Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).
(57/1021)
Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2. (+info)
Crystal structure of diisopropylfluorophosphatase from Loligo vulgaris.
(58/1021)
BACKGROUND: Phosphotriesterases (PTE) are enzymes capable of detoxifying organophosphate-based chemical warfare agents by hydrolysis. One subclass of these enzymes comprises the family of diisopropylfluorophosphatases (DFPases). The DFPase reported here was originally isolated from squid head ganglion of Loligo vulgaris and can be characterized as squid-type DFPase. It is capable of hydrolyzing the organophosphates diisopropylfluorophosphate, soman, sarin, tabun, and cyclosarin. RESULTS: Crystals were grown of both the native and the selenomethionine-labeled enzyme. The X-ray crystal structure of the DFPase from Loligo vulgaris has been solved by MAD phasing and refined to a crystallographic R value of 17.6% at a final resolution of 1.8 A. Using site-directed mutagenesis, we have structurally and functionally characterized essential residues in the active site of the enzyme. CONCLUSIONS: The crystal structure of the DFPase from Loligo vulgaris is the first example of a structural characterization of a squid-type DFPase and the second crystal structure of a PTE determined to date. Therefore, it may serve as a structural model for squid-type DFPases in general. The overall structure of this protein represents a six-fold beta propeller with two calcium ions bound in a central water-filled tunnel. The consensus motif found in the blades of this beta propeller has not yet been observed in other beta propeller structures. Based on the results obtained from mutants of active-site residues, a mechanistic model for the DFP hydrolysis has been developed. (+info)
Reflective properties of iridophores and fluorescent 'eyespots' in the loliginid squid Alloteuthis subulata and Loligo vulgaris.
(59/1021)
Observations were made of the reflective properties of the iridophore stripes of the squid Alloteuthis subulata and Loligo vulgaris, and the likely functions of these stripes are considered in terms of concealment and signalling. In both species, the mantle muscle is almost transparent. Stripes of iridophores run along the length of each side of the mantle, some of which, when viewed at normal incidence in white light, reflect red, others green or blue. When viewed obliquely, the wavebands best reflected move towards the blue/ultraviolet end of the spectrum and their reflections are almost 100% polarised. These are properties of quarter-wavelength stacks of chitin and cytoplasm, predicted in theoretical analyses made by Sir A. F. Huxley and Professor M. F. Land. The reflecting surfaces of the individual iridophores are almost flat and, in a given stripe, these surfaces are within a few degrees of being parallel. Both species of squid have conspicuous, brightly coloured reflectors above their eyes. These 'eyespots' have iridescent layers similar to those found on the mantle but are overlaid by a green fluorescent layer that does not change colour or become polarised as it is viewed more obliquely. In the sea, all reflections from the iridophore stripes will be largely confined to the blue-green parts of the spectrum and all reflections in other wavebands, such as those in the red and near ultraviolet, will be weak. The functions of the iridophores reflecting red at normal incidence must be sought in their reflections of blue-green at oblique angles of incidence. These squid rely for their camouflage mainly on their transparency, and the ventral iridophores and the red, green and blue reflective stripes must be used mainly for signalling. The reflectivities of some of these stripes are relatively low, allowing a large fraction of the incident light to be transmitted into the mantle cavity. Despite their low reflectivities, the stripes are very conspicuous when viewed from some limited directions because they reflect light from directions for which the radiances are much higher than those of the backgrounds against which they are viewed. The reflective patterns seen, for example, by neighbouring squid when schooling depend on the orientation of the squid in the external light field and the position of the squid relative to these neighbours. (+info)
Fitness of the marine parasitic nematode Anisakis simplex s. str. in temperate waters of the NE Atlantic.
(60/1021)
In temperate waters of the NE Atlantic, third-stage larvae of Anisakis simplex sensu stricto collected from 3 paratenic host species were identified by restriction fragment length polymorphisms. The condition of wild larval infrapopulations was assessed by examining morphometric and growth characteristics. The differentiation patterns and the excretory/secretory products of larvae grown in vitro were also examined. An extensive morphometric, growth and differentiation variability was found between parasite larvae collected from different paratenic host sources. Nematode infrapopulation larvae from the squid comprise those smaller individuals with the lowest values of survival rates and moult success. It may be then concluded that the fitness of A. simplex s. str. larvae is not the best possible in the squid, which impaired the competitiveness of the parasite and its chances of developing into an adult. This suggests that the microenvironments impaired by the paratenic host may provide larval infrapopulations with unique ecological factors probably influencing its recruitment to the final host populations. (+info)
Organogenesis in Cephalopoda: further evidence of blastodisc-bound developmental information.
(61/1021)
The basic mechanisms of organ differentiation in the Cephalopod embryo (telolecithal egg, discoidal cleavage) are studied. The results of ligation experiments, performed in early cleavage stages, confirm earlier conclusions of the author, drawn from transplantation/explantation and heat-shock experiments. The developmental information for cellular differentiation is shown to reside in the blastodisc; the yolk syncytium, in which a large part of the original egg cortex is incorporated, acts as as nutritive substrate for the cellular material involved in organogenesis. On the basis of these results, Arnold's induction model supposing an undisplaceable determining informational pattern laid down in the uncleaved egg cortex must be rejected. (+info)
Potassium and sodium ion current noise in the membrane of the squid giant axon.
(62/1021)
1. The spectral density of current noise power from 20 mm segments of giant axons of the squid Loligo vulgaris has been measured under space-clamp and voltage-clamp conditions. From 4 to 1000 Hz the measured noise is larger by several orders of magnitude than the theoretical thermal noise. The amplifier's noise, which may yield appreciable contributions above 200 Hz, could be evaluated and subtracted from the total noise using direct measurements of the membrane impedance... (+info)
Long duration responses in squid giant axons injected with 134cesium sulfate solutions.
(63/1021)
Giant axons from the squid were injected with 1.5 M cesium sulfate solutions containing the radioactive isotopes 42K and 134Cs. These axons, when stimulated, gave characteristic long duration action potentials lasting between 5 and 45 msec. The effluxes of 42K and 134Cs were measured both under resting conditions and during periods of repetitive stimulation. During the lengthened responses there were considerable increases in potassium efflux but only small increases in cesium efflux. The selectivity of the delayed rectification process was about 9 times greater for potassium ions than for cesium ions. The data suggest that internal cesium ions inhibit the outward potassium movement occurring during an action potential. The extra potassium effluxes taking place during excitation appear to be reduced in the presence of cesium ions to values between 7 and 22% of those expected in the absence of cesium inhibition. (+info)
Voltage clamp studies on the effect of internal cesium ion on sodium and potassium currents in the squid giant axon.
(64/1021)
Isolated and cleaned giant axons of Loligo pealii were internally perfused with solutions containing cesium sulfate and potassium fluoride. Membrane currents obtained as a function of clamped membrane potentials indicated a severe depression of the delayed outward current component normally attributed to potassium ion movement. Steady-state currents showed a negative slope in the potential range from -45 to -5 mv which corresponded to the negative slope for the peak sodium current relation vs. membrane potential which suggested long duration sodium currents. Using sodium-free sea water externally, sodium currents were separated from total currents and these persisted for longer times than normal. This result suggested that internal cesium ion delays the sodium conductance turnoff. The separated nonsodium currents showed an abnormal rectification as compared with those predicted by the independence principle, such that while potassium permeability appeared normal at the resting potential, its value decreased progressively with increasing depolarization. (+info)