Conotoxins of the O-superfamily affecting voltage-gated sodium channels

The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While ω-conotoxins and κ-conotoxins block Ca²⁺ and K⁺ channels, respectively, the closely related δ- and μO-conotoxins affect voltage-gated Na⁺ channels (Na_v channels). δ-conotoxins mainly remove the fast inactivation of Na_v channels and, thus, functionally resemble long-chain scorpion α-toxins. μO-conotoxins are functionally similar to μ-conotoxins, since they inhibit the ion flow through Na_v channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Na_v channels. Received 27 December 2006; received after revision 30 January 2007; accepted 19 February 2007     

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.     

Aquaporins with selectivity for unconventional permeants

The aquaporin protein family generally seems to be designed for the selective passage of water or glycerol. Charged molecules, metal ions and even protons are strictly excluded. Recently, particular aquaporin isoforms were reported to conduct unconventional permeants, i.e., the unpolar gases carbon dioxide and nitric oxide, the polar gas ammonia, the oxidative oxygen species hydrogen peroxide, and the metalloids antimonite, arsenite and silicic acid. Here, we summarize the available data on permeability properties and physiological settings of these aquaporins and we analyze which structural features might be connected to permeability for non-water, non-glycerol solutes. Received 31 March 2007; received after revision 3 May 2007; accepted 23 May 2007     

Acyl-CoA thioesterase 9 (ACOT9) in mouse may provide a novel link between fatty acid and amino acid metabolism in mitochondria

Acyl-CoA thioesterase (ACOT) activities are found in prokaryotes and in several compartments of eukaryotes where they hydrolyze a wide range of acyl-CoA substrates and thereby regulate intracellular acyl-CoA/CoA/fatty acid levels. ACOT9 is a mitochondrial ACOT with homologous genes found from bacteria to humans and in this study we have carried out an in-depth kinetic characterization of ACOT9 to determine its possible physiological function. ACOT9 showed unusual kinetic properties with activity peaks for short-, medium-, and saturated long-chain acyl-CoAs with highest V _max with propionyl-CoA and (iso) butyryl-CoA while K _cat/K _m was highest with saturated long-chain acyl-CoAs. Further characterization of the short-chain acyl-CoA activity revealed that ACOT9 also hydrolyzes a number of short-chain acyl-CoAs and short-chain methyl-branched CoA esters that suggest a role for ACOT9 in regulation also of amino acid metabolism. In spite of markedly different K _ms, ACOT9 can hydrolyze both short- and long-chain acyl-CoAs simultaneously, indicating that ACOT9 may provide a novel regulatory link between fatty acid and amino acid metabolism in mitochondria. Based on similar acyl-CoA chain-length specificities of recombinant ACOT9 and ACOT activity in mouse brown adipose tissue and kidney mitochondria, we conclude that ACOT9 is the major mitochondrial ACOT hydrolyzing saturated C₂-C₂₀-CoA in these tissues. Finally, ACOT9 activity is strongly regulated by NADH and CoA, suggesting that mitochondrial metabolic state regulates the function of ACOT9.     

Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins

A subclass of aquaporin (AQP) water channels, termed aquaglyceroporins, are also able to transport glycerol and perhaps urea and other small solutes. Although extensive data exist on the physiological roles of aquaporin-facilitated water transport, until recently the biological significance of glycerol transport by the mammalian aquaglyceroporins has been unknown. There is now compelling evidence for involvement of aquaglyceroporin- facilitated glycerol transport in skin hydration and fat cell metabolism. Mice deficient in AQP3 have dry skin, reduced skin elasticity and impaired epidermal biosynthesis. Mice lacking AQP7 manifest progressive adipocyte fat accumulation and hypertrophy. These skin and fat phenotypes are attributable to impaired glycerol transport. A potential implication of these findings is the possibility of modulation of aquaglyceroporin expression or function in the therapy of skin diseases and obesity. Received 20 January 2006; received after revision 21 February 2006; accepted 20 March 2006     

Function and evolution of channels and transporters in photosynthetic membranes

Chloroplasts from land plants and algae originated from an endosymbiotic event, most likely involving an ancestral photoautotrophic prokaryote related to cyanobacteria. Both chloroplasts and cyanobacteria have thylakoid membranes, harboring pigment-protein complexes that perform the light-dependent reactions of oxygenic photosynthesis. The composition, function and regulation of these complexes have thus far been the major topics in thylakoid membrane research. For many decades, we have also accumulated biochemical and electrophysiological evidence for the existence of solute transthylakoid transport activities that affect photosynthesis. However, research dedicated to molecular identification of the responsible proteins has only recently emerged with the explosion of genomic information. Here we review the current knowledge about channels and transporters from the thylakoid membrane of Arabidopsis thaliana and of the cyanobacterium Synechocystis sp. PCC 6803. No homologues of these proteins have been characterized in algae, although similar sequences could be recognized in many of the available sequenced genomes. Based on phylogenetic analyses, we hypothesize a host origin for most of the so far identified Arabidopsis thylakoid channels and transporters. Additionally, the shift from a non-thylakoid to a thylakoid location appears to have occurred at different times for different transport proteins. We propose that closer control of and provision for the thylakoid by products of the host genome has been an ongoing process, rather than a one-step event. Some of the proteins recruited to serve in the thylakoid may have been the result of the increased specialization of its pigment-protein composition and organization in green plants.     

TRPM7 is regulated by halides through its kinase domain

Transient receptor potential melastatin 7 (TRPM7) is a divalent-selective cation channel fused to an atypical α-kinase. TRPM7 is a key regulator of cell growth and proliferation, processes accompanied by mandatory cell volume changes. Osmolarity-induced cell volume alterations regulate TRPM7 through molecular crowding of solutes that affect channel activity, including magnesium (Mg²⁺), Mg-nucleotides and a further unidentified factor. Here, we assess whether chloride and related halides can act as negative feedback regulators of TRPM7. We find that chloride and bromide inhibit heterologously expressed TRPM7 in synergy with intracellular Mg²⁺ ([Mg²⁺]_i) and this is facilitated through the ATP-binding site of the channel’s kinase domain. The synergistic block of TRPM7 by chloride and Mg²⁺ is not reversed during divalent-free or acidic conditions, indicating a change in protein conformation that leads to channel inactivation. Iodide has the strongest inhibitory effect on TRPM7 at physiological [Mg²⁺]_i. Iodide also inhibits endogenous TRPM7-like currents as assessed in MCF-7 breast cancer cells, where upregulation of SLC5A5 sodium-iodide symporter enhances iodide uptake and inhibits cell proliferation. These results indicate that chloride could be an important factor in modulating TRPM7 during osmotic stress and implicate TRPM7 as a possible molecular mechanism contributing to the anti-proliferative characteristics of intracellular iodide accumulation in cancer cells.     

Differential sensitivity of amphibian nodal and paranodal K+ channels to 4-aminopyridine and TEA

Summary Voltage-dependent K⁺ channels are blocked by several drugs, including 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP is most widely used to localize K⁺ channels in mammalian and non-mammalian nerve fibers, but 4-AP and TEA alter various K⁺ channels and/or preparations in specific ways. The reason is not known, in part because dissociation constants for 4-AP and TEA have not been measured for nodal and internodal K⁺ channels in the same fibers. Smith and Schauf showed that the density of nodal versus paranodal K⁺ channels in frog nerves depends on fiber diameter. This size dependence was used to determine the relative sensitivity of nodal and internodal K⁺ channels to 4-AP and TEA, and to compare voltage- and time-dependent activation. The results show nodal and internodal K⁺ channels activate similarly. However, internodal channels are selectivity blocked by 4-AP while TEA is more effective on nodal channels. A high sensitivity of internodal K⁺ channels may explain why 4-AP improves symptoms in diseases such as multiple sclerosis.     

A study of macromolecular diffusion through native porcine mucus

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueus and mucus layers. Over the molecular weight range of solutes investigated (126–186 000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (>30 000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.     

Molecular regulation of CRAC channels and their role in lymphocyte function

Calcium (Ca²⁺) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca²⁺ entry (SOCE) through Ca²⁺ release-activated Ca²⁺ (CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins that form the channel pore and are activated by stromal interaction molecules (STIM) 1 and 2. Located in the membrane of the endoplasmic reticulum, STIM1 and STIM2 have the dual function of sensing the intraluminal Ca²⁺ concentration in the ER and to activate CRAC channels. A decrease in the ER’s Ca²⁺ concentration induces STIM multimerization and translocation into puncta close to the plasma membrane where they bind to and activate ORAI channels. Since the identification of ORAI and STIM genes as the principal mediators of CRAC channel function, substantial advances have been achieved in understanding the molecular regulation and physiological role of CRAC channels in cells of the immune system and other organs. In this review, we discuss the mechanisms that regulate CRAC channel function and SOCE, the role of recently identified proteins and mechanisms that modulate the activation of ORAI/STIM proteins and the consequences of CRAC channel dysregulation for lymphocyte function and immunity.     

ATP-induced Ca<Superscript>2+</Superscript>-signalling mechanisms in the regulation of mesenchymal stem cell migration

The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca²⁺ is a primary second messenger in the control and regulation of a wide range of cell functions including cell migration. Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca²⁺ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca²⁺ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca²⁺ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), albeit with different kinetics and capacity. Reduction in the ER Ca²⁺ level following the P2Y receptor activation can further induce store-operated Ca²⁺ entry as a distinct Ca²⁺ influx pathway that contributes in ATP-induced increase in the [Ca²⁺]_c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca²⁺ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.     

The phosphoinositide PI(3,5)P2 mediates activation of mammalian but not plant TPC proteins: functional expression of endolysosomal channels in yeast and plant cells

Two-pore channel proteins (TPC) encode intracellular ion channels in both animals and plants. In mammalian cells, the two isoforms (TPC1 and TPC2) localize to the endo-lysosomal compartment, whereas the plant TPC1 protein is targeted to the membrane surrounding the large lytic vacuole. Although it is well established that plant TPC1 channels activate in a voltage- and calcium-dependent manner in vitro, there is still debate on their activation under physiological conditions. Likewise, the mode of animal TPC activation is heavily disputed between two camps favoring as activator either nicotinic acid adenine dinucleotide phosphate (NAADP) or the phosphoinositide PI(3,5)P₂. Here, we investigated TPC current responses to either of these second messengers by whole-vacuole patch-clamp experiments on isolated vacuoles of Arabidopsis thaliana. After expression in mesophyll protoplasts from Arabidopsis tpc1 knock-out plants, we detected the Arabidopsis TPC1-EGFP and human TPC2-EGFP fusion proteins at the membrane of the large central vacuole. Bath (cytosolic) application of either NAADP or PI(3,5)P₂ did not affect the voltage- and calcium-dependent characteristics of AtTPC1-EGFP. By contrast, PI(3,5)P₂ elicited large sodium currents in hTPC2-EGFP-containing vacuoles, while NAADP had no such effect. Analogous results were obtained when PI(3,5)P₂ was applied to hTPC2 expressed in baker’s yeast giant vacuoles. Our results underscore the fundamental differences in the mode of current activation and ion selectivity between animal and plant TPC proteins and corroborate the PI(3,5)P₂-mediated activation and Na⁺ selectivity of mammalian TPC2.     

Arachidonic acid signaling is involved in the mechanism of imidazoline-induced K<Subscript>ATP</Subscript> channel-independent stimulation of insulin secretion

The mechanism by which the novel, pure glucose-dependent insulinotropic, imidazoline derivative BL11282 promotes insulin secretion in pancreatic islets has been investigated. The roles of K_ATP channels, α₂-adrenoreceptors, the I₁-receptor-phosphatidylcholine-specific phospholipase (PC-PLC) pathway and arachidonic acid signaling in BL11282 potentiation of insulin secretion in pancreatic islets were studied. Using SUR1^(-/-) deficient mice, the previous notion that the insulinotropic activity of BL11282 is not related to its interaction with K_ATP channels was confirmed. Insulinotropic activity of BL11282 was not related to its effect on α₂-adrenoreceptors, I₁-imidazoline receptors or PC-PLC. BL11282 significantly increased [³H]arachidonic acid production. This effect was abolished in the presence of the iPLA₂ inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, which is independent of concomitant changes in cytoplasmic free Ca²⁺ concentration, involves release of arachidonic acid by iPLA₂ and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway. Received 5 July 2007; received after revision 18 September 2007; accepted 20 September 2007     

The regulation of ion channels and transporters by glycolytically derived ATP

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na⁺/K⁺-ATPase, the H⁺-ATPase, various types of Ca²⁺-ATPases, the Na⁺/H⁺ exchanger, the ATP-sensitive K⁺ channel, cation channels, Na⁺ channels, Ca²⁺ channels and other channels involved in intracellular Ca²⁺ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches. Received 20 July 2007; received after revision 30 July 2007; accepted 17 August 2007     

Voltage-gated proton channels

The history of research on voltage-gated proton channels is recounted, from their proposed existence in dinoflagellates by Hastings in 1972 and their demonstration in snail neurons by Thomas and Meech in 1982 to the discovery in 2006 (after a decade of controversy) of genes that unequivocally code for proton channels. Voltage-gated proton channels are perfectly selective for protons, conduct deuterons half as well, and the conductance is strongly temperature dependent. These properties are consistent with a conduction mechanism involving hydrogen-bonded-chain transfer, in which the selectivity filter is a titratable amino acid residue. Channel opening is regulated stringently by pH such that only outward current is normally activated. Main functions of proton channels include acid extrusion from cells and charge compensation for the electrogenic activity of the phagocyte NADPH oxidase. Genetic approaches hold the promise of rapid progress in the near future.     

Apolipoprotein CIII hyperactivates β cell CaV1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src

Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca²⁺ (Ca_V) channels in the pancreatic β cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates β cell Ca_V channels. We now demonstrate that ApoCIII increased Ca_V1 channel open probability and density. ApoCIII enhanced whole-cell Ca²⁺ currents and the Ca_V1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of β1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating β cell Ca_V channels. These data reveal that ApoCIII hyperactivates β cell Ca_V1 channels through SR-BI/β1 integrin-dependent coactivation of PKA and Src.     

A study of macromolecular diffusion through native porcine mucus.

A diffusion chamber technique based on time-lag analysis for the estimation of effective diffusion coefficients of radiolabelled macromolecules of varying molecular weights through native mucus gel is reported. For all solutes studied, a reduction in effective diffusion coefficients was observed with a retardation of solute flux in both aqueous and mucus layers. Over the molecular weight range of solutes investigated (126-186,000 Daltons), a consistent effect of molecular weight was evident with regard to the retarding effect of mucus. No apparent or absolute molecular weight cut-off for macromolecular transfer was exhibited. However, at high molecular weights (greater than 30,000 Daltons) the retardation was greatly enhanced. The results confirm that mucus can be regarded as a gel with finite pores, but that it does not constitute an absolute barrier to even high molecular weight solutes.     

Diversity of Cl− Channels

Cl⁻ channels are widely found anion pores that are regulated by a variety of signals and that play various roles. On the basis of molecular biologic findings, ligand-gated Cl⁻ channels in synapses, cystic fibrosis transmembrane conductors (CFTRs) and ClC channel types have been established, followed by bestrophin and possibly by tweety, which encode Ca²⁺-activated Cl⁻ channels. The ClC family has been shown to possess a variety of functions, including stabilization of membrane potential, excitation, cellvolume regulation, fluid transport, protein degradation in endosomal vesicles and possibly cell growth. The molecular structure of Cl⁻ channel types varies from 1 to 12 transmembrane segments. By means of computer-based prediction, functional Cl⁻ channels have been synthesized artificially, revealing that many possible ion pores are hidden in channel, transporter or unidentified hydrophobic membrane proteins. Thus, novel Cl⁻-conducting pores may be occasionally discovered, and evidence from molecular biologic studies will clarify their physiologic and pathophysiologic roles. Received 28 July 2005; received after revision 25 August 2005; accepted 21 September 2005     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules

Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5_{wild type} in MDCK-II cells—but not that of mutants R145A, Y148A, Y158A or E159Q—increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5_Y148A, _Y158A or _E159Q enhanced permeability of FITC-dextran_10 kDa, which was unchanged in cells expressing claudin-5_{wild type} or claudin-5_R145A. In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.