A gastropod toxin selectively slows early transitions in the Shaker K channel's activation pathway

A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.     

Suppressive subtractive hybridisation transcriptomics provides a novel insight into the functional role of the hypobranchial gland in a marine mollusc

The hypobranchial gland present in gastropods is an organ whose function is not clearly understood. Involved in mucus production, within members of the family Muricidae it is also the source of the ancient dye Tyrian purple and its bioactive precursors. To gain further insights into hypobranchial gland biology, suppressive subtractive hybridisation was performed on hypobranchial gland and mantle tissue from the marine snail Dicathais orbita creating a differentially expressed cDNA library. 437 clones were randomly sequenced, analysed and annotated and 110 sequences had their functions putatively identified. Importantly this approach identified a putative gene involved in Tyrian purple biosynthesis, an arylsulphatase gene. Confirmation of the upregulation of arylsulphatase in the hypobranchial gland compared to the mantle was demonstrated using quantitative real-time PCR. Other genes identified as playing an important role in the hypobranchial gland were those involved in mucus protein synthesis, choline ester regulation, protein and energy production. This study confirms that the hypobranchial gland is involved in the production of mucus secretion and also identifies it as a site of chemical interaction and biosynthesis. This study lays the foundation for a better understanding of the enzymatic production of Tyrian purple precursors within the gland.     

Binding of a gating modifier toxin induces intersubunit cooperativity early in the Shaker K channel's activation pathway

Potassium currents from voltage-gated Shaker K channels activate with a sigmoid rise. The degree of sigmoidicity in channel opening kinetics confirms that each subunit of the homotetrameric Shaker channel undergoes more than one conformational change before the channel opens. We have examined effects of two externally applied gating modifiers that reduce the sigmoidicity of channel opening. A toxin from gastropod mucus, 6-bromo-2-mercaptotryptamine (BrMT), and divalent zinc are both found to slow the same conformational changes early in Shaker's activation pathway. Sigmoidicity measurements suggest that zinc slows a conformational change independently in each channel subunit. Analysis of activation in BrMT reveals cooperativity among subunits during these same early steps. A lack of competition with either agitoxin or tetraethylammonium indicates that BrMT binds channel subunits outside of the external pore region in an allosterically cooperative fashion. Simulations including negatively cooperative BrMT binding account for its ability to induce gating cooperativity during activation. We conclude that cooperativity among K channel subunits can be greatly altered by experimental conditions.     

Observations on the production of purple pigments in the egg capsules,hypobranchial and reproductive glands from seven species of Muricidae (Gastropoda: Mollusca)

Summary

Tyrian purple is a well-known colourant that can be obtained from the hypobranchial glands of muricids. Here we investigate the presence of purple and related pigments in the hypobranchial glands, reproductive glands and egg capsules of three Rapaninae, three Ocenebrinae and one Muricinae species. Observations on the dissected specimens revealed the presence of purple pigmentation in the hypobranchial glands of all species. All of the Rapaninae and the Muricinae, but only some species of Ocenebrinae, appear to transfer the pigment precursors to their egg capsules. This provides evidence that the precursors of Tyrian purple are not produced for the sole purpose of protecting the egg masses as has been previously suggested. In all the subfamilies, the hypobranchial and reproductive (capsule and prostate) glands lie adjacent to one another. Colour changes in the reproductive glands, indicating the presence of dye precursors, were observed in two species of Rapaninae. In Dicathais orbita, colour changes could be seen in both the capsule glands of females and prostrate glands of males. The final colour was more red in the reproductive glands than the purple observed in the hypobranchial glands. Preliminary observations of detached hypobranchial gland sections in both D. orbita and Pterynotus triformis indicate a different suite of colour reactions occur when compared to sections that are intact and attached to the reproductive glands. This demonstrates that the reproductive glands can influence the chemical conversions of dye precursors synthesized by these species. Further studies on the secondary metabolism occurring within the hypobranchial glands and reproductive organs of Muricidae are ongoing, including histological sectioning and confirmation of the chemistry behind the colours observed and functional analysis of Tyrian purple precursors.     

Synthesis of the non-peptidic snail toxin 6-bromo-2-mercaptotryptamine dimer (BrMT)2, its lower and higher thio homologs and their ability to modulate potassium ion channels

The first synthesis of the non-peptidic snail toxin 6-bromo-2-mercaptotryptamine dimer (BrMT)₂ is described, along with the preparation of its lower and higher thio homologs. The synthetic (BrMT)₂ and its derivatives reported herein are all capable of slowing the activation of the K_v1.1 potassium ion channel. Only the monosulfide variant shows significant slowing of the deactivation process. This synthetic strategy can now be applied to creating a more extensive set of compounds that vary in the length of the linker connecting the two monomers, the substituents on the indole ring core, and terminal amine.     

Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita.     

Vibrio harveyi Adheres to and Penetrates Tissues of the European Abalone Haliotis tuberculata within the First Hours of Contact

Vibrio harveyi is a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abalone Haliotis tuberculata. The aim of this study was to identify the portal of entry and the dynamics of infection of V. harveyi in the European abalone. The results indicate that the duration of contact between V. harveyi and the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized by V. harveyi. Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain of V. harveyi in abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact. V. harveyi was also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such as V. harveyi in mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration of V. harveyi in European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry of V. harveyi in the European abalone is the area situated between the gills and the hypobranchial gland.     

Intracellular chloride in submucosal gland cells

The chloride ion concentration within isolated tracheal submucosal gland cells was studied micro-spectrofluorometrically using a fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), that is quenched by Cl-. Cells from normal weanling swine and from a cystic fibrosis (CF) patient were used. Ion substitution experiments showed that cell fluorescence increased in both cell types when bath Cl- was replaced with the impermeant anion glucuronate. Following a Donnan-type ion substitution that kept the product of the bath K+ and Cl- concentrations constant, reducing bath chloride had little effect on fluorescence for normal cells, but caused a marked increase for CF cells. Thus, K+ and Cl- ions have approximately the same Nernst potential in control submucosal gland cells; in contrast, cells from a CF patient concentrated Cli, resulting in a Cl- Nernst potential that was more positive than the K+ Nernst potential. This finding is consistent with the hypothesis that CF submucosal gland cells have a decreased Cl- permeability.     

Side-chain ionization states in a potassium channel

KcsA is a bacterial K+ channel that is gated by pH. Continuum dielectric calculations on the crystal structure of the channel protein embedded in a low dielectric slab suggest that side chains E71 and D80 of each subunit, which lie adjacent to the selectivity filter region of the channel, form a proton-sharing pair in which E71 is neutral (protonated) and D80 is negatively charged at pH 7. When K+ ions are introduced into the system at their crystallographic positions the pattern of proton sharing is altered. The largest perturbation is for a K+ ion at site S3, i.e., interacting with the carbonyls of T75 and V76. The presence of multiple K+ ions in the filter increases the probability of E71 being ionized and of D80 remaining neutral (i.e., protonated). The ionization states of the protein side chains influence the potential energy profile experienced by a K+ ion as it is translated along the pore axis. In particular, the ionization state of the E71-D80 proton-sharing pair modulates the shape of the potential profile in the vicinity of the selectivity filter. Such reciprocal effects of ion occupancy on side-chain ionization states, and of side-chain ionization states on ion potential energy profiles will complicate molecular dynamics simulations and related studies designed to calculate ion permeation energetics.     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

The buccal gland of Lampetra japonica is a source of diverse bioactive proteins

The parasitic phase lampreys (Lampetra japonica) are bloodsuckers in the marine, and their buccal gland secretion (lamphredin) contains various regulators such as anticoagulants, ion channel blockers, and immune suppressors like those from leeches, insects, ticks, vampire bats, and snakes. This review focuses on the functions and characteristics of the active proteins from the buccal gland of L. japonica for the first time, and provides new insights into the parasitic mechanisms of lampreys and the possibilities of developing drugs such as novel anticoagulants, thrombolytic agents, local anesthetics, and immunosuppressants.     

The ammocoetes endostyle: its oxidative enzymes as an evidence of its homology with the thyroid of higher chordates.

Histochemical methods were used for the demonstration of activity of the following intracellular oxidative enzymes, unstudied hitherto, in the epithelial cells of the endostyle of the river lampre (Lampertr aluviatilis L.) ammocoetes: reduced NAD dehydrogenase (NADD), lactate dehydrogenase (LD), cytochrome oxidase (CO), succinate dehydrogenase (SD), alpha-glycerophosphate dehydrogenase (alphaGPD) and glucose-6-phosphate dehydrogenase (G6PD). The activities of NADD and LD in the iodophil and throidogenic cells of type 3, then of subtype 2c and partly types 4 and 5 of the endostylar epithelium and the hypobranchial duct-lining epithelium were particularly ithe larva proves the possibility of their participation in the formation of the thyroid gland in the period of metamorphosis. In type 1 cells of the ammocoetes, despite their fairly strong enzymatic reactivity, the oxidative activity does not change significantly during the ontogenetic stages examined. The data obtained make it possible to modify the present views on the genesis of the thyroid gland of the adult lamprey, namely, they indicate the participation of the type 6 cells of the hypobranchial duct-lining epithelium in the process of thyroidogenesis.     

Evidence for a role of Na+,K(+)-ATPase in the hydration of Atlantic croaker and spotted seatrout oocytes during final maturation.

During final maturation the oocytes of many marine teleosts swell four to five times their original size due to uptake of water. The involvement of active inorganic ion transport and Na+,K(+)-ATPase in oocyte hydration in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), marine teleosts which spawn pelagic eggs, was investigated by examining changes in the inorganic ion content of ovarian follicles containing mainly oocytes, by performing in vitro incubations of the follicles with ion channel blockers, and by assaying membrane preparations of ovaries containing hydrating and non-hydrating oocytes for Na+,K(+)-ATPase activity and content. There were marked increases in the contents of K+, Mg++, and Ca++, but not Na+, in oocytes of M. undulatus and C. nebulosus during hydration. Incubation of follicle-enclosed oocytes in K(+)-free medium or with ouabain or amiloride, inhibitors of Na+,K(+)-ATPase and Na+ channels, respectively, blocked gonadotropin-induced oocyte hydration in M. undulatus. In addition, Na+,K(+)-ATPase activity increased threefold and the concentration of the enzyme increased 50% in ovarian tissue during oocyte hydration. These results strongly suggest a major role for active ion regulation by a ouabain-sensitive Na+,K(+)-ATPase system in oocyte hydration in two species of sciaenid fishes.     

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.     

Mechanisms of signal transduction in photo-stimulated secretion in Phaeocystis globosa

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca(2+) signal. However, the source of Ca(2+) in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca(2+) oscillator. Optical tomography reveals that photo-stimulation induces InsP(3)-triggered periodic lumenal [Ca(2+)] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca(2+)] that trigger exocytosis. This Ca(2+) dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca(2+)-sensitive ion channels located on the granule membrane: an InsP(3)-receptor-Ca(2+) channel, and an apamin-sensitive K(+) channel.     

Ultrastructural studies and Na+,K+-ATPase immunolocalization in the antennal urinary glands of the lobster Homarus gammarus (Crustacea, Decapoda).

Unlike in crustacean freshwater species, the structure and ultrastructure of the excretory antennal gland is poorly documented in marine species. The general organization and ultrastructure of the cells and the localization of Na(+),K(+)-ATPase were examined in the antennal gland of the adult lobster Homarus gammarus. Each gland is composed of a centrally located coelomosac surrounded ventrally by a labyrinth divided into two parts (I and II) and dorsally by a voluminous bladder. There is no differentiated nephridal tubule between them. The labyrinth and bladder cells have in common a number of ultrastructural cytological features, including basal membrane infoldings associated with mitochondria, apical microvilli, and cytoplasmic extrusions, and a cytoplasm packed with numerous vacuoles, vesicles, lysosome-like bodies, and swollen mitochondria. Each type of cell also presents distinctive characters. Na(+),K(+)-ATPase was detected through immunofluorescence in the basal part of the cells of the labyrinth and in the bladder cells with an increasing immunostaining from labyrinth I to the bladder. No immunoreactivity was detected in the coelomosac. The cells of the labyrinth and of the bladder present morphological and enzymatic features of ionocytes. The antennal glands of the lobster thus possess active ion exchanges capabilities.     

Modifications of alamethicin ion channels by substitution of Glu-7 for Gln-7

To evaluate the role of charged residues facing a pore lumen in stability of channel structure and ion permeation, we studied electrical properties of ion channels formed by synthesized native alamethicins (Rf50 (alm-Q7Q18) and Rf30 (alm-Q7E18)) and their analogs with Glu-7 (alm-E7Q18 and alm-E7E18). The single-channel currents were measured over a pH range of 3.5 to 8.7 using planar bilayers of diphytanoyl PC. The peptides all showed multi-level current fluctuations in this pH range. At pH 3.5 the channels formed by the four peptides were similar to each other irrespective of the side chain differences at positions 7 and 18. The ionization of Glu-7 (E7) and Glu-18 (E18) above neutral pH reduced the relative probabilities of low-conductance states (levels 1 and 2) and increased those of high-conductance states (levels 4-6). The channel conductance of the peptides with E7 and/or E18, which was distinct from that of alm-Q7Q18, showed a marked pH-dependence, especially for low-conductance states. The ionization of E7 further reduced the stability of channel structure, altered the current-voltage curve from a superlinear relation to a sublinear one, and enhanced cation selectivity. These results indicate that ionized E7 strongly influences the channel structure and the ion permeation, in contrast to ionized E18.     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.     

Phosphorus-31 nuclear magnetic resonance of phosphoenzymes of sodium- and potassium-activated and of calcium-activated adenosinetriphosphatase

Prior studies identified phosphoenzyme intermediates in the turnover of sodium- and potassium-activated adenosinetriphosphatase [(Na,K)ATPase] from several sources and of the calcium-activated adenosinetriphosphatase [(Ca)-ATPase] of skeletal muscle sarcoplasmic reticulum. In both cases, the transphosphorylation is to a beta-aspartyl carboxyl group at the active site. We now report observation of a K+-sensitive phosphorylated intermediate of purified (Na,-K)ATPase from the salt gland of the duck using high-field 31P nuclear magnetic resonance. Addition of ATP to a suspension of this enzyme in the presence of Mg2+ and Na+ produced a resonance at about +17 ppm relative to 85% phosphoric acid. Addition of inorganic phosphate and Mg2+ to (Na,K)ATPase also produced a resonance at about +17 ppm which was enhanced in the presence of a saturating concentration of the inhibitor, ouabain; again, addition of K+ made this resonance disappear. These findings are consistent with earlier kinetic characterization of an acid-stable (Na,K)ATPase phosphoenzyme intermediate by 32P-labeled phosphate incorporation into a denatured precipitate of the enzyme. We attribute the +17-ppm resonance to formation of an acyl phosphate at an aspartyl residue of the catalytic site of (Na,K)ATPase. This is supported by our finding of a similar resonance at +17 ppm after phosphorylation of another membrane-bound cation transport enzyme, sarcoplasmic reticulum (Ca)ATPase, as well as by a similar resonance at about +17 ppm after phosphorylation of the model dipeptide L-seryl-L-aspartate.