Quantitative comparison of dipole models for steady-state currents in excitable membranes.
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A group of homologous, nonmethylene-interrupted dienoic fatty acids (NMID) recently reported in oysters has been found in several other shellfish species and also in other marine phyla. The distribution of most other "normal" fatty acids among several species of shellfish is basically similar but mantle lipids from two other molluscan species, both squid, differ radically. The squid mantle fatty acids do not include NMID, suggesting that in molluscs NMID may accumula-e primarily in filter-feeders or herbovores. The distribution of these anomalous fatty acid components in higher species suggests that they reflect invertebrates in the diet and are biochemically inert.
The study of visual transduction has given invaluable insight into the mechanisms of signal transduction by heptahelical receptors that act via guanine nucleotide binding proteins (G-proteins). However, the cyclic-GMP second messenger system seen in vertebrate photoreceptor cells is not widely used in other cell types. In contrast, the retina of higher invertebrates, such as squid, offers an equally accessible transduction system, which uses the widespread second messenger chemistry of an increase in cytosolic calcium caused by the production of inositol-(1,4,5)-trisphosphate (InsP3) by the enzyme phospholipase C, and which may be a model for store-operated calcium influx. In this article, we highlight some key aspects of invertebrate visual transduction as elucidated from the combination of biochemical techniques applied to cephalopods, genetic techniques applied to flies, and electrophysiology applied to the horseshoe crab. We discuss the importance and applicability of ideas drawn from these model systems to the understanding of some general processes in signal transduction, such as the integration of the cytoskeleton into the signal transduction process and the possible modes of regulation of store-operated calcium influx.
The transport mechanism of Na ions within the nerve cell was studied by measuring the radioactivity distribution profile of 22Na that had been intracellularly injected into the giant axon. Specifically, we tested whether or not the movement of Na ions is coupled with the process of "fast axonal transport." Results of our measurements indicate that the intracellular transport of Na+ and the fast axonal transport are two independent processes. Very few Na ions are irreversibly sequestered into the axoplasmic vesicles involved in axonal transport. The movement of Na+ inside the axon can be modeled by a one-dimension diffusion. The effective diffusion coefficient of the intracellular Na+ was determined in this study.
PURPOSE: To measure the dynamic penetration process of lidocaine, lidocaine concentration (Ci) and pH (pHi) in squid giant axon, and to determine the times and Ci of disappearance and reappearance of action potentials (AP). METHODS: Lidocaine solutions adjusted to four different pHs (pH = 5.5, 6.8, 7.8 and 9.0) were externally administered to the axon and Ci and pHi were measured using lidocaine and pH microsensors. The times and Ci when the AP just disappeared and reappeared were recorded. In addition, for comparison with Ci, the lidocaine content in the whole axon (Cw) was measured with high-performance liquid chromatography (HPLC). RESULTS: The Ci (charged plus uncharged) was 1.5 times greater than the uncharged form of administered lidocaine. The changes in pHi depended on the increase in Ci. The AP disappeared only after administration of high pH lidocaine solutions (pH = 7.8, 9.0) and reappeared by washing out the solution in the chamber. Nerve block occurred more rapidly at pH 9.0 than at pH 7.8, and the time after washing out the lidocaine was longer at pH 9.0 than at pH 7.8. The mean Ci and charged lidocaine concentration in the axoplasm, when the AP disappeared or reappeared, were lower at pH 9.0 than at pH 7.8 (P < 0.05). CONCLUSION: Uncharged lidocaine penetrates the axon membrane to the axoplasm where it changes to the charged form and is concentrated in the axon membrane and axoplasm. External application of uncharged lidocaine plays a role in modulating nerve conduction.
Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages [3, 8, 24]. The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont [1]. Infection by intraspecific V. fischeri symbionts exhibited preferential colonization over interspecific V. fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains. Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids. These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host.
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The nucleotide sequence of a 9240-base pair DNA fragment of the mitochondrial (mt) genome of a squid, Loligo bleekeri, was determined, in which 8 protein and 14 tRNA genes were identified. The gene organization of the mt-genome exhibits a greater resemblance to the gene organization of arthropods and a chiton, Katharina tunicata, than to those of a mussel, Mytilus edulis, and land snails. A cloverleaf-like structure was observed between the genes for subunits 4 and 5 of NADH dehydrogenase (ND4 and -5), which is considered to have originated from histidine tRNA. It is presumed that this structure functions as a transcriptional punctuation signal for the maturation of the ND4 and ND5 mRNAs.
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Inactivation of the sapABCDF genes results in a loss of virulence in several bacterial pathogens of animals and plants. The role of this locus in the growth physiology of Vibrio fischeri, and in the symbiotic colonization of the squid Euprymna scolopes was investigated. In rich medium, a V. fischeri sapA insertion mutant grew at only 85% the rate of its wild-type parent. While a similar effect has been attributed to a potassium-transport defect in sap mutants of enteric bacteria, the V. fischeri mutant grew more slowly regardless of the potassium concentration of the medium. Similarly, the growth-rate defect was independent of the source of either carbon, nitrogen, or phosphorous, indicating that the V. fischeri sap genes do not encode functions required for the transport of a specific form of any of these nutrients. Finally, while a delay in colonizing the nascent light organ of the squid could be accounted for by the lower growth rate of the mutant, a small but statistically significant reduction in its final population size in the host, but not in medium, suggests that the sap genes play another role in the symbiosis. All of these phenotypic defects could be genetically complemented in trans by the sapABCDF genes, but not by the sapA gene alone, indicating that the insertion in sapA is polar to the four downstream genes in the locus. Thus, while the sap locus is important to the normal growth of V. fischeri, it plays different physiological roles in growth and tissue colonization than it does in enteric pathogens.
The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.
Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K(+) and HCO(3)(-) out of the cell, tending to lower intracellular pH (pH(i)). With an inwardly directed K/HCO(3) gradient, the cotransporter mediates a net uptake of alkali (i.e., K(+) and HCO(3)(-) influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA(+)) inhibit the inwardly directed cotransporter by interacting at the intracellular K(+) site. We computed the equivalent HCO(3)(-) influx (J(HCO3)) mediated by the cotransporter from the rate of pH(i) increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH(i) 8.0, using a dialysis fluid (DF) free of K(+), Na(+) and Cl(-). Our standard artificial seawater (ASW) also lacked Na(+), K(+) and Cl(-). After halting dialysis, we introduced an ASW containing 437 mm K(+) and 0.5% CO(2)/12 mm HCO(3)(-), which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO(2) influx, followed by a slower plateau-phase pH(i) increase, due to inward cotransport of K(+) and HCO(3)(-). With no QA(+) in the DF, J(HCO3) was approximately 58 pmole cm(-2) sec(-1). With 400 mm tetraethylammonium (TEA(+)) in the DF, J(HCO3) was virtually zero. The apparent K(i) for intracellular TEA(+) was approximately 78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K(+) channels. Introducing 100 mm inhibitor into the DF reduced J(HCO3) to approximately 20 pmole cm(-2) sec(-1) for tetramethylammonium (TMA(+)), approximately 24 for TEA(+), approximately 10 for tetrapropylammonium (TPA(+)), and virtually zero for tetrabutylammonium (TBA(+)). The apparent K(i) value for TBA(+) is approximately 0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA(+)), with an apparent K(i) of approximately 91 microm. Thus, trans-side quaternary ammonium ions inhibit K/HCO(3) influx in the potency sequence PPTEA(+) > TBA(+) > TPA(+) > TEA(+) congruent with TMA(+). The identification of inhibitors of the K/HCO(3) cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.
Raising the intracellular pH (pHi) above 7.7 in intracellularly perfused squid giant axons causes spontaneous firing of action potentials. The firing frequency ranged from 20 Hz at 0 degrees C to 200 Hz at 23 degrees C. Above 23 degrees C, the axons were quiescent. They were bistable for 13 <T <23 degrees C. That is, they were either quiescent or spontaneously firing. Below 13 degrees C, spontaneous firing was the only stable element. The primary effects of changes in temperature on the underlying ionic currents were on gating of the delayed rectifier potassium channel IK, and the sodium ion channel INa. The kinetics of IK had a Q10 of 3.63. The effect of T on INa was more complicated in that the peak INa amplitude increased with T, as demonstrated in earlier reports. This effect, as well as the changes in INa kinetics produced by changes in T, were mimicked in the context of a model of INa gating in which activation and inactivations are coupled. Electrical activity was simulated in a model of the action potential with appropriate temperature-dependent modifications for INa and IK. The model predicts a change from monostability (spontaneous firing) at relatively low temperatures to bistability (quiescence and spontaneous firing) as the temperature is raised, followed by change back to monostability (quiescence) as the temperature is further increased, which is consistent with the experimental results.
A ouabain sensitive inward current occurs in Xenopus oocytes in Na+ and K(+)-free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pHo) produces a conducting pathway through the Na+/K+ pump that is permeable to H+ and blocked by [Na+]o. An alternative suggestion is that the current is mediated by an electrogenic H(+)-ATPase. Here we investigate the effect of pHo and [Na+]o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pHo the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pHo, as predicted by a model in which protonation of the Na+/K+ pump reduces the energy barrier between the internal solution and the Na+ occluded state. The model also predicts that acidic pHo increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is approximately 6, suggesting that histidine is involved in induction of the conductance pathway. 22Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pHo suggest that both Na+ and H+ are permeant. The acid-induced inward current is reduced by high [Na+]o, consistent with block by Na+. A least squares analysis predicts that H+ is four orders of magnitude more permeant than Na+, and that block occurs when 3 Na+ ions occupy a low affinity binding site (K(0.5) = 130 +/- 30 m M) with a dielectric coefficient of 0.23 +/- 0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na+ and H+ through the Na+/K+ pump.
SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) alpha-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (IK) if cultured at the standard postinfection temperature of 27 degrees C. At 18 degrees C, less SqKv1A protein is produced than at 27 degrees C, but cells with IK currents are much more numerous and can survive for at least 20 days postinfection (vs. approximately 5 days at 27 degrees C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (approximately 3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of IK inactivation in 20 mM external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL IK is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.