Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.
Calcium signaling is one of the most extensively employed signal transduction mechanisms in life. As life evolved into increasingly complex organisms, Ca(2+) acquired more extensive and varied functions. Here, we compare genes encoding proteins that govern Ca(2+) entry and exit across cells or organelles within organisms of early eukaryotic evolution into fungi, plants, and animals. Recent phylogenomics analyses reveal a complex Ca(2+) signaling machinery in the apusozoan protist Thecamonas trahens, a putative unicellular progenitor of Opisthokonta. We compare T. trahens Ca(2+) signaling to that in a marine bikont protist, Aurantiochytrium limacinum, and demonstrate the conservation of key Ca(2+) signaling molecules in the basally diverging alga Cyanophora paradoxa. Particularly, our findings reveal the conservation of the CatSper channel complex in Au. limacinum and C. paradoxa, suggesting that the CatSper complex likely originated from an ancestral Ca(2+) signaling machinery at the root of early eukaryotic evolution prior to the unikont/bikont split.
Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.
The recent discovery of genes encoding the mitochondrial calcium (Ca(2+)) uniporter has revealed new opportunities for studying how abnormal Ca(2+) signals cause disease. Ca(2+) transport across the mitochondrial inner membrane is highly regulated, and the uniporter is the channel that acts as a major portal for Ca(2+) influx. Low amounts of mitochondrial Ca(2+) can boost ATP synthesis, but excess amounts, such as following cytoplasmic Ca(2+) overload in heart failure, triggers mitochondrial failure and cell death. In fact, precisely because mitochondrial Ca(2+) transport is so tightly regulated, a fundamental understanding of how the uniporter functions is necessary. Two key uniporter features allow Ca(2+) influx without mitochondrial damage during normal physiology. First, the channel is significantly more selective than other known Ca(2+) channels. This prevents the permeation of other ions and uncoupling of the electrochemical gradient. Second, the uniporter becomes active at only high Ca(2+) concentrations, preventing a resting leak of cytoplasmic Ca(2+) itself. Now possessing the identities of the various proteins forming the uniporter, we can proceed with efforts to define the molecular determinants of permeation, selectivity and Ca(2+)-regulation.
Peripheral neuropathic pain, typified by the development of spontaneous pain or pain hypersensitivity following injury to the peripheral nervous system, is common, greatly impairs quality of life, and is inadequately treated with available drugs. Maladaptive changes in chloride homeostasis due to a decrease in the functional expression of the potassium-chloride cotransporter KCC2 in spinal cord dorsal horn neurons are a major contributor to the central disinhibition of ?-aminobutyric acid type A receptor- and glycine receptor-mediated signaling that characterizes neuropathic pain. A compelling novel analgesic strategy is to restore spinal ionotropic inhibition by enhancing KCC2-mediated chloride extrusion. We review the data on which this theory of alternative analgesia is based, discuss recent high-throughput screens that have searched for small-molecule activators of KCC2, and propose other strategies of KCC2 activation based on recent developments in the basic understanding of KCC2's functional regulation. Exploiting the chloride-dependent functional plasticity of the ?-aminobutyric acid and glycinergic system by targeting KCC2 may be a tenable method of restoring ionotropic inhibition not only in neuropathic pain but also in other "hyperexcitable" diseases of the nervous system such as seizures and spasticity.
Transient receptor potential (TRP) channels are abundant in the brain where they regulate transmission of sensory signals. The expression patterns of different TRPC subunits (TRPC1, 4, and 5) are consistent with their potential role in fear-related behaviors. Accordingly, we found recently that mutant mice lacking a specific TRP channel subunit, TRPC5, exhibited decreased innate fear responses. Both TRPC5 and another member of the same subfamily, TRPC4, form heteromeric complexes with the TRPC1 subunit (TRPC1/5 and TRPC1/4, respectively). As TRP channels with specific subunit compositions may have different functional properties, we hypothesized that fear-related behaviors could be differentially controlled by TRPCs with distinct subunit arrangements. In this study, we focused on the analysis of mutant mice lacking the TRPC4 subunit, which, as we confirmed in experiments on control mice, is expressed in brain areas implicated in the control of fear and anxiety. In behavioral experiments, we found that constitutive ablation of TRPC4 was associated with diminished anxiety levels (innate fear). Furthermore, knockdown of TRPC4 protein in the lateral amygdala via lentiviral-mediated gene delivery of RNAi mimicked the behavioral phenotype of constitutive TRPC4-null (TRPC4(-/-)) mouse. Recordings in brain slices demonstrated that these behavioral modifications could stem from the lack of TRPC4 potentiation in neurons in the lateral nucleus of the amygdala through two G?q/11 protein-coupled signaling pathways, activated via Group I metabotropic glutamate receptors and cholecystokinin 2 receptors, respectively. Thus, TRPC4 and the structurally and functionally related subunit, TRPC5, may both contribute to the mechanisms underlying regulation of innate fear responses.
Caspase-11 is a highly inducible caspase that controls both inflammatory responses and cell death. Caspase-11 controls interleukin 1? (IL-1?) secretion by potentiating caspase-1 activation and induces caspase-1-independent pyroptosis downstream of noncanonical NLRP3 inflammasome activators such as lipopolysaccharide (LPS) and Gram-negative bacteria. However, we still know very little about the downstream mechanism of caspase-11 in regulating inflammation because the known substrates of caspase-11 are only other caspases. Here, we identify the cationic channel subunit transient receptor potential channel 1 (TRPC1) as a substrate of caspase-11. TRPC1 deficiency increases the secretion of IL-1? without modulating caspase-1 cleavage or cell death in cultured macrophages. Consistently, trpc1(-/-) mice show higher IL-1? secretion in the sepsis model of intraperitoneal LPS injection. Altogether, our data suggest that caspase-11 modulates the cationic channel composition of the cell and thus regulates the unconventional secretion pathway in a manner independent of caspase-1.
Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel (CatSpers) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.
TRPM7 is a ubiquitous ion channel and kinase, a unique "chanzyme," required for proper early embryonic development. It conducts Zn(2+), Mg(2+), and Ca(2+) as well as monovalent cations and contains a functional serine/threonine kinase at its carboxyl terminus. Here, we show that in normal tissues and cell lines, the kinase is proteolytically cleaved from the channel domain in a cell-type-specific manner. These TRPM7 cleaved kinase fragments (M7CKs) translocate to the nucleus and bind multiple components of chromatin-remodeling complexes, including Polycomb group proteins. In the nucleus, the kinase phosphorylates specific serines/threonines of histones. M7CK-dependent phosphorylation of H3Ser10 at promoters of TRPM7-dependent genes correlates with their activity. We also demonstrate that cytosolic free [Zn(2+)] is TRPM7 dependent and regulates M7CK binding to transcription factors containing zinc-finger domains. These findings suggest that TRPM7-mediated modulation of intracellular Zn(2+) concentration couples ion-channel signaling to epigenetic chromatin covalent modifications that affect gene expression patterns. PAPERCLIP:
Vaccine-induced seropositivity (VISP) or seroreactivity (VISR), defined as the reaction of antibodies elicited by HIV vaccines with antigens used in HIV diagnostic immunoassays, can result in reactive assay results for vaccinated but uninfected individuals, with subsequent misclassification of their infection status. The eventual licensure of a vaccine will magnify this issue and calls for the development of mitigating solutions in advance. An immunoassay that discriminates between antibodies elicited by vaccine antigens and those elicited by infection has been developed to address this laboratory testing need. The HIV Selectest is based on consensus and clade-specific HIV peptides that are omitted in many HIV vaccine constructs. The assay was redesigned to enhance performance across worldwide clades and to simplify routine use via a standard kit format. The redesigned assay was evaluated with sera from vaccine trial participants, HIV-infected and uninfected individuals, and healthy controls. The HIV Selectest exhibited specificities of 99.5% with sera from uninfected recipients of 6 different HIV vaccines and 100% with sera from normal donors, while detecting HIV-1 infections, including intercurrent infections, with 95 to 100% sensitivity depending on the clade, with the highest sensitivities for clades A and C. HIV Selectest sensitivity decreased in very early seroconversion specimens, which possibly explains the slightly lower sensitivity observed for asymptomatic blood donors than for clinical HIV cases. Thus, the HIV Selectest provides a new laboratory tool for use in vaccine settings to distinguish the immune response to HIV vaccine antigens from that due to true infection.
The identification and cloning of full-length homologs of circadian clock genes from Picea abies represent a first step to study the function and evolution of the circadian clock in gymnosperms. Phylogenetic analyses suggest that the sequences of key circadian clock genes are conserved between angiosperms and gymnosperms though fewer homologous copies were found for most gene families in P. abies. We detected diurnal cycling of circadian clock genes in P. abies using quantitative RT-PCR; however, cycling appeared to be rapidly dampened under free-running conditions. Given the unexpected absence of transcriptional cycling during constant conditions we employed a complementary method to assay circadian rhythmic outputs and measured delayed fluorescence in seedlings of Norway spruce. None of the two approaches to study circadian rhythms in Norway spruce could detect robust ~24h cycling behaviour under constant conditions. These data suggest gene conservation but fundamental differences in clock function between gymnosperms and other plant taxa.
Although animals eliminate apoptotic cells using macrophages, plants use cell corpses throughout development and disassemble cells in a cell-autonomous manner by vacuolar cell death. During vacuolar cell death, lytic vacuoles gradually engulf and digest the cytoplasmic content. On the other hand, acute stress triggers an alternative cell death, necrosis, which is characterized by mitochondrial dysfunction, early rupture of the plasma membrane, and disordered cell disassembly. How both types of cell death are regulated remains obscure. In this paper, we show that vacuolar death in the embryo suspensor of Norway spruce requires autophagy. In turn, activation of autophagy lies downstream of metacaspase mcII-Pa, a key protease essential for suspensor cell death. Genetic suppression of the metacaspase-autophagy pathway induced a switch from vacuolar to necrotic death, resulting in failure of suspensor differentiation and embryonic arrest. Our results establish metacaspase-dependent autophagy as a bona fide mechanism that is responsible for cell disassembly during vacuolar cell death and for inhibition of necrosis.
The leucine zipper, EF hand-containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca(2+) and K(+) homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca(2+) fluorophore and (45)Ca(2+)-based assays, we demonstrate directly that Letm1 is a Ca(2+) transporter, with apparent affinities of cations in the sequence of Ca(2+) ? Mn(2+) > Gd(3+) ? La(3+) > Sr(2+) > Ba(2+), Mg(2+), K(+), Na(+). Kinetic analysis yields a Letm1 turnover rate of 2 Ca(2+)/s and a Km of ?25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca(2+)/2 H(+) antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H(+)-dependent Ca(2+) transport mechanism identified in intact mitochondria.
Massive endocytosis is initiated by a series of steps that involve a sudden influx of calcium ions, changes in mitochondria, and modification of surface proteins by lipids. A better understanding of this process could lead to new approaches to reducing the tissue damage that is caused by heart attacks.
The mitochondrial uniporter is a highly selective calcium channel in the organelles inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10-kilodalton, metazoan-specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium-conducting role of MCU.
During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca(2+). The elevation of Ca(2+) is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K(+) current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca(2+) current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1-4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K(+) current present in the Slo3 (-/-) sperm arise from CATSPER? Second, can any additional membrane K(+) currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 (-/-) sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca(2+) influx in response to alkalization.
The timing of bud set, as one determinant of the annual growth rhythm, is critical for local adaptation of the conifer Norway spruce (Picea abies). Previous gene expression and population genetic studies have suggested a role for P. abies FLOWERING LOCUS T/TERMINAL FLOWER1-Like2 (PaFTL2) in the control of growth cessation and bud set in Norway spruce as well as in local adaptation resulting in clinal variation for timing of bud set. Using transgenic plants with PaFTL2 driven by an inducible promoter, we found that PaFTL2 indeed induces bud set and most probably also growth cessation. PaFTL2 shows high expression around the procambium and vascular tissue and in the crown region in buds of both seedlings and older trees. Furthermore, PaFTL2 expression is induced in vegetative shoots and all bud types in late summer, when growth cessation occurs. This supports the notion that PaFTL2 is involved in growth cessation. A close paralog to PaFTL2, PaFTL1, is strongly expressed in meristems during the summer, possibly to repress meristem activity and the formation of needle primordia during this period. The temporal and spatial expression of PaFTL1 and PaFTL2 largely complement each other, which suggests that they act in concert to control perennial growth in Norway spruce.
Sick sinus syndrome and atrioventricular block are common clinical problems, often necessitating permanent pacemaker placement, yet the pathophysiology of these conditions remains poorly understood. Here we show that Transient Receptor Potential Melastatin 7 (TRPM7), a divalent-permeant channel-kinase of unknown function, is highly expressed in embryonic myocardium and sinoatrial node (SAN) and is required for cardiac automaticity in these specialized tissues. TRPM7 disruption in vitro, in cultured embryonic cardiomyocytes, significantly reduces spontaneous Ca(2+) transient firing rates and is associated with robust down-regulation of Hcn4, Cav3.1, and SERCA2a mRNA. TRPM7 knockdown in zebrafish, global murine cardiac Trpm7 deletion (KO(?MHC-Cre)), and tamoxifen-inducible SAN restricted Trpm7 deletion (KO(HCN4-CreERT2)) disrupts cardiac automaticity in vivo. Telemetered and sedated KO(?MHC-Cre) and KO(HCN4-CreERT2) mice show episodes of sinus pauses and atrioventricular block. Isolated SAN from KO(?MHC-Cre) mice exhibit diminished Ca(2+) transient firing rates with a blunted diastolic increase in Ca(2+). Action potential firing rates are diminished owing to slower diastolic depolarization. Accordingly, Hcn4 mRNA and the pacemaker current, I(f), are diminished in SAN from both KO(?MHC-Cre) and KO(HCN4-CreERT2) mice. Moreover, heart rates of KO(?MHC-Cre) mice are less sensitive to the selective I(f) blocker ivabradine, and acute application of the recently identified TRPM7 blocker FTY720 has no effect on action potential firing rates of wild-type SAN cells. We conclude that TRPM7 influences diastolic membrane depolarization and automaticity in SAN indirectly via regulation of Hcn4 expression.
Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein-activated K(+) current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4(-/-) mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4(-/-) mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4(-/-) animals. Although the relative extent of heart rate regulation of Girk4(-/-) mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological ?-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4(-/-) animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct ?-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.
In mammals, calcium influx is required for oocyte maturation and egg activation. The molecular identities of the calcium-permeant channels that underlie the initiation of embryonic development are not established. Here, we describe a transient receptor potential (TRP) ion channel current activated by TRP agonists that is absent in TrpV3(-/-) eggs. TRPV3 current is differentially expressed during oocyte maturation, reaching a peak of maximum density and activity at metaphase of meiosis II (MII), the stage of fertilization. Selective activation of TRPV3 channels provokes egg activation by mediating massive calcium entry. Widely used to activate eggs, strontium application is known to yield normal offspring in combination with somatic cell nuclear transfer. We show that TRPV3 is required for strontium influx, because TrpV3(-/-) eggs failed to conduct Sr(2+) or undergo strontium-induced activation. We propose that TRPV3 is a major mediator of calcium influx in mouse eggs and is a putative target for artificial egg activation.
Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9?Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.
Transient receptor potential (TRP) channels are a superfamily of broadly expressed ion channels with diverse physiological roles. TRPC1, TRPC3, and TRPC6 are believed to contribute to cardiac hypertrophy in mouse models. Human mutations in TRPM4 have been linked to progressive familial heart block. TRPM7 is a divalent-permeant channel and kinase of unknown function, recently implicated in the pathogenesis of atrial fibrillation; however, its function in ventricular myocardium remains unexplored.
Mitochondrial metabolism, respiration, and ATP production necessitate ion transport across the inner mitochondrial membrane. Leucine zipper-EF-hand containing transmembrane protein 1 (Letm1), one of the genes deleted in Wolf-Hirschhorn syndrome, encodes a putative mitochondrial Ca(2+)/H(+) antiporter. Cellular Letm1 knockdown reduced Ca(2+)mito uptake, H(+)mito extrusion and impaired mitochondrial ATP generation capacity. Homozygous deletion of Letm1 in mice resulted in embryonic lethality before day 6.5 of embryogenesis and ~50% of the heterozygotes died before day 13.5 of embryogenesis. The surviving heterozygous mice exhibited altered glucose metabolism, impaired control of brain ATP levels, and increased seizure activity. We conclude that loss of Letm1 contributes to the pathology of Wolf-Hirschhorn syndrome in humans and may contribute to seizure phenotypes by reducing glucose oxidation and other specific metabolic alterations.
Basic guidance on the photostability testing of pharmaceuticals, designed to cover manufacturing and storage over shelf life, has long been established within ICH Q1(ICH,B(10) , but the guideline does not cover the photostability of drugs during or after administration (i.e., under conditions of use). To date, there has been a paucity of guidance covering the additional testing that would be of value during the clinical preparation and use of products. This commentary suggests a systematic approach, based on realistic "worst case" photoexposure scenarios and the existing ICH Option 1 and 2 light sources, to provide valuable data to pharmaceutical manufacturers and compounding pharmacists for the safe and effective use of photosensitive injection products.
The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channels full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.
A primary cilium is a solitary, slender, non-motile protuberance of structured microtubules (9+0) enclosed by plasma membrane. Housing components of the cell division apparatus between cell divisions, primary cilia also serve as specialized compartments for calcium signalling and hedgehog signalling pathways. Specialized sensory cilia such as retinal photoreceptors and olfactory cilia use diverse ion channels. An ion current has been measured from primary cilia of kidney cells, but the responsible genes have not been identified. The polycystin proteins (PC and PKD), identified in linkage studies of polycystic kidney disease, are candidate channels divided into two structural classes: 11-transmembrane proteins (PKD1, PKD1L1 and PKD1L2) remarkable for a large extracellular amino terminus of putative cell adhesion domains and a G-protein-coupled receptor proteolytic site, and the 6-transmembrane channel proteins (PKD2, PKD2L1 and PKD2L2; TRPPs). Evidence indicates that the PKD1 proteins associate with the PKD2 proteins via coiled-coil domains. Here we use a transgenic mouse in which only cilia express a fluorophore and use it to record directly from primary cilia, and demonstrate that PKD1L1 and PKD2L1 form ion channels at high densities in several cell types. In conjunction with an accompanying manuscript, we show that the PKD1L1-PKD2L1 heteromeric channel establishes the cilia as a unique calcium compartment within cells that modulates established hedgehog pathways.
Primary cilia are solitary, non-motile extensions of the centriole found on nearly all nucleated eukaryotic cells between cell divisions. Only ?200-300?nm in diameter and a few micrometres long, they are separated from the cytoplasm by the ciliary neck and basal body. Often called sensory cilia, they are thought to receive chemical and mechanical stimuli and initiate specific cellular signal transduction pathways. When activated by a ligand, hedgehog pathway proteins, such as GLI2 and smoothened (SMO), translocate from the cell into the cilium. Mutations in primary ciliary proteins are associated with severe developmental defects. The ionic conditions, permeability of the primary cilia membrane, and effectiveness of the diffusion barriers between the cilia and cell body are unknown. Here we show that cilia are a unique calcium compartment regulated by a heteromeric TRP channel, PKD1L1-PKD2L1, in mice and humans. In contrast to the hypothesis that polycystin (PKD) channels initiate changes in ciliary calcium that are conducted into the cytoplasm, we show that changes in ciliary calcium concentration occur without substantially altering global cytoplasmic calcium. PKD1L1-PKD2L1 acts as a ciliary calcium channel controlling ciliary calcium concentration and thereby modifying SMO-activated GLI2 translocation and GLI1 expression.
Sperm intracellular pH and calcium concentration ([Ca(2+)]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca(2+)] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels.
A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an ?-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.
In sea urchins, spermatozoan motility is altered by chemotactic peptides, giving rise to the assumption that mammalian eggs also emit chemotactic agents that guide spermatozoa through the female reproductive tract to the mature oocyte. Mammalian spermatozoa indeed undergo complex adaptations within the female (the process of capacitation) that are initiated by agents ranging from pH to progesterone, but these factors are not necessarily taxic. Currently, chemotaxis, thermotaxis, and rheotaxis have not been definitively established in mammals.
The application of the potentiometric multisensor system (electronic tongue, ET) for quantification of the bitter taste of structurally diverse active pharmaceutical ingredients (API) is reported. The measurements were performed using a set of bitter substances that had been assessed by a professional human sensory panel and the in vivo rat brief access taste aversion (BATA) model to produce bitterness intensity scores for each substance at different concentrations. The set consisted of eight substances, both inorganic and organic - azelastine, caffeine, chlorhexidine, potassium nitrate, naratriptan, paracetamol, quinine, and sumatriptan. With the aim of enhancing the response of the sensors to the studied APIs, measurements were carried out at different pH levels ranging from 2 to 10, thus promoting ionization of the compounds. This experiment yielded a 3 way data array (samples×sensors×pH levels) from which 3wayPLS regression models were constructed with both human panel and rat model reference data. These models revealed that artificial assessment of bitter taste with ET in the chosen set of APIs is possible with average relative errors of 16% in terms of human panel bitterness score and 25% in terms of inhibition values from in vivo rat model data. Furthermore, these 3wayPLS models were applied for prediction of the bitterness in blind test samples of a further set of APIs. The results of the prediction were compared with the inhibition values obtained from the in vivo rat model.
Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosomes membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cells metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.
Mitochondrial calcium (Ca(2+)) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca(2+) uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca(2+) channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca(2+) current (I MiCa ), whereas overexpression increases it. Additionally, a classic feature of I MiCa , its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel. DOI:http://dx.doi.org/10.7554/eLife.00704.001.
Global disruption of transient receptor potential-melastatin-like 7 (Trpm7) in mice results in embryonic lethality before embryonic day 7. Using tamoxifen-inducible disruption of Trpm7 and multiple Cre recombinase lines, we show that Trpm7 deletion before and during organogenesis results in severe tissue-specific developmental defects. We find that Trpm7 is essential for kidney development from metanephric mesenchyme but not ureteric bud. Disruption of neural crest Trpm7 at early stages results in loss of pigment cells and dorsal root ganglion neurons. In contrast, late disruption of brain-specific Trpm7 after embryonic day 10.5 does not alter normal brain development. We developed induced pluripotent stem cells and neural stem (NS) cells in which Trpm7 disruption could be induced. Trpm7(-/-) NS cells retained the capacities of self-renewal and differentiation into neurons and astrocytes. During in vitro differentiation of induced pluripotent stem cells to NS cells, Trpm7 disruption prevents the formation of the NS cell monolayer. The in vivo and in vitro results demonstrate a temporal requirement for the Trpm7 channel kinase during embryogenesis.
The exceptionally high temperature sensitivity of certain transient receptor potential (TRP) family ion channels is the molecular basis of hot and cold sensation in sensory neurons. The laws of thermodynamics dictate that opening of these specialized TRP channels must involve an unusually large conformational standard-state enthalpy, ?H(o): positive ?H(o) for heat-activated and negative ?H(o) for cold-activated TRPs. However, the molecular source of such high-enthalpy changes has eluded neurobiologists and biophysicists. Here we offer a general, unifying mechanism for both hot and cold activation that recalls long-appreciated principles of protein folding. We suggest that TRP channel gating is accompanied by large changes in molar heat capacity, ?C(P). This postulate, along with the laws of thermodynamics and independent of mechanistic detail, leads to the conclusion that hot- and cold-sensing TRPs operate by identical conformational changes.
Specialized proteins in the plasma membrane, endoplasmic reticulum (ER), and mitochondria tightly regulate intracellular calcium. A unique mechanism called store-operated calcium entry is activated when ER calcium is depleted, serving to restore intra-ER calcium levels. An ER calcium sensor, stromal interaction molecule 1 (STIM1), translocates within the ER membrane upon store depletion to the juxtaplasma membrane domain, where it interacts with intracellular domains of a highly calcium-selective plasma membrane ion channel, Orai1. STIM1 gates Orai1, allowing calcium to enter the cytoplasm, where it repletes the ER store via calcium-ATPases pumps. Here, we performed affinity purification of Orai1 from Jurkat cells to identify partner of STIM1 (POST), a 10-transmembrane-spanning segment protein of unknown function. The protein is located in the plasma membrane and ER. POST-Orai1 binding is store depletion-independent. On store depletion, the protein binds STIM1 and moves within the ER to localize near the cell membrane. This protein, TMEM20 (POST), does not affect store-operated calcium entry but does reduce plasma membrane Ca(2+) pump activity. Store depletion promotes STIM1-POST complex binding to smooth ER and plasma membrane Ca(2+) ATPases (SERCAs and PMCAs, respectively), Na/K-ATPase, as well as to the nuclear transporters, importins-? and exportins.
Detection and adaptation to cold temperature is crucial to survival. Cold sensing in the innocuous range of cold (>10-15 °C) in the mammalian peripheral nervous system is thought to rely primarily on transient receptor potential (TRP) ion channels, most notably the menthol receptor, TRPM8. Here we report that TRP cation channel, subfamily C member 5 (TRPC5), but not TRPC1/TRPC5 heteromeric channels, are highly cold sensitive in the temperature range 37-25 °C. We found that TRPC5 is present in mouse and human sensory neurons of dorsal root ganglia, a substantial number of peripheral nerves including intraepithelial endings, and in the dorsal lamina of the spinal cord that receives sensory input from the skin, consistent with a potential TRPC5 function as an innocuous cold transducer in nociceptive and thermosensory nerve endings. Although deletion of TRPC5 in 129S1/SvImJ mice resulted in no temperature-sensitive behavioral changes, TRPM8 and/or other menthol-sensitive channels appear to underpin a much larger component of noxious cold sensing after TRPC5 deletion and a shift in mechanosensitive C-fiber subtypes. These findings demonstrate that highly cold-sensitive TRPC5 channels are a molecular component for detection and regional adaptation to cold temperatures in the peripheral nervous system that is distinct from noxious cold sensing.
Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology.
Sperm cells acquire hyperactivated motility as they ascend the female reproductive tract, which enables them to overcome barriers and penetrate the cumulus and zona pellucida surrounding the egg. This enhanced motility requires Ca(2+) entry via cation channel of sperm (CatSper) Ca(2+)-selective ion channels in the sperm tail. Ca(2+) entry via CatSper is enhanced by the membrane hyperpolarization mediated by Slo3, a K(+) channel also present in the sperm tail. To date, no transmitter-mediated currents have been reported in sperm and no currents have been detected in the head or midpiece of mature spermatozoa. We screened a number of neurotransmitters and biomolecules to examine their ability to induce ion channel currents in the whole spermatozoa. Surprisingly, we find that none of the previously reported neurotransmitter receptors detected by antibodies alone are functional in mouse spermatozoa. Instead, we find that mouse spermatozoa have a cation-nonselective current in the midpiece of spermatozoa that is activated by external ATP, consistent with an ATP-mediated increase in intracellular Ca(2+) as previously reported. The ATP-dependent current is not detected in mice lacking the P2X2 receptor gene (P2rx2(-/-)). Furthermore, the slowly desensitizing and strongly outwardly rectifying ATP-gated current has the biophysical and pharmacological properties that mimic heterologously expressed mouse P2X2. We conclude that the ATP-induced current on mouse spermatozoa is mediated by the P2X2 purinergic receptor/channel. Despite the loss of ATP-gated current, P2rx2(-/-) spermatozoa have normal progressive motility, hyperactivated motility, and acrosome reactions. However, fertility of P2rx2(-/-) males declines with frequent mating over days, suggesting that P2X2 receptor adds a selection advantage under these conditions.
Ridaforolimus is a nonprodrug rapamycin analogue that potently inhibits mTOR and has shown significant activity in patients with metastatic sarcoma and endometrial cancer, two diseases where high unmet need remains. Here, we evaluated the activity of ridaforolimus in preclinical models of these tumor types and used these models to explore molecular correlates of sensitivity. The in vitro sensitivity of a panel of sarcoma and endometrial cancer cell lines was established by measuring the effect of ridaforolimus on cell proliferation rate, revealing broad inhibition at low nanomolar concentrations. Additional benefit was found when ridaforolimus was combined with agents used to treat sarcoma and endometrial cancer patients. In vivo, potent antitumor activity of ridaforolimus associated with inhibition of mTOR signaling was observed in sarcoma and endometrial xenograft models. Immunoblot analysis was conducted to assess the expression and activation state of multiple signaling proteins in the phosphoinositide-3-kinase/AKT/mTOR and cell-cycle pathways. In endometrial but not sarcoma cell lines, the absence of PTEN or elevated levels of phosphorylated or total AKT was associated with greater sensitivity. However, in both tumor types, the proportion of cells in the G(0)-G(1) phase before treatment correlated significantly with ridaforolimus sensitivity. Consistent with this, expression of several G(1) phase cell-cycle proteins, notably p21 and p27, was higher in more sensitive lines. These results underscore the promise of ridaforolimus as a single agent or combination treatment of these tumor types and suggest novel potential predictive biomarkers of sensitivity to an mTOR inhibitor based on cell-cycle status.
Animals and fungi diverged from a common unicellular ancestor of Opisthokonta, yet they exhibit significant differences in their components of Ca2+ signaling pathways. Many Ca2+ signaling molecules appear to be either animal-specific or fungal-specific, which is generally believed to result from lineage-specific adaptations to distinct physiological requirements. Here, by analyzing the genomic data from several close relatives of animals and fungi, we demonstrate that many components of animal and fungal Ca2+ signaling machineries are present in the apusozoan protist Thecamonas trahens, which belongs to the putative unicellular sister group to Opisthokonta. We also identify the conserved portion of Ca2+ signaling molecules in early evolution of animals and fungi following their divergence. Furthermore, our results reveal the lineage-specific expansion of Ca2+ channels and transporters in the unicellular ancestors of animals and in basal fungi. These findings provide novel insights into the evolution and regulation of Ca2+ signaling critical for animal and fungal biology.
TRPM7 is a ubiquitously expressed nonselective cation channel fused to a C-terminal alpha kinase. TRPM7 current is typically small at physiological magnesium concentrations, but large outwardly rectifying currents develop in low-magnesium extracellular solution when cells are dialyzed with magnesium free solutions during whole-cell patch clamp recordings. In addition to regulation by magnesium, TRPM7 current is potentiated by low extracellular pH and inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) during phospholipase C mediated signaling events. A diverse body of literature has implicated TRPM7 in fundamental cellular processes including death, survival, proliferation, cell cycle progression, magnesium homeostasis and responses to shear stress and oxidative stress. Global deletion of TRPM7 in mouse results in embryonic lethality and a thymocyte-restricted conditional knockout exhibits defective thymopoeisis, suggesting a role for TRPM7 in development and organogenesis. In disease states, TRPM7 has been linked to Guamanian amyotrophic lateral sclerosis and parkinsonian dementia (ALS/PD), various forms of neoplasia, hypertension and delayed neuronal death following cerebral ischemia.
Calcium signalling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and the acrosome reaction are all mediated by increases in intracellular Ca(2+). Cation channels of sperm proteins (CATSPERS1-4) form an alkalinization-activated Ca(2+)-selective channel required for the hyperactivated motility of spermatozoa and male fertility. Each of the CatSper1-4 genes encodes a subunit of a tetramer surrounding a Ca(2+)-selective pore, in analogy with other six-transmembrane ion channel ? subunits. In addition to the pore-forming proteins, the sperm Ca(2+) channel contains auxiliary subunits, CATSPER? and CATSPER?. Here, we identify the Tmem146 gene product as a novel subunit, CATSPER?, required for CATSPER channel function. We find that mice lacking the sperm tail-specific CATSPER? are infertile and their spermatozoa lack both Ca(2+) current and hyperactivated motility. We show that CATSPER? is an essential element of the CATSPER channel complex and propose that CATSPER? is required for proper CATSPER channel assembly and/or transport.
Transient receptor potential (TRP) channels are a large family of ion channel proteins, surpassed in number in mammals only by voltage-gated potassium channels. TRP channels are activated and regulated through strikingly diverse mechanisms, making them suitable candidates for cellular sensors. They respond to environmental stimuli such as temperature, pH, osmolarity, pheromones, taste, and plant compounds, and intracellular stimuli such as Ca(2+) and phosphatidylinositol signal transduction pathways. However, it is still largely unknown how TRP channels are activated in vivo. Despite the uncertainties, emerging evidence using TRP channel knockout mice indicates that these channels have broad function in physiology. Here we review the recent progress on the physiology, pharmacology and pathophysiological function of mammalian TRP channels.
Nanoparticle (NP)-mediated drug delivery typically relies on cargo release to occur passively or in response to environmental stimuli. Here we present a delivery method based on light-activated disruption of intracellular vesicles after internalization of biofunctionalized mesoporous silica nanoparticles loaded with cargo. This method combines the power of targeted delivery with the spatiotemporal control of light activation. As an example, we delivered a cell-impermeable fluorescent compound exclusively to the cytosol of multidrug resistant cancer cells in a mixed population.
This mini-review discusses relevant aspects of gastro-intestinal transit in different ages of paediatric patients with an attempt to highlight factors which should be considered in oral dosage form design, in particular multi-particulate dosage forms. This emphasis is due to multi-particulates possessing many of the benefits of liquid oral formulations (such as ease of swallowing and dose adaptability) without many of their drawbacks (such as stability issues and lack of enteric or modified release functionalities). It is commonly stated that children are not merely small adults with regards to medicines. However, there has been very little research regarding how different dosage forms transit through the gastro-intestinal tract in children compared to adults, due to both ethical and practical hurdles. Due to this lack of studies on dosage form transit in children, information which was available on the transit of food, milk and liquids (often dependent upon the age of the patient) has been used to look at how various aspects of transit vary with age and, where possible, when they reach adult values and how these may affect the fate of dosage forms in vivo: swallowability, oesophageal transit, gastric emptying and pH, intestinal and colonic transit are discussed.
A plethora of growth factors regulate keratinocyte proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that keratinocyte TRPV3, a member of the transient receptor potential (TRP) family of Ca(2+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(2+)-dependent crosslinking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.
Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H(+) flux and are gated by both voltage and pH gradients. Selective H(+) transfer in membrane proteins is commonly achieved by Grotthuss proton hopping in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H(+) currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.
Mitochondria are integral components of cellular calcium (Ca2+) signaling. Calcium stimulates mitochondrial adenosine 5-triphosphate production, but can also initiate apoptosis. In turn, cytoplasmic Ca2+ concentrations are regulated by mitochondria. Although several transporter and ion-channel mechanisms have been measured in mitochondria, the molecules that govern Ca2+ movement across the inner mitochondrial membrane are unknown. We searched for genes that regulate mitochondrial Ca2+ and H+ concentrations using a genome-wide Drosophila RNA interference (RNAi) screen. The mammalian homolog of one Drosophila gene identified in the screen, Letm1, was found to specifically mediate coupled Ca2+/H+ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified Letm1 protein demonstrate that Letm1 is a mitochondrial Ca2+/H+ antiporter.
Magnesium (Mg(2+)) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg(2+) transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg(2+) influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg(2+). Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg(2+) concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg(2+) or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg(2+) transport system.
TRPM1 (melastatin), which encodes the founding member of the TRPM family of transient receptor potential (TRP) ion channels, was first identified by its reduced expression in a highly metastatic mouse melanoma cell line. Clinically, TRPM1 is used as a predictor of melanoma progression in humans because of its reduced abundance in more aggressive forms of melanoma. Although TRPM1 is found primarily in melanin-producing cells and has the molecular architecture of an ion channel, its function is unknown. Here we describe an endogenous current in primary human neonatal epidermal melanocytes and mouse melanoma cells that was abrogated by expression of microRNA directed against TRPM1. Messenger RNA analysis showed that at least five human ion channel-forming isoforms of TRPM1 could be present in melanocytes, melanoma, brain, and retina. Two of these isoforms are encoded by highly conserved splice variants that are generated by previously uncharacterized exons. Expression of these two splice variants in human melanoma cells generated an ionic current similar to endogenous TRPM1 current. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures. Plasma membrane TRPM1 currents are small, raising the possibility that their primary function is intracellular, or restricted to specific regions of the plasma membrane. In neonatal human epidermal melanocytes, TRPM1 expression correlates with melanin content. We propose that TRPM1 is an ion channel whose function is critical to normal melanocyte pigmentation and is thus a potential target for pigmentation disorders.
TRPC5 is a calcium (Ca(2+))-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner ( approximately 5-fold at positive potentials and approximately 25-fold at negative potentials). The reversal potential, doubly rectifying current-voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca(2+): replacement by Ba(2+) or Mg(2+) abolishes it, whereas the addition of 10 mM Ca(2+) accelerates it. The site of action for Ca(2+) is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at approximately 1 microM [Ca(2+)]. This potentiation is prevented when intracellular Ca(2+) is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca(2+)]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca(2+)] led to an approximately 10-20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca(2+)-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.
Granulocytes generate a "respiratory burst" of NADPH oxidase-dependent superoxide anion (O(2)(-*)) production that is required for efficient clearance of bacterial pathogens. Hv1 mediates a voltage-gated H(+) channel activity that is proposed to serve a charge-balancing role in granulocytic phagocytes such as neutrophils and eosinophils. Using mice in which the gene encoding Hv1 is replaced by beta-Geo reporter protein sequence, we show that Hv1 expression is required for measurable voltage-gated H(+) current in unstimulated phagocytes. O(2)(-*) production is substantially reduced in the absence of Hv1, suggesting that Hv1 contributes a majority of the charge compensation required for optimal NADPH oxidase activity. Despite significant reduction in superoxide production, Hv1(-/-) mice are able to clear several types of bacterial infections.
Activation of plasma membrane calcium channels by depletion of endoplasmic reticulum (ER) calcium stores is important for calcium entry in many cell types. Park et al. (2009) now show that direct binding of the ER protein STIM to tetramers of the Orai1 calcium channel in the plasma membrane triggers opening of this channel.
The transient receptor potential channel 5 (TRPC5) is predominantly expressed in the brain where it can form heterotetrameric complexes with TRPC1 and TRPC4 channel subunits. These excitatory, nonselective cationic channels are regulated by G protein, phospholipase C-coupled receptors. Here, we show that TRPC5(-/-) mice exhibit diminished innate fear levels in response to innately aversive stimuli. Moreover, mutant mice exhibited significant reductions in responses mediated by synaptic activation of Group I metabotropic glutamate and cholecystokinin 2 receptors in neurons of the amygdala. Synaptic strength at afferent inputs to the amygdala was diminished in P10-P13 null mice. In contrast, baseline synaptic transmission, membrane excitability, and spike timing-dependent long-term potentiation at cortical and thalamic inputs to the amygdala were largely normal in older null mice. These experiments provide genetic evidence that TRPC5, activated via G protein-coupled neuronal receptors, has an essential function in innate fear.
This protocol details methods to identify and record from cutaneous primary afferent axons in an isolated mammalian skin-saphenous nerve preparation. The method is based on extracellular recordings of propagated action potentials from single-fiber receptive fields. Cutaneous nerve endings show graded sensitivities to various stimulus modalities that are quantified by adequate and controlled stimulation of the superfused skin with heat, cold, touch, constant punctate pressure or chemicals. Responses recorded from single-fibers are comparable with those obtained in previous in vivo experiments on the same species. We describe the components and the setting-up of the basic equipment of a skin-nerve recording station (few days), the preparation of the skin and the adherent saphenous nerve in the mouse (15-45 min) and the isolation and recording of neurons (approximately 1-3 h per recording). In addition, stimulation techniques, protocols to achieve single-fiber recordings, issues of data acquisition and action potential discrimination are discussed in detail.
NaChBac is a bacterial voltage-gated sodium (Nav) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Nav channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of NavRh, a marine bacterial NaChBac ortholog. Two Na(+) binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na(+) ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca(2+) ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na(+) and Ca(2+) to the vestibule of the SF. In addition, Na(+) permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Nav orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na(+) over Ca(2+) in mammalian Nav channels.
Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A(+)) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce.
Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.
When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Cl(swell)) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Cl(swell) channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila Cl(swell) channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Cl(swell) current (I(Clswell)). We further demonstrate the essential contribution of dBest1 to Drosophila I(Clswell) with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous I(Clswell). We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila-like I(Clswell). In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Cl(swell) channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.
PGC1? is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1? expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1?, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.
Bacterial mechanosensitive channels are some of the largest pores in nature. In particular, MscL, with a pore diameter >25 Å, allows passage of large organic ions and small proteins. Functional MscL reconstitution into lipids has been proposed for applications in vesicular-based drug release. Here we show that these channels can be functionally expressed in mammalian cells to afford rapid controlled uptake of membrane-impermeable molecules. We first demonstrate that MscL gating in response to increased membrane tension is preserved in mammalian cell membranes. Molecular delivery is controlled by adopting an established method of MscL charge-induced activation. We then determine pore size limitations using fluorescently labelled model cargoes. Finally, we activate MscL to introduce the cell-impermeable bi-cyclic peptide phalloidin, a specific marker for actin filaments, into cells. We propose that MscL will be a useful tool for gated and controlled delivery of bioactive molecules into cells.
Transient receptor potential melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7-/- T cells are deficient in Fas-receptor-induced apoptosis and that TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor after receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage, with wide-ranging implications for cell death and differentiation.
Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05?Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr?178 and Leu?179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser?181 and Glu?183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an inactivated conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.
Calorimetry is a general method for determination of the rates of zero-order processes, but analysis of the data for the rate constant and reaction enthalpy is difficult because these occur as a product in the rate equation so evaluation of one requires knowledge of the other. Three methods for evaluation of both parameters, without prior knowledge, are illustrated with examples and compared with literature data. Method 1 requires the reaction to be studied in two buffers with different enthalpies of ionization. Method 2 is based on calculation of reaction enthalpy from group additivity functions. Method 3 applies when reaction progresses to completion. The methods are applied to the enzymatic hydrolysis of urea, the hydrolysis of acetylsalicylic acid, and the photodegradation of nifedipine, respectively.
The nonselective cation channel transient receptor potential canonical (TRPC)5 is found predominantly in the brain and has been proposed to regulate neuronal processes and growth cones. Here, we demonstrate that semaphorin 3A-mediated growth cone collapse is reduced in hippocampal neurons from TRPC5 null mice. This reduction is reproduced by inhibition of the calcium-sensitive protease calpain in wild-type neurons but not in TRPC5(-/-) neurons. We show that calpain-1 and calpain-2 cleave and functionally activate TRPC5. Mutation of a critical threonine at position 857 inhibits calpain-2 cleavage of the channel. Finally, we show that the truncated TRPC5 predicted to result from calpain cleavage is functionally active. These results indicate that semaphorin 3A initiates growth cone collapse via activation of calpain that in turn potentiates TRPC5 activity. Thus, TRPC5 acts downstream of semaphorin signaling to cause changes in neuronal growth cone morphology and nervous system development.
Phagocytic cell NADPH oxidase (NOX) generates reactive oxygen species (ROS) as part of innate immunity. Unfortunately, ischemia can also induce this pathway and inflict damage on native cells. The voltage-gated proton channel Hv1 enables NOX function by compensating cellular loss of electrons with protons. Accordingly, we investigated whether NOX-mediated brain damage in stroke can be inhibited by suppression of Hv1. We found that mouse and human brain microglia, but not neurons or astrocytes, expressed large Hv1-mediated currents. Hv1 was required for NOX-dependent ROS generation in brain microglia in situ and in vivo. Mice lacking Hv1 were protected from NOX-mediated neuronal death and brain damage 24 h after stroke. These results indicate that Hv1-dependent ROS production is responsible for a substantial fraction of brain damage at early time points after ischemic stroke and provide a rationale for Hv1 as a therapeutic target for the treatment of ischemic stroke.
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