity of BSA for hydrophobic A23187 could explain this recovery. However, the question as to whether these spermatozoa are capable of pursuing their physiological function (fertilization) remained unanswered. We report here that mouse spermatozoa treated with ionophore A23178 did indeed become immotile, but soon after the ionophore was removed, they began to move vigorously (hyperactivated) and quickly fertilized oocytes. More surprisingly, ionophore-treated spermatozoa fertilize oocytes under the constant presence of H89, a PKA inhibitor, and also in the absence of HCO₃⁻ in the medium, an ion that is absolutely necessary for fertilization under normal in vitro conditions (27).     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

The inositol 1,4,5-trisphosphate receptor (IP₃R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP₃Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP₃ at a distance. Defective allostery in IP₃R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP₃R type 1 (IP₃R1) and negatively regulates IP₃R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP₃R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.Ligand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ligand-gated ion channels that release calcium ions (Ca²⁺) from the endoplasmic reticulum (ER) (2, 3). IP₃Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3) are structurally divided into three domains: the IP₃-binding domain (IBD), the regulatory domain, and the channel domain (3–6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca²⁺ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP₃R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP₃R (IP₃R1) leads to perturbations in long-term potentiation/depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP₃R1 gene (13–15). Dysregulation of IP₃R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16–18) and Alzheimer’s disease (AD) (19–22). IP₃Rs also control fundamental cellular processes—for example, mitochondrial energy production (23, 24), autophagy regulation (24–27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophage inflammasomes (31). On the other hand, excessive IP₃R function promotes cell death processes including apoptosis by activating mitochondrial or calpain pathways (2, 17). Considering these versatile roles of IP₃Rs, appropriate IP₃R structure and function are essential for living systems, and aberrant regulation of IP₃R closely relates to various diseases.Several factors such as cytosolic molecules, interacting proteins, and posttranslational modifications control the IP₃-induced Ca²⁺ release (IICR) through allosteric sites in IP₃Rs. Cytosolic Ca²⁺ concentrations strictly control IICR in a biphasic manner with activation at low concentrations and inhibition at higher concentrations. The critical Ca²⁺ sensor for activation is conserved among the three isoforms of IP₃ and ryanodine receptors, and this sensor is located in the regulatory domain outside the IBD and the channel domain (32). A putative ATP regulatory region is deleted in opisthotonos mice, and IICR is also regulated by this mutation in the regulatory domain (33). Various interacting proteins, such as cytochrome c, Bcl-2-family proteins, ataxin-3, huntingtin (Htt) protein, Htt-associated protein 1A (HAP1A), and G-protein–coupled receptor kinase-interacting protein 1 (GIT1), target allosteric sites in the carboxyl-terminal tail (3–5). The regulatory domain and the carboxyl-terminal tail also undergo phosphorylation by the protein kinases A/G and B/Akt and contain the apoptotic cleavage sites for the protease caspase-3 (4, 5). These factors allosterically regulate IP₃R structure and function to control cellular fates; therefore, understanding the allosteric coupling of the IBD to channel gating will elucidate the regulatory mechanism of these factors.Transglutaminase (TG) catalyses protein cross-linking between a glutamine (Gln) residue and a lysine (Lys) residue via an N^ε-(γ-glutamyl)lysine isopeptide bond (34, 35). TG type 2 (TG2) is a Ca²⁺-dependent enzyme with widespread distribution and is highly inducible by various stimulations such as oxidative stress, cytokines, growth factors, and retinoic acid (RA) (34, 35). TG2 is considered a significant disease-modifying factor in neurodegenerative diseases including HD, AD, and Parkinson’s diseases (PD) (34, 36–45) because TG2 might enzymatically stabilize aberrant aggregates of proteins implicated in these diseases—that is, mutant Htt, β-amyloid, and α-synuclein; however, the causal role of TG2 in Ca²⁺ signaling in brain pathogenesis has been unclear. Ablation of TG2 in HD mouse models is associated with increased lifespan and improved motor function (46, 47). However, TG2 knockout mice do not show impaired Htt aggregation, suggesting that TG2 may play a causal role in these disorders rather than TG2-dependent cross-links in aberrant protein aggregates (47, 48).In this study, we discovered a new mode of chronic and irreversible allosteric regulation in IP₃R1 in which covalent modification of the receptor at Gln2746 is catalyzed by TG2. We demonstrate that up-regulation of TG2 modifies IP₃R1 structure and function in HD models and propose an etiologic role of this modification in the reduction of neuronal signaling and subsequent processes during the prodromal state of the neurodegenerative disease.     

Dynamic modulation of ANO1/TMEM16A HCO3? permeability by Ca2+/calmodulin

Anoctamin 1 (ANO1)/transmembrane protein 16A (TMEM16A) is a calcium-activated anion channel that may play a role in HCO₃⁻ secretion in epithelial cells. Here, we report that the anion selectivity of ANO1 is dynamically regulated by the Ca²⁺/calmodulin complex. Whole-cell current measurements in HEK 293T cells indicated that ANO1 becomes highly permeable to HCO₃⁻ at high [Ca²⁺]_i. Interestingly, this result was not observed in excised patches, indicating the involvement of cytosolic factors in this process. Further studies revealed that the direct association between ANO1 and calmodulin at high [Ca²⁺]_i is responsible for changes in anion permeability. Calmodulin physically interacted with ANO1 in a [Ca²⁺]_i-dependent manner, and addition of recombinant calmodulin to the cytosolic side of excised patches reversibly increased P_HCO3/P_Cl. In addition, the high [Ca²⁺]_i-induced increase in HCO₃⁻ permeability was reproduced in mouse submandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. These results indicate that the HCO₃⁻ permeability of ANO1 can be dynamically modulated and that ANO1 may play an important role in cellular HCO₃⁻ transport, especially in transepithelial HCO₃⁻ secretion.Calcium-activated chloride channels (CaCCs) mediate a number of important physiological functions including sensory transduction, regulation of vascular tone, and fluid secretion (1). In the secretory epithelium of airways and exocrine organs, such as intestines, pancreas, and salivary glands, CaCCs control apical efflux of anions, which is essential for the vectorial transport of water and electrolytes in these organs (2, 3). Recently, members of anoctamin (ANO; also known as TMEM16) family, in particular ANO1/TMEM16A and ANO2/TMEM16B, were shown to function as CaCCs in the gut, trachea, salivary glands, and olfactory organs (2–6).In general, Cl⁻ channels have nonspecific anion selectivity and permeate other anions in addition to Cl⁻. In fact, halide ions larger than Cl⁻, such as I⁻ and Br⁻, are more readily permeable to most Cl⁻ channels. For example, the anion selectivity sequence of both endogenous CaCCs and heterologously expressed ANO1 is I⁻ > Br⁻ > Cl⁻ > HCO₃⁻ > F⁻ (3, 7). However, in physiological conditions, the two most abundant anions that can be the charge carrier of anion channels are Cl⁻ and HCO₃⁻. Although the permeation and conduction mechanisms of Cl⁻ via anion channels are fairly well characterized, those of HCO₃⁻ are poorly understood. It has been demonstrated that a significant proportion of transepithelial HCO₃⁻ transport is mediated by electrodiffusive pathways, suggesting that anion channels are involved in this process (8–10). Up to this point, neither molecular nor physiological experiments have demonstrated the presence of bona fide selective HCO₃⁻ channels. Therefore, it is generally believed that nonspecific anion channels mediate electrodiffusive HCO₃⁻ transport.HCO₃⁻, as a major component of the CO₂/HCO₃⁻ buffer system, is an indispensible ingredient in our body fluids that guards against toxic intracellular and extracellular fluctuations in pH (11). In addition, as a chaotropic ion, HCO₃⁻ facilitates the solubilization of macromolecules in biological fluids and stimulates mucin secretion (11, 12). Indeed, recent progress in epithelial pathophysiology has indicated that aberrant HCO₃⁻ secretion is associated with a spectrum of diseases in the respiratory, gastrointestinal, and genitourinary systems, such as cystic fibrosis, pancreatitis, and infertility (10, 11, 13, 14). In the present study, we provide evidence that Ca²⁺/calmodulin dynamically regulates the anion selectivity and HCO₃⁻ permeability of ANO1 by using integrated molecular and physiological approaches. These results offer insight into how transepithelial HCO₃⁻ transport is activated, in particular in response to cytosolic Ca²⁺ signaling, and offer a therapeutic strategy for the treatment of diseases derived from aberrant HCO₃⁻ secretion.     

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O₂^•−) dismutase-like properties yet were inert to nitric oxide (NO^•) as well as peroxynitrite (ONOO⁻). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO₂ and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O₂^•− to O₂ by PEG-HCCs at >20,000 s⁻¹. The major products of this catalytic turnover are O₂ and H₂O₂, making the PEG-HCCs a biomimetic superoxide dismutase.Reactive oxygen species (ROS), such as superoxide (O₂^•−), hydrogen peroxide (H₂O₂), organic peroxides, and hydroxyl radical (^•OH), are a consequence of aerobic metabolism (1, 2). These ROS are necessary for the signaling pathways in biological processes (3, 4) such as cell migration, circadian rhythm, stem cell proliferation, and neurogenesis (5). In healthy systems, ROS are efficiently regulated by the defensive enzymes superoxide dismutase (SOD) and catalase, and by antioxidants such as glutathione, vitamin A, ascorbic acid, uric acid, hydroquinones, and vitamin E (6). When the production of ROS overwhelms the scavenging ability of the defense system, oxidative stress occurs, causing dysfunctions in cell metabolism (7–16).In addition to ROS, reactive nitrogen species (RNS) such as nitric oxide (NO^•), nitrogen dioxide, and dinitrogen trioxide can be found in all organisms. NO^• can act as an oxidizing or reducing agent depending on the environment (17), is more stable than other radicals (half-life 4–15 s) (18), and is synthesized in small amounts in vivo (17–22). NO^• is a potent vasodilator and has an important role in neurotransmission and cytoprotection (17, 18, 22, 23). Owing to its biological importance and the low concentration found normally in vivo, it is often important to avoid alteration of NO^• levels in biological systems to prevent aggravation of acute pathologies including ischemia and reperfusion.One way to treat these detrimental pathologies is to supply antioxidant molecules or particles that renormalize the disturbed oxidative condition. We recently developed a biocompatible carbon nanoparticle, the poly(ethylene glycolated) hydrophilic carbon cluster (PEG-HCC), which has shown ability to scavenge oxyradicals and protect against oxyradical damage in rodent models and thus far has demonstrated no in vivo toxicity in laboratory rodents (24–27). The carbon cores of PEG-HCCs are ∼3 nm wide and range from 30 to 40 nm long. Based on these data, we estimate that there are 2,000–5,000 sp² carbon atoms on each PEG-HCC core. We have demonstrated the efficacy of PEG-HCCs for normalizing in vivo O₂^•− in models of traumatic brain injury with concomitant hypotension. Simultaneously, we observed normalization in NO^• levels in these experiments (26, 27). A better understanding of these materials is necessary to potentially translate these therapeutic findings to the clinic.In the present work, we evaluated antioxidant properties of PEG-HCCs. Using spin-trap EPR spectroscopy, we demonstrate that PEG-HCCs scavenge O₂^•− with high efficiency. X-ray photoelectron spectroscopy (XPS) indicates that covalent addition of ROS to the PEG-HCCs is not responsible for the observed activity. Direct measurement of O₂^•− concentration using freeze-trap EPR demonstrates that PEG-HCCs behave as catalysts, and measurements made with a Clark oxygen electrode during the reaction reveal that the rate of production of O₂ is above that expected due to self-dismutation of O₂^•− in water. An equivalent amount of H₂O₂ is also simultaneously produced. Finally, selectivity for ROS is confirmed using a hemoglobin and a pyrogallol red assay; PEG-HCCs are unreactive to both NO^• and ONOO⁻. These results clarify the fundamental processes involved in the previously observed in vivo protection against oxygen damage (26, 27).     

Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability

Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channels in the endoplasmic reticulum to modulate Ca²⁺ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-x_L activates single InsP₃R channels with a biphasic concentration dependence. The Bcl-x_L Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-x_L activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca²⁺ signaling and cell viability.The inositol trisphosphate receptors (InsP₃R) are a family of intracellular cation channels that release Ca²⁺ from the endoplasmic reticulum (ER) in response to a variety of extracellular stimuli (1). Three InsP₃R isoforms are ubiquitously expressed and regulate diverse cell processes, including cell viability (1). Activation of the channels by InsP₃ elicits changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that provide versatile signals to regulate molecular processes with high spatial and temporal fidelity (1). Regions of close proximity to mitochondria enable localized Ca²⁺ release events to be transduced to mitochondria (2, 3). Ca²⁺ released from the ER during cell stimulation modulates activities of effector molecules and is taken up by mitochondria to stimulate oxidative phosphorylation and enhance ATP production (4–6) to match energetic supply with enhanced demand. In addition, cells in vivo are constantly exposed to low levels of circulating hormones, transmitters, and growth factors that bind to plasma membrane receptors to provide a background level of cytoplasmic InsP₃ (7) that generates low-level stochastic InsP₃R-mediated localized or propagating [Ca²⁺]_i signals (8–10). Such signals also play an important role in maintenance of cellular bioenergetics (8). Nevertheless, under conditions of cell stress the close proximity of mitochondria to Ca²⁺ release sites may result in mitochondrial Ca²⁺ overload and initiate Ca²⁺-dependent forms of cell death, including necrosis and apoptosis (11–13). It has been suggested that high levels of ER Ca²⁺ (14–16) and enhanced activity of the InsP₃R (17–19) promote cell death by providing a higher quantity of released Ca²⁺ to mitochondria (3, 20, 21).Protein interactions modulate the magnitude and quality of InsP₃R-mediated [Ca²⁺]_i signals that regulate apoptosis and cell viability. Notable in this regard is the Bcl-2 protein family. Proapoptotic Bcl-2–related proteins Bax and Bak initiate cytochrome C release from mitochondria in response to diverse apoptotic stimuli, whereas antiapoptotic Bcl-2–related proteins, including Bcl-2 and Bcl-x_L, antagonize Bax/Bak by forming heterodimers that prevent their oligomerization and apoptosis initiation (22, 23). Heterodimerization is mediated by interactions of proapoptotic Bcl-2 homology 3 (BH3) domains with a hydrophobic groove on the surface of antiapoptotic Bcl-2 proteins (23) that is a therapeutic target in diseases, including cancer (22). Whereas a central feature of molecular models of apoptosis is the control of outer mitochondrial membrane permeability by Bcl-2–related proteins, a substantial body of evidence has demonstrated that these proteins localize to the ER (24, 25), bind to InsP₃Rs (26–32) and, by modulating InsP₃R-mediated Ca²⁺ release, regulate ER-mediated cell death and survival (15, 27, 32–34). Nevertheless, a unified understanding of the detailed molecular mechanisms by which Bcl-2 family proteins interact with and regulate InsP₃R channel activity is lacking. The Bcl-2 family member homolog NrZ interacts with the amino-terminal InsP₃-binding region via its helix 1 BH4 domain and inhibits Ca²⁺ release (28). Bcl-2 also interacts with the InsP₃R (26) via its BH4 domain (35), but in contrast it associates with a region in the central coupling domain (35). Whereas this interaction also inhibits Ca²⁺ release (26), Bok interacts with the channel 500 residues C-terminal to the Bcl-2 binding sequence via its BH4 domain but does not affect Ca²⁺ release (29). Conversely, the Bcl-x_L BH4 domain may lack this interaction (36). Inhibition of the Bcl-2 BH4 domain interaction with the channel enhanced InsP₃R-mediated Ca²⁺ signals and apoptosis sensitivity in white blood cells (18, 35, 37). However, it is unclear if Bcl-2 inhibits Ca²⁺ signaling directly by binding to the channel or if it acts indirectly, as a hub in a protein complex that influences channel phosphorylation (38). Conversely, we demonstrated that Bcl-x_L, Bcl-2, and Mcl-1 bind to the carboxyl (C)-terminus of all three InsP₃R isoforms, and showed that these interactions activated single InsP₃R channels and promoted InsP₃R-mediated Ca²⁺ release and apoptosis resistance (27, 31, 32). Furthermore, Bcl-x_L mediates an interaction of oncogenic K-RAS with the InsP₃R C terminus that regulates its biochemical and functional interaction and cell survival (39). However, the molecular details of the interactions of antiapoptotic protein with the InsP₃R C terminus are unknown. Furthermore, the relationship between Bcl-2 family protein binding in the coupling domain and C terminus is unclear. Thus, the mechanisms whereby Bcl-2 and Bcl-x_L affect InsP₃R activity and the effects of this modulation on cell viability remain to be determined.Here, we used single-channel electrophysiology of native ER membranes to explore the detailed mechanisms of the effects of Bcl-x_L on the InsP₃R, and the role of this interaction on cell viability. Surprisingly, our results reveal that whereas Bcl-x_L activates the channel at low concentrations, it inhibits it at higher concentrations, resulting in a biphasic response of channel activation on [Bcl-x_L]. Remarkably, the Bcl-x_L BH3 domain-binding pocket is required for both effects. Low [Bcl-x_L] activates the channel by simultaneous binding to two BH3 domain-like helices in the channel C terminus, whereas channel inhibition at high [Bcl-x_L] requires binding to only one of them and to a site previously identified as the Bcl-2 binding site in the channel-coupling domain. Disruption of these interactions diminishes cell viability. Our results provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca²⁺ signaling and cell viability.     

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

Stimulating presynaptic terminals can increase the proton concentration in synapses. Potential receptors for protons are acid-sensing ion channels (ASICs), Na⁺- and Ca²⁺-permeable channels that are activated by extracellular acidosis. Those observations suggest that protons might be a neurotransmitter. We found that presynaptic stimulation transiently reduced extracellular pH in the amygdala. The protons activated ASICs in lateral amygdala pyramidal neurons, generating excitatory postsynaptic currents. Moreover, both protons and ASICs were required for synaptic plasticity in lateral amygdala neurons. The results identify protons as a neurotransmitter, and they establish ASICs as the postsynaptic receptor. They also indicate that protons and ASICs are a neurotransmitter/receptor pair critical for amygdala-dependent learning and memory.Although homeostatic mechanisms generally maintain the brain’s extracellular pH within narrow limits, neural activity can induce transient and localized pH fluctuations. For example, acidification may occur when synaptic vesicles, which have a pH of ∼5.2–5.7 (1–3), release their contents into the synapse. Studies of mammalian cone photoreceptors showed that synaptic vesicle exocytosis rapidly reduced synaptic cleft pH by an estimated 0.2–0.6 units (4–6). Transient synaptic cleft acidification also occurred with GABAergic transmission (7). Some, but not all, studies also reported that high-frequency stimulation (HFS) transiently acidified hippocampal brain slices, likely as a result of the release of synaptic vesicle contents (8, 9). Neurotransmission also induces a slower, more prolonged alkalinization (10, 11). In addition to release of synaptic vesicle protons, neuronal and glial H⁺ and HCO₃⁻ transporters, channels, H⁺-ATPases, and metabolism might influence extracellular pH (10–12).ASICs are potential targets of reduced extracellular pH. ASICs are Na⁺-permeable and, to a lesser extent, Ca²⁺-permeable channels that are activated by extracellular acidosis (13–19). In the brain, ASICs consist of homotrimeric and heterotrimeric complexes of ASIC1a, ASIC2a, and ASIC2b. The ASIC1a subunit is required for acid-activation in the physiological range (>pH 5.0) (20, 21). Several observations indicate that ASIC are located postsynaptically. ASICs are located on dendritic spines. Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20, 22–24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20, 24–29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20, 24, 30).Although these observations raised the possibility that protons might be a neurotransmitter, postsynaptic ASIC currents have not been detected in cultured hippocampal neurons (31, 32), and whether localized pH transients might play a signaling role in neuronal communication remains unclear. In previous studies of hippocampal brain slices, extracellular field potential recordings suggested impaired hippocampal long-term potentiation (LTP) in ASIC1a^−/− mice (20), although another study did not detect an effect of ASIC1a (33). Another study using microisland cultures of hippocampal neurons suggested that the probability of neurotransmitter release increased in ASIC1a^−/− mice (32).Here, we tested the hypothesis that protons are a neurotransmitter and that ASICs are the receptor. Criteria to identify substances as neurotransmitters have been proposed (34). Beg and colleagues (35) used these criteria to conclude that protons are a transmitter released from Caenorhabditis elegans intestine to cause muscle contraction. Key questions about whether protons meet criteria for a neurotransmitter are: Does presynaptic stimulation increase the extracellular proton concentration? Do protons activate currents in postsynaptic cells? Can exogenously applied protons reproduce effects of endogenous protons? What is the postsynaptic proton receptor? We studied lateral amygdala brain slices because amygdala-dependent fear-related behavior depends on a pH reduction (36). In addition, ASICs are abundantly expressed there, and ASIC1a^−/− mice have impaired fear-like behavior (36–38).     

Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms

Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na⁺ (Na_V) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial Na_V channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.General anesthetic agents have been in use for more than 160 y. However, we still understand relatively little about their mechanisms of action, which greatly limits our ability to design safer and more effective general anesthetic agents. Ion channels of the central nervous system are known to be key targets of general anesthetic agents, as their modulation can account for the endpoints and side effects of general anesthesia (1–4). Many families of ion channels are modulated by general anesthetic agents, including ligand-gated, voltage-gated, and nongated ion channels (2, 5–7). Mammalian voltage-gated Na⁺ (Na_V) channels, which mediate the upstroke of the action potential, are regulated by numerous inhaled general anesthetic agents (8–14), which generally cause inhibition. Previous work showed that inhaled general anesthetic agents, including sevoflurane, isoflurane, desflurane, and halothane, mediate inhibition by increasing the rate of Na⁺ channel inactivation, hyperpolarizing steady-state inactivation, and slowing recovery from inactivation (11, 15–18). Inhibition of presynaptic Na_V channels in the spinal cord is proposed to lead to inhibition of neurotransmitter release, facilitating immobilization—one of the endpoints of general anesthesia (14, 19, 20). Despite the importance of Na_V channels as general anesthetic targets, little is known about interaction sites or the mechanisms of action.What is known about anesthetic sites in Na_V channels comes primarily from the local anesthetic field. Local anesthetic agent binding to Na_V channels is well characterized. These amphiphilic drugs enter the channel pore from the intracellular side, causing open-channel block (21). Investigating molecular mechanisms of mammalian Na_V channel modulation by general anesthetic agents has been complicated by the lack of high-resolution structures of these channels as a result of their large size and pseudotetrameric organization. However, the recent discovery of the smaller, tetrameric bacterial Na⁺ channel family has provided an invaluable tool to characterize the structural features of Na_V channels and investigate their interactions with general anesthetic agents at the molecular level (22, 23). Several bacterial Na⁺ channels have been crystallized (24–27). These channels have a classical domain structure in which helices S1–S4 form the voltage sensor domain (VSD), S5 and S6 form the pore, and the S4–S5 linker connects the voltage sensor to the pore domain. One notable structural feature is the presence of “fenestrations” or hydrophobic tunnels through the pore domain (24).Although crystal structures are not yet reported, the bacterial Na⁺ channel NaChBac has been extensively characterized by electrophysiology (22, 28–36). Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15, 37). These anesthetic agents reduce peak current and accelerate current decay, making it conceivable that local and general anesthetic agents could share a site of action in NaChBac. The local anesthetic binding site identified in the central cavity of the mammalian Na_V1.2 channel, which mediates open channel block, is partially conserved in NaChBac (37, 38). A recent molecular dynamics (MD) modeling study found that isoflurane, which inhibits NaChBac (15), interacts with multiple regions of this channel, including the pore, the selectivity filter, and the S4–S5 linker/S6 interface (39). Although the importance of these interactions on the modulation of mammalian Na_V channels remains to be determined, the available data indicate that NaChBac is currently one of the best starting points to investigate the mechanisms of action of sevoflurane.Here, we investigated NaChBac to gain structural insight into the mechanisms of inhaled anesthetic modulation of Na_V channels. The focus of this work is sevoflurane because this anesthetic is commonly used in clinical settings and is a known inhibitor of several mammalian Na_V channels (Na_V 1.4, 1.7, and 1.8) (11, 13). A three-pronged approach incorporating MD simulation, whole-cell patch-clamp electrophysiology, and kinetic modeling suggests that sevoflurane acts on multiple sites to alter gating and permeation. Whereas the effect on gating results from modulating activation and inactivation gating at low concentrations (0.2 mM), the permeation effect is apparent at high concentrations (2 mM) and results from open channel block (2 mM). Although the net inhibitory effect of these multisite interactions is consistent with anesthetic-induced reduction of neuronal firing, general anesthesia does not simply result from a global reduction in firing. General anesthesia depends on complex mechanisms throughout the brain, which include increases and decreases in firing (3). Thus, precisely how Na⁺ channel activation by sevoflurane fits into the global effects of anesthesia remains to be seen. The present work helps elucidate the molecular mechanism of sevoflurane action on Na_V channels.     

Tumor-derived hydrogen sulfide,produced by cystathionine-β-synthase,stimulates bioenergetics,cell proliferation,and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H₂S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H₂S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H₂S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H₂S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H₂S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H₂S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H₂S as a tumor growth factor and anticancer drug target.The endogenous gasotransmitter hydrogen sulfide (H₂S) is a stimulator of vasorelaxation (1–3), angiogenesis (3–5), and cellular bioenergetics (6, 7). H₂S is generated from l-cysteine by two pyridoxal-5′-phospate–dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) (8–10). H₂S exerts its cellular actions via multiple mechanisms (1–15), including activation of potassium channels (1–3), stimulation of kinase pathways (4, 11, 12), and inhibition of phosphodiesterases (3, 15).Both ATP generation and angiogenesis are vital factors for the growth and proliferation of tumors (16–19). Using human colon cancer tissues and cancer-derived cell lines, we have now conducted a series of in vitro and in vivo studies to explore whether endogenous, tumor cell-derived H₂S plays a role as a tumor-derived survival factor. The results show that CBS is selectively overexpressed in colon cancer, and that H₂S produced by it serves to maintain the tumor''s cellular bioenergetics and to promote tumor angiogenesis.     

High-pressure study of lithium amidoborane using Raman spectroscopy and insight into dihydrogen bonding absence

One of the major obstacles to the use of hydrogen as an energy carrier is the lack of proper hydrogen storage material. Lithium amidoborane has attracted significant attention as hydrogen storage material. It releases ∼10.9 wt% hydrogen, which is beyond the Department of Energy target, at remarkably low temperature (∼90 °C) without borazine emission. It is essential to study the bonding behavior of this potential material to improve its dehydrogenation behavior further and also to make rehydrogenation possible. We have studied the high-pressure behavior of lithium amidoborane in a diamond anvil cell using in situ Raman spectroscopy. We have discovered that there is no dihydrogen bonding in this material, as the N—H stretching modes do not show redshift with pressure. The absence of the dihydrogen bonding in this material is an interesting phenomenon, as the dihydrogen bonding is the dominant bonding feature in its parent compound ammonia borane. This observation may provide guidance to the improvement of the hydrogen storage properties of this potential material and to design new material for hydrogen storage application. Also two phase transitions were found at high pressure at 3.9 and 12.7 GPa, which are characterized by sequential changes of Raman modes.Hydrogen economy has been considered as potentially efficient and environmental friendly alternative energy solution (1). However, one of the most important scientific and technical challenges facing the “hydrogen economy” is the development of safe and economically viable on-board hydrogen storage for fuel cell applications, especially to the transportation sector. Ammonia borane (BH₃NH₃), a solid state hydrogen storage material, possesses exceptionally high hydrogen content (19.6 wt%) and in particular, it contains a unique combination of protonic and hydridic hydrogen, and on this basis, offers new opportunities for developing a practical source for generating molecular dihydrogen (2–5). Stepwise release of H₂ takes place through thermolysis of ammonia borane, yielding one-third of its total hydrogen content (6.5 wt%) in each heating step, along with emission of toxic borazine (6–8). Recently, research interests are focusing on how to improve discharge of H₂ from ammonia borane, including lowering the dehydrogenation temperature and enhancing hydrogen release rate using different techniques, e.g., nanoscaffolds (9), ionic liquids (10), acid catalysis (11), base metal catalyst (12), or transition metal catalysts (13, 14). More recently, significant attention is given to chemical modification of ammonia borane through substitution of one of the protonic hydrogen atoms with an alkali or alkaline–earth element (15–21). Lithium amidoborane (LiNH₂BH₃) has been successfully synthesized by ball milling LiH with NH₃BH₃ (15–18). One of the driving forces suggested for the formation of LiNH₂BH₃ is the chemical potential of the protonic H^δ+ in NH₃ and the hydridic H^δ− in alkali metal hydrides making them tend to combine, producing H₂ + LiNH₂BH₃. LiNH₂BH₃ exhibits significantly different and improved dehydrogenation characteristics from its parent compound ammonia borane. It releases more than 10 wt% of hydrogen at around 90 °C without borazine emission. Also, the dehydrogenation process of lithium amidoborane is much less exothermic (∼3–5 kJmole⁻¹ H₂) (15–17) than that of NH₃BH₃ (∼22.5 kJmole⁻¹ H₂) (6–8), which greatly enhances the search for suitable regeneration routes (prerequisite for a hydrogen storage material). Although the rationale behind the improved dehydrogenation behavior is still unclear, these improved property modifications evidently originate from the substitution of one H in the NH₃ group by the more electron-donating Li, which exerts influences on the bonding characteristics, especially on the dihydrogen bonding, which is one of the characteristic bonds of ammonia borane (15). So, it is essential to understand details about the bonding behavior of this potential material.High-pressure study of molecular crystals can provide unique insight into the intermolecular bonding forces, such as hydrogen bonding and phase stability in hydrogen storage materials and thus provides insight into the improvement of design (22–30). For instance, Raman spectroscopic study of ammonia borane at high pressure provided insight about its phase transition behavior and the presence of dihydrogen bonding in its structure (25–30). We have investigated LiNH₂BH₃ at high pressure using Raman spectroscopy. We have found that, other than in NH₃BH₃, dihydrogen bonding is absent in lithium amidoborane structure and LiNH₂BH₃ shows two phase transitions at high pressure.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.