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.     

Hybrid-fuel bacterial flagellar motors in Escherichia coli

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H⁺ or Na⁺ ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H⁺- and Na⁺-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na⁺ concentration ([Na⁺]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na⁺ and H⁺ stator components expressed at high and medium levels, respectively, Na⁺ stator units dominate at high [Na⁺] and are replaced by H⁺ units when Na⁺ is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H⁺ and Na⁺ stator components sharing a common rotor.Molecular motors are tiny machines that perform a wide range of functions in living cells. Typically each motor generates mechanical work using a specific chemical or electrochemical energy source. Linear motors such as kinesin on microtubules or myosin on actin filaments and rotary motors such as F₁-ATPase, the soluble part of ATP-synthase, run on ATP, whereas the rotary bacterial flagellar motor embedded in the bacterial cell envelope is driven by the flux of ions across the cytoplasmic membrane (1–4). Coupling ions are known to be either protons (H⁺) or sodium ions (Na⁺) (5, 6).The bacterial flagellar motor consists of a rotor ∼50 nm in diameter surrounded by multiple stator units (7–10). Each unit contains two types of membrane proteins forming ion channels: MotA and MotB in H⁺ motors in neutrophiles (e.g., Escherichia coli and Salmonella) and PomA and PomB in Na⁺ motors in alkalophiles and Vibrio species (e.g., Vibrio alginolyticus) (1, 11). Multiple units interact with the rotor to generate torque independently in a working motor (9, 10, 12, 13). The structure and function of H⁺ and Na⁺ motors are very similar, to the extent that several functional chimeric motors have been made containing different mixtures of H⁺- and Na⁺-motor components (11). One such motor that runs on Na⁺ in E. coli combines the rotor of the H⁺-driven E. coli motor with the chimeric stator unit PomA/PotB, containing PomA from V. alginolyticus and a fusion protein between MotB from E. coli and PomB from V. alginolyticus (14).In most flagellated bacteria, motors are driven by ion-specific rotor–stator combinations. However, some species (e.g., Bacillus subtilis and Shewanella oneidensis) combine a single set of rotor genes with multiple sets of stator genes encoding both H⁺ and Na⁺ stator proteins, and it has been speculated that these stator components may interact with the rotor simultaneously, allowing a single motor to use both H⁺ and Na⁺. An appealing hypothesis that the mixture of stator components is controlled dynamically depending on the environment has arisen from the observation that the localization of both stator components depends upon Na⁺ (15). However, despite some experimental effort there is as yet no direct evidence of both H⁺ and Na⁺ stator units interacting with the same rotor (16).The rotation of single flagellar motors can be monitored in real time by light microscopy of polystyrene beads (diameter ∼1 μm) attached to truncated flagellar filaments (17). Under these conditions, the E. coli motor torque and speed are proportional to the number of stator units in both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB (17–19) motors. The maximum number of units that can work simultaneously in a single motor has been shown to be at least 11 by “resurrection” experiments, in which newly produced functional units lead to restoration of motor rotation in discrete speed increments in an E. coli strain lacking functional stator proteins (19). Stator units are not fixed permanently in a motor: Each dissociates from the motor with a typical rate of ∼2 min⁻¹, exchanging between the motor and a pool of diffusing units in the cytoplasmic membrane (20). Removal of the relevant ion gradient inactivates both H⁺ and Na⁺ stator units, most likely leading to dissociation from the motor into the membrane pool (2, 21, 22).Here we demonstrate a hybrid-fuel motor containing both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB stator components, sharing a common rotor in E. coli. We control the expression level of each stator type by induced expression from plasmids, and the affinity of Na⁺-driven stator units for the motor by external [Na⁺]. Units of each type compete for spaces around the rotor, and the motor torque is simply the sum of the independent contributions, with no evidence of direct interaction between units. Thus, we demonstrate the possibility of modularity in the E. coli flagellar motor, with ion selectivity determined by the choice of stator modules interacting with a common rotor. Our artificial hybrid motor demonstrates that species with multiple types of stator gene and a single set of rotor genes could contain natural hybrid motors that work on a similar principle (15, 16, 23).     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na⁺-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na⁺/leucine transporter LeuT, our results suggest that Na⁺ binding at the conserved second Na⁺ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.Secondary active transporters harness the energy of ion gradients to power the uphill movement of solutes across membranes. Mitchell (1) and others (2, 3) proposed and elaborated “alternating access” mechanisms wherein the transporter transitions between two conformational states that alternately expose the substrate binding site to the two sides of the membrane. The LeuT class of ion-coupled symporters consists of functionally distinct transporters that share a conserved scaffold of two sets of five transmembrane helices related by twofold symmetry around an axis nearly parallel to the membrane (4). Ions and substrates are bound near the middle of the membrane stabilized by electrostatic interactions with unwound regions of transmembrane helix (TM) 1 and often TM6 (4). The recurrence of this fold in transporters that play critical roles in fundamental physiological processes (5, 6) has spurred intense interest in defining the principles of alternating access.Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7–11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4, 7, 9–12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outward-facing, or occluded on the basis of the accessibility of the substrate binding site (7–11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).Mhp1, an Na⁺-coupled symporter of benzyl-hydantoin (BH) from Microbacterium liquefaciens, was the first LeuT-fold member to be characterized by crystal structures purported to represent outward-facing, inward-facing, and outward-facing/occluded conformations of an alternating access cycle (8, 15). In these structures, solvent access to ligand-binding sites is defined by the relative orientation between a 4-helix bundle motif and a 4-helix scaffold motif (8). In Mhp1, alternating access between inward- and outward-facing conformations, was predicted from a computational analysis based on the inverted repeat symmetry of the LeuT fold and is referred to as the rocking-bundle model (16). The conservation of the inverted symmetry prompted proposal of the rocking-bundle mechanism as a general model for LeuT-fold transporters (16). Subsequent crystal structures of other LeuT-fold transporters (7, 9, 10) tempered this prediction because the diversity of the structural rearrangements implicit in these structures is seemingly inconsistent with a conserved conformational cycle.Another outstanding question pertains to the ion-coupling mechanism and the driving force of conformational changes. The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na⁺ ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) simulations (20, 21) and electron paramagnetic resonance (EPR) analysis (13, 22) of LeuT demonstrated that Na⁺ binding favors an outward-facing conformation although it is unclear which Na⁺ site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na⁺ in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.Here, we used site-directed spin labeling (SDSL) (24) and double electron-electron resonance (DEER) spectroscopy (25) to elucidate the conformational changes underlying alternating access in Mhp1 and define the role of ion and substrate binding in driving transition between conformations. This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13, 26–32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra- and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward- and outward-facing conformations. A major finding is that this transition is driven by substrate but not Na⁺ binding. Although the amplitudes of the observed distance changes are in overall agreement with the rocking-bundle model deduced from the crystal structures of Mhp1 (8, 15) and predicted computationally (16), we present evidence that relative movement of bundle and scaffold deviate from strict rigid body. Comparative analysis of LeuT and Mhp1 alternating access reveal how the conserved LeuT fold harnesses the energy of the Na⁺ gradient through two distinct coupling mechanisms and supports divergent conformational cycles to effect substrate binding and release.     

From the Cover: Widespread convergence in toxin resistance by predictable molecular evolution

The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na⁺/K⁺-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na⁺/K⁺-ATPase H1–H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na⁺/K⁺-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.Convergent evolution is the process by which phenotypic similarities evolve independently among disparate species (1–3). Although convergence is sometimes distinguished from parallel evolution, both are part of a continuum, which often makes attempting to distinguish between them problematic and potentially misleading (4); here, we simply use the term “convergence” throughout. Convergence has strong bearing on a fundamental and heated debate on the predictability of evolution. Epitomized by the writings of Gould (5) and Conway Morris (6), this debate centers on whether evolution is stochastic and unpredictable (5) or subject to constraints that limit the available options for evolution, resulting in frequent convergence and a degree of predictability (6). However, evidence of convergence at the genetic level, where similar molecular changes confer the same change in phenotype, is currently limited to only a few taxonomically restricted examples (7–11).Cardiac glycosides offer an ideal model system to investigate the extent to which evolution can be constrained to predictable changes throughout the animal kingdom. These organic compounds are highly toxic molecules that inhibit the sodium-potassium-pump (Na⁺/K⁺-ATPase), which disrupts ion transport and thereby perturbs membrane potentials, often resulting in lethal cardiotoxicity (12, 13). Cardiac glycosides are produced independently by a number of plants and bufonid toads as secondary metabolites and are used for defense against natural enemies. For example, milkweed, foxglove, and oleander plants produce “cardenolides” (e.g., ouabain) that protect against herbivorous insects (13), whereas bufonid toads secrete structurally and functionally similar defensive compounds, “bufotoxins” (e.g., bufalin), from their parotoid glands (14).Despite the toxicity of these molecules, natural resistance exists in many different herbivores and predators and is predominately mediated by molecular alterations to the H1–H2 extracellular domain of the Na⁺/K⁺-ATPase, resulting in target-site insensitivity to cardiac glycosides (9, 15, 16). For example, we recently showed that two amino acid replacements in the Na⁺/K⁺-ATPase α3 subunit (previously denoted α1) are responsible for a 3,000-fold increased resistance to bufalin in toad-feeding African and Asian varanid lizards, compared with toad toxin-susceptible Australian varanids (17). However, until now, comparisons of the molecular basis of resistance and mechanisms of action across the diverse range of resistant taxa reported (9, 15, 17, 18) have been lacking. Here we present an analysis of convergent molecular changes to the Na⁺/K⁺-ATPase H1–H2 domain in cardiac glycoside-resistant invertebrates and vertebrates. Our results support the view that molecular evolution in the animal kingdom can be heavily constrained, resulting in convergent processes canalizing evolution along highly predictable pathways.     

Nonfunctional NaV1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura. Mutations causing FHM type 3 have been identified in SCN1A, the gene encoding the Na_v1.1 Na⁺ channel, which is also a major target of epileptogenic mutations and is particularly important for the excitability of GABAergic neurons. However, functional studies of Na_V1.1 FHM mutations have generated controversial results. In particular, it has been shown that the Na_V1.1-L1649Q mutant is nonfunctional when expressed in a human cell line because of impaired plasma membrane expression, similarly to Na_V1.1 mutants that cause severe epilepsy, but we have observed gain-of-function effects for other Na_V1.1 FHM mutants. Here we show that Na_V1.1-L1649Q is nonfunctional because of folding defects that are rescuable by incubation at lower temperatures or coexpression of interacting proteins, and that a partial rescue is sufficient for inducing an overall gain of function because of the modifications in gating properties. Strikingly, when expressed in neurons, the mutant was partially rescued and was a constitutive gain of function. A computational model showed that 35% rescue can be sufficient for inducing gain of function. Interestingly, previously described folding-defective epileptogenic Na_V1.1 mutants show loss of function also when rescued. Our results are consistent with gain of function as the functional effect of Na_V1.1 FHM mutations and hyperexcitability of GABAergic neurons as the pathomechanism of FHM type 3.Epilepsy and migraine are common neurologic disorders that may have pathophysiological links (1–3). Mutations have been identified for some rare types of epilepsy and migraine (1, 4–6), opening a window for investigating their pathogenic mechanisms, which may provide useful information also about more common forms. The Na⁺ channel α subunit Na_V1.1, encoded by the SCN1A gene, is the target of hundreds of epileptogenic mutations (7–9), and of mutations causing familial hemiplegic migraine type 3 (FHM-3), a rare subtype of migraine with aura characterized by hemiplegia during the attacks, which can also be caused by mutations of Ca_V2.1 Ca²⁺ channels and the α2 subunit of the Na⁺/K⁺ ATPase (FHM types 1 and 2) (1, 6). The results of most studies suggest that epileptogenic Na_V1.1 mutations cause variable degrees of loss of function of Na_V1.1, leading to reduced Na⁺ current and excitability in GABAergic neurons, and resulting in decreased inhibition in neuronal networks (10–14). The most severe phenotypes (e.g., Dravet syndrome, an extremely severe epileptic encephalopathy) are in general caused by mutations that induce complete Na_V1.1 loss of function, leading to haploinsufficiency (15). Thus, it has been hypothesized that a more severe loss of function would cause more severe epilepsy (8). Functional studies of Na_V1.1 FHM mutations have generated more confusing results (1). For instance, we have reported gain-of-function effects for the mutant Q1489K causing pure FHM (16), and modulable gain-/loss-of-function effects for the mutant T1174S associated with FHM or mild epilepsy in different branches of the family (17). Overall, our results are consistent with a gain of function of Na_V1.1 as the cause of FHM, which might induce cortical spreading depression (CSD), a probable pathomechanism of migraine, because of hyperexcitability of GABAergic interneurons (16). However, a study has reported loss of function for FHM hNa_V1.1 mutants expressed in the human cell line tsA-201—in particular, complete loss of function for the L1649Q mutant because of lack of cell surface expression (18). L1649Q has been identified in a four-generation family with eight members presenting with FHM, without epilepsy or other neurologic symptoms (19); this is a puzzling result more consistent with a phenotype of severe epilepsy (7, 8). We have found that Na_V1.1 epileptogenic mutations can induce loss of function by causing folding defects (20), which can be partially rescued by incubation of the transfected cells at lower temperatures (≤30 °C) or by molecular interactions (21, 22), as recently confirmed also for other epileptogenic Na_V1.1 mutants (23, 24). We report here that L1649Q is a folding-defective mutant that, when partially rescued, is characterized by an overall gain of function, consistent with our hypothesis of FHM type 3 pathomechanism (16).     

Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na⁺ symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na⁺-dependent. The transport activity of R262 mutants increased by 30–40% in the presence of a membrane potential, indicating substrate/Na⁺ cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1′ helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na⁺-coupled substrate symporters. We propose that a similar mechanism is operative in other Na⁺/H⁺-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.The carnitine/γ-butyrobetaine antiporter CaiT belongs to the betaine/carnitine/choline transporter family of secondary transporters that transfer substrates containing a quaternary ammonium group (1, 2) in and out of the cell. In Escherichia coli and other enterobacteria, such as Proteus mirabilis, carnitine is taken up by CaiT and converted to γ-butyrobetaine via the reaction intermediate crotonobetaine (3–5), which serves as an external electron acceptor under anaerobic growth conditions (4). Biochemical studies of E. coli CaiT (EcCaiT) have shown that it is a constitutively active, Na⁺/H⁺-independent antiporter (6).Crystal structures of CaiT from P. mirabilis (PmCaiT) and E. coli (EcCaiT) were recently determined with and without bound substrate (7, 8). These structures revealed a trimeric assembly of CaiT, as previously found (9). The protein was in an inward-facing conformation with two substrate molecules bound per EcCaiT monomer: one in the central transport site and another in an external binding site (7). Fluorescent binding assays with the protein reconstituted into liposomes indicated that substrate binding was cooperative. This suggested a regulatory role for the external binding site, which was proposed to increase substrate affinity and initiate substrate transport (7). Strikingly, the crystal structures revealed that CaiT adopts a fold similar to that of the LeuT-type transporters (7, 10, 11). This places it in the amino acid–polyamine-organocation (APC) superfamily (12), which shares a conserved architecture of two inverted repeats of five transmembrane (TM) helices each, implying common mechanistic principles. Among the APC transporters, the leucine transporter LeuT from the neurotransmitter/sodium symporter family (10), the betaine transporter BetP from the betaine/choline/carnitine transporter family (13), the benzyl-hydantoin transporter Mhp1 of the nucleobase/cation symport 1 family, and the Na⁺/galactose symporter vSGLT of the solute/sodium symporter family are substrate/sodium symporters (14, 15). Although the substrate to sodium stoichiometry varies for each Na⁺-dependent LeuT-type transporter, most of them possess a conserved sodium-binding site (Na2 site) at which the binding and dissociation of a sodium ion is proposed to facilitate structural changes that lead to substrate transport (16–18). Although an additional sodium-binding site (Na1 site) exists in transporters such as LeuT and BetP, the position or even the presence of this site is not strictly conserved among the Na⁺-dependent LeuT-type transporters (14, 15, 19–21).A small number of LeuT-type transporters, namely, AdiC (arginine/agmatine antiporter), ApcT (broad-specificity amino acid transporter), and CaiT, are Na⁺-independent (6, 22–25). The crystal structure of ApcT revealed that a lysine residue (K158) occupies a position equivalent to the Na2 site in LeuT. This lysine is proposed to undergo a protonation/deprotonation event that leads to conformational changes facilitating substrate transport (25). In CaiT, a methionine residue (M331) occupies a position equivalent to Na1 in LeuT, whereas a positively charged arginine residue (R262) occupies the Na2 site (7). Previously, we have shown that mutating M331 reduces transport activity but does not induce Na⁺ dependence in CaiT (7). Here we report that point mutations of R262 render CaiT inactive. Strikingly, the transport activity was partially restored by the addition of sodium, thus making these mutants Na⁺-dependent. Unlike wild-type, the transport activity of R262 mutants increased by 30–40% when a membrane potential was applied, suggesting that Na⁺ and substrate were cotransported. To find out whether and how the mutation affects the Na2 site, we determined the crystal structure of CaiT R262E. Although we did not find any major changes in the mutant protein, comparison with the substrate-bound EcCaiT wild-type protein revealed that the γ-butyrobetaine substrate adopts a different orientation at the central binding site, in which it directly interacts with the unwound part of TM1′. (To make the nomenclature consistent, we adopt the same helix numbering as in LeuT. Because CaiT has two extra helices at the N terminus in comparison with LeuT, TM3 in CaiT corresponds to TM1 in LeuT and is denoted as TM1′, and the remainder of the CaiT helices follow suit.) Because R262 is known to play a role in stabilizing the unwound part of TM1′ (7), we propose that R262 is crucial for substrate binding, similar to Na⁺ in the Na2 site of LeuT and BetP (17, 19). Indeed, our fluorescent binding assays using R262 mutants showed markedly decreased substrate affinity. Modeling CaiT in various conformations with BetP as a template revealed that R262 undergoes an oscillatory movement, contributing to different hydrogen bond networks in each conformation. We suggest that this movement of the positively charged R262 sidechain in CaiT mimics Na⁺ binding/unbinding in Na⁺-dependent LeuT-type transporters and plays a central role in the transport mechanism.     

Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H⁺ along with extracellular superoxide, and the intracellular H⁺ must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na⁺/H⁺ exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia–reperfusion was likewise prevented by inhibiting Na⁺/H⁺ exchange and by reduced expression of the Na⁺/H⁺ exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na⁺/H⁺ exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.Many metabolic processes generate hydrogen ions, and hydrogen ions in turn influence cell metabolism and survival (1). Cerebral ischemia in particular produces acidosis of variable degree, depending upon blood glucose levels, degree of blood flow reduction, and other factors. Severe acidosis, below pH 6.4, exacerbates ischemic injury (2) by mechanisms involving protein denaturation, acid-sensing calcium channels, and release of ferrous iron (3–5). Conversely, lesser degrees of acidosis, in the range of 7.0–6.5, reduce both ischemic injury (6) and glutamate-induced neuronal death (7). These neuroprotective effects have been attributed to an inhibitory effect of hydrogen ions on NMDA receptor activation (8–10), but a causal link has not been demonstrated.Excessive activation of N-methyl-D-aspartate (NMDA) type glutamate receptors leads to excitotoxic cell death in stroke and other neurological disorders (11, 12). Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic cell death (13–19). NADPH oxidase exists as several isoforms, of which NOX2 is the one most abundantly expressed in CNS neurons. NOX2 is also the isoform most abundantly expressed in phagocytes, microglia, and other immune cells, in which its regulation and function have been extensively characterized (20). NOX2 is composed of three cytosolic subunits, p47^phox, p67^phox, and p40^phox, which when phosphorylated bind with two membrane-bound subunits, p22^phox and gp91^phox (the catalytic unit), to form an active transmembrane enzyme complex. The transmembrane complex generates superoxide in the extracellular space and hydrogen ions in the intracellular space: 2O₂ + NADPH → 2O₂⁻ + NADP⁺ + H⁺. In immune cells, H⁺ concentration influences the phosphorylation status of the NOX2 p47^phox subunit, and the H⁺ generated by NOX2 must be transferred to the extracellular space to sustain NOX2 activity (21–24).Together, the pH sensitivity of NOX2 and the role of NOX2 in NMDA receptor-mediated cell death suggest the possibility that reduced intracellular pH might limit neurotoxicity by dissociating NMDA receptor activation from superoxide production. Findings presented here confirm that both the superoxide production and cell death resulting from neuronal NMDA receptor activation are highly pH sensitive. We show that neurons use Na⁺/H⁺ exchange as a major route of proton efflux during NOX2 activation, and either genetic or pharmacologic inhibition of neuronal Na⁺/H⁺ exchange prevent both excitotoxic superoxide production and cell death.     

On the principle of ion selectivity in Na+/H+-coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na⁺ or H⁺, thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na⁺/H⁺ selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H⁺ selective, although not sufficiently to be functionally coupled to H⁺, owing to the large excess of Na⁺ in physiological settings. The functional Na⁺ specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H⁺ selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na⁺- and H⁺-coupled systems in other families of membrane transporters and enzymes.Gradients in the electrochemical potential of H⁺ or Na⁺ across biological membranes sustain a wide range of essential cellular process. The resulting proton or sodium motive forces (pmf, smf) are the predominant energy source for secondary-active membrane transporters, which mediate the uptake of many substances required by the cell (1–3), and also enable pathogenic bacteria to protect themselves from human-made antibiotics and other toxic compounds (4–6). Downhill membrane permeation of Na⁺ and H⁺ across the membrane also powers the ATP synthase (7), which produces most of cellular ATP, and energizes the rotation of bacterial flagella (8). Thus, the importance of this mode of energy transduction in cells cannot be overstated. Nevertheless, little is known about the factors that control and modulate the specificity for Na⁺ or H⁺ in most of these processes.It seems clear, although, that there is no correlation between function type and ion specificity; that is, the same process in different species can be coupled to either Na⁺ or H⁺ (2, 5–7, 9–11). Organism-specific environmental factors, such as temperature or pH, also do not provide a consistent rationale; for example, ATP synthases from thermoalkaliphilic bacteria use a H⁺ gradient despite the scarcity of H⁺ and the potentially greater degree of H⁺ leakage across the membrane at high temperatures, compared with Na⁺ (12, 13). Indeed, pmf- and smf-driven systems are often found within the same organism, and sometimes with the same or similar function; for example, malate uptake in Bacillus subtilis is mediated by the H⁺-coupled symporter CimH (14) and by the Na⁺-coupled MaeN (15). Moreover, specific membrane transporters and enzymes are sometimes coupled to both Na⁺ and H⁺, either concurrently (using multiple binding sites), such as the multidrug efflux pump NorM of Vibrio cholera (16) and the Methanosarcina acetivorans ATP synthase (17), or alternately (using a single binding site), such as the Escherichia coli melibiose permease (18) and some membrane-integral pyrophosphatases (19).At the molecular level, the architecture of pmf- and smf-driven membrane proteins within the same family has consistently been found to be largely conserved (20–28). Thus, it appears as if the Na⁺ or H⁺ specificity of these systems is dictated by localized variations in their amino acid sequence, rather than by major structural or mechanistic adaptations. This conclusion leads to an intriguing dilemma. Under most physiological conditions, the concentration of Na⁺ exceeds that of protons by many orders of magnitude (for example, a millionfold in mitochondria, or a billionfold in alkaline environments). Thus, in membrane-protein families with members that are driven by the smf or the pmf, the latter must have evolved amino acid adaptions that result in an extreme H⁺ selectivity, so as to counter the large Na⁺ concentration excess. Conversely, specific coupling to the smf would not actually require a strong Na⁺ selectivity; weak H⁺ selectivity or nonselectivity would be sufficient for Na⁺-coupling, physiologically. How can variation changes at the amino acid level result in such extreme variations in the H⁺ selectivity of a protein structure, spanning 10 or more orders of magnitude?In previous theoretical studies, we have addressed this question for the family of ion-motive ATPases (17, 29, 30), which comprises eukaryotic and prokaryotic ATP synthases, as well as vacuolar ion pumps. In these multicomponent enzymes, a membrane-embedded substructure known as the rotor ring can revolve around its axis, relative to the rest of the protein’s membrane domain. This rotational motion enables the ring to capture Na⁺ or H⁺ ions as they enter the protein via an access channel, and to shuttle them to a separate exit channel, in a sequential manner. Because the entry and exit channels are not colinear, there is a strict correspondence between the direction of ion permeation and the sense of rotation of the ring (which in turn determines the type of activity of the catalytic sector of the enzyme, i.e., ATP synthesis or hydrolysis).The principle of ion selectivity emerging from the abovementioned computational studies posits that rotor rings are universally H⁺ selective, owing to the fact that ion binding is consistently mediated by a conserved carboxylic side chain (the intrinsic H⁺/Na⁺ selectivity of a carboxylic group in solution is 10,000-fold), and that this inherent H⁺ selectivity is enhanced or suppressed by multiple orders of magnitude, depending on the balance between hydrophobic and polar groups lining the ion-binding sites (aside from the key carboxyl side chain); geometric factors may additionally fine-tune the selectivity of these sites, for a given chemical composition.In this study, we challenge the validity of this theory through experimental measurements and further computational analyses. Specifically, we set out to experimentally determine the thermodynamic ion selectivity of a rotor ring from a Na⁺-driven ATP synthase, and to compare this ring with others physiologically driven by either Na⁺ or H⁺. We find that the results of this analysis are qualitatively and quantitatively consistent with the theory of ion selectivity proposed previously, and provide novel insights into the influence of conformational factors.     

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).     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2^−/− mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.Due to its unique chemistry, the redox-active metal ion copper (Cu) is an essential element for human growth and development (1–3). Defects in Cu metabolism are associated with pathologies that include Alzheimer’s disease, peripheral neuropathy, anemia, neutropenia, cardiomyopathy, Menkes disease, and Wilson’s disease (4–9). Although many of the components responsible for Cu uptake, intracellular distribution, detoxification, and efflux have been identified, the mechanisms by which these proteins are regulated are not well understood.The copper transporter 1 (Ctr1) protein is a high-affinity Cu⁺ transporter that functions in copper accumulation in organisms ranging from yeast to mammals (10–16). In mammals Ctr1 localizes to both the plasma membrane and to intracellular vesicles (17–19). Mice bearing a systemic Ctr1 deletion fail to survive gestation, whereas tissue-specific ablation of Ctr1 in the intestinal epithelium, liver, or heart cause a range of phenotypes that include peripheral Cu deficiency, hepatic iron accumulation, and lethal cardiac hypertrophy, respectively (20–24). Moreover, both yeast and mammalian Ctr1 function in acquisition of the chemotherapeutic agent cisplatin (25–29) and Ctr1 expression levels have been correlated to the efficacy of chemotherapy and patient survival (30). The regulation of Ctr1 function and abundance is of great significance to both normal growth and development as well as to the efficacy of platinum-based chemotherapy.The general structure and function of Ctr1 is conserved from yeast to humans, with three membrane-spanning domains and a Met-X₃-Met motif in the second transmembrane domain that is essential for Cu⁺ import (16). The human and mouse protein contains a short ecto-domain with clusters of Met and His. Mutagenic and truncation studies in the context of intact yeast or human Ctr1 indicate that the ecto-domain in general, and the Met residues in particular, play an important role in high-affinity cellular Cu⁺ import, yet all but one key Met near the first transmembrane domain appear to be dispensable for function in cellular Cu⁺ import (31). Studies using model peptides suggest that the Ctr1 Met residues are direct ligands for both Cu⁺ and cisplatin (32–34). In contrast to Cu⁺ uptake, the Met-rich ecto-domain of yeast Ctr1 is required for cisplatin import (27). Moreover, as Ctr1 M-X₃-M mutants are competent for cisplatin uptake, but not Cu⁺ (35), studies suggest that Ctr1-mediated cisplatin uptake may occur via an ecto-domain–dependent receptor-mediated endocytosis mechanism, rather than as an ion channel as for Cu⁺ (27). Ctr1 has been observed in both cell lines and mouse tissues as a full-length glycosylated form and a lower-molecular-weight form, which has been reported to lack a portion of the Cu⁺ and cisplatin-binding ecto-domain (17, 36). However, neither the physiological significance of this truncated form of Ctr1, nor its mode of biogenesis, have been elucidated.The Ctr2 protein is structurally related to Ctr1 and is encoded by a linked gene in both the mouse and the human genome. Recent studies suggest that Ctr2 functions as a low-affinity Cu⁺ importer, a lysosomal Cu⁺ exporter, or as a regulator of cellular macropinocytosis (37–39). However, these studies have been performed in cultured cells, and the physiological role of Ctr2 in animals has not been reported. Here we demonstrate that Ctr2 interacts with Ctr1 in vivo and that Ctr2 knockout mice show increased levels of total copper in several tissues. Mice and mouse embryonic fibroblasts lacking Ctr2 accumulate copper in endosomal compartments and have lower levels of the truncated form of Ctr1 lacking the metal-binding ecto-domain. Whereas truncation of the Ctr1 ecto-domain reduces Cu⁺ import at the plasma membrane, truncated Ctr1 stimulates the mobilization of Cu⁺ from endosomal compartments. These studies demonstrate a critical role for Ctr2 in modulating the accumulation of Ctr1 lacking the Cu⁺ and cisplatin-binding ecto-domain of Ctr1 and, as a consequence, in the regulation of cellular copper uptake and intracellular mobilization. Given the fundamental role for Ctr1 in Cu⁺ import and cisplatin acquisition, the action of Ctr2 represents an important mechanism for the regulation of Ctr1 function.     

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.     

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K⁺ reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K⁺ secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)—components of an E3 ubiquitin ligase complex that targets WNKs for degradation—cause constitutively increased renal salt reabsorption and impaired K⁺ secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3^S433 phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K⁺ secretion in the setting of volume depletion.Hypertension affects 1 billion people worldwide and is a major risk factor for death from stroke, myocardial infarction, and congestive heart failure. The study of Mendelian forms of hypertension has demonstrated the key role of increased renal salt reabsorption in disease pathogenesis (1–4). Observational and intervention trials (5, 6) also indicate that increased dietary K⁺ lowers blood pressure; however, the mechanism of this effect has been unclear.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man no. 145260), featuring hypertension and hyperkalemia, has revealed a previously unrecognized mechanism that regulates the balance between renal salt reabsorption and K⁺ secretion in response to aldosterone (7). Aldosterone is produced by the adrenal glomerulosa in volume depletion, in response to angiotensin II (AII), and in hyperkalemia via membrane depolarization (8). In volume depletion, aldosterone maximizes renal salt reabsorption, whereas in hyperkalemia, aldosterone promotes maximal renal K⁺ secretion. Volume depletion increases both the NaCl cotransporter (NCC) (9) and electrogenic Na⁺ reabsorption via the epithelial Na⁺ channel (ENaC) (10). The lumen-negative potential produced by ENaC activity provides the electrical driving force for paracellular Cl⁻ reabsorption (11). In hyperkalemia, the lumen-negative potential promotes K⁺ secretion via the K⁺ channel Kir1.1 (renal outer medullary K⁺ channel ROMK), reducing plasma K⁺ level (12, 13). Additionally, recent studies have implicated aldosterone signaling in intercalated cell transcellular Cl⁻ flux (14). In these cells, hyperkalemia induces phosphorylation of the mineralocorticoid receptor (MR) ligand-binding domain, making it incapable of ligand binding and activation. AII signaling induces dephosphorylation, and activation of the MR by aldosterone then induces transcellular Cl⁻ flux, which is required for defense of intravascular volume (14, 15). Because electrogenic Cl⁻ reabsorption and K⁺ secretion both dissipate the lumen-negative potential produced by ENaC, maximal Cl⁻ reabsorption inhibits K⁺ secretion and vice versa.Patients with PHAII have constitutive reabsorption of NaCl with concomitant inhibition of K⁺ secretion, resulting in hypertension and hyperkalemia, despite normal levels of aldosterone (7). Dominant mutations in the serine–threonine kinases with-no-lysine 4 (WNK4) or WNK1, or in CUL3 or KLHL3, elements of a ubiquitin ligase complex, cause this disease (2, 4). WNK4 modulates the activities of NCC, ENaC, Kir1.1, and MR (14, 16–21), and WNK4 function can be modulated by phosphorylation (21). CUL3/KLHL3 has been shown to target WNK4 and WNK1 for ubiquitination and degradation, and disease-causing mutations impair this binding and degradation (22–24). In particular, dominant mutations in the Kelch domain of KLHL3 prevent binding to WNKs; reciprocally, disease-causing point mutations in WNK4 also prevent WNK4–KLHL3 binding.These findings suggest that regulation of WNK degradation by CUL3/KLHL3 is highly regulated and that disease-causing mutations might phenocopy a state in which WNKs are normally turned off, producing constitutive salt reabsorption and inhibited K⁺ secretion. We now demonstrate that this inference is correct and implicate AII signaling in this process.     

Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways

The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung’s innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na⁺ channel (ENaC)-dependent Na⁺ absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na⁺ absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.The epithelial Na⁺ channel (ENaC) is the rate-determining step for Na⁺ absorption across the colon, kidney, and lung (1). ENaC is a heterotrimer consisting of α-, β-, and γ-subunits (2). The extracellular domains of the α- and γ-ENaC subunits must be proteolytically cleaved by serine proteases, such as trypsin or neutrophil elastase, or by intracellular furin-type convertases in order for the channel to become active and to conduct Na⁺ (2, 3). In contrast, the β-subunit is highly glycosylated and not cleaved but may form a regulatory subunit that governs ENaC surface densities (1, 4). In some cases, ENaC may bypass the steps required for proteolysis and be inserted into the plasma membrane as near-silent, inactive channels (5). Abnormal ENaC activity has been linked with the pathogenesis of several diseases, including cystic fibrosis (CF), Liddle syndrome, and salt-sensitive hypertension (6). In CF airways, the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane causes ENaC hyperactivity, and the resulting excessive Na⁺ absorption contributes to airway surface liquid (ASL) dehydration, mucus stasis, and bacterial infections (7). Similar lung disorders have been observed in transgenic mice either overexpressing β-ENaC or exhibiting altered regulation of ENaC by the ubiquitin protein ligase NEDD4L, thus linking Na⁺ hyperabsorption and ASL volume depletion to the development of pulmonary disease (4, 8). However, whether or not ENaC activity is up-regulated in CF airways is currently controversial (9). Part of this problem may lie in the lack of an identified mechanism for ENaC hyperactivity in CF airways. Thus, although ENaC has been shown to be up-regulated in vivo, in freshly isolated human airway tissues, and in cell culture, no mechanism for this up-regulation has been discovered (10–13).Normal but not CF airway cultures autoregulate ASL volume by coordinating CFTR and ENaC activity via soluble “reporter molecules” that, by virtue of their dilution and concentration, transmit information on ASL volume to the epithelia (14). The short palate lung and nasal epithelial clone 1 (SPLUNC1), the most abundant secreted protein in the airways, is one such reporter molecule and is absolutely required for limiting ENaC activity and maintaining normal ASL homeostasis (14, 15). We have previously shown that SPLUNC1 binds to ENaC, causing it to be internalized, thus protecting ENaC from proteolytic cleavage and activation (15). SPLUNC1 is also a secreted protein that has been proposed to share homology with the N-terminal domain of bacterial permeability-increasing protein (BPI) (16).Despite the presence of SPLUNC1 in CF ASL (17, 18), CF airway cultures are unable to regulate ENaC (19). Importantly, CF ENaC is not fully dysfunctional and is sensitive to exogenous protease inhibitors, suggesting that the defect lies elsewhere (19). CFTR conducts HCO₃⁻, which maintains ASL at near-neutral pH, and when CFTR is defective (20), the ASL becomes more acidic (21). Because SPLUNC1–ENaC interactions are extracellular, we hypothesized that an acidified ASL prevented SPLUNC1 from inhibiting ENaC. To test this hypothesis, we examined the relationship between SPLUNC1-dependent regulation of ENaC and the ASL pH. To explore this interaction further, we resolved the crystal structure of SPLUNC1 to ∼2.8 Å and used this structure to understand better how ENaC is regulated in CF airway cultures.