Crayfish Tail Muscle Research Articles

Integrating transcriptomic and metabolomic analysis to understand muscle qualities of red swamp crayfish (Procambarus clarkii) under transport stress

This study investigated the transport stress (crowding stress and duration) on the physicochemical properties, energy metabolism and antioxidant enzyme activities of the red swamp crayfish (Procambarus clarkii) tail muscle (CTM). Besides, transcriptomic and metabolomic were conducted to elucidate the possible mechanism of CTM alternations during transport stress. The survival rate of crayfish gradually decreased with the external crowding stress and crowding time increasing. The transport stress also led to the increased distance among muscle fibers, water mobility and energy consumption, and the decreased of water holding capacity (WHC), hardness of CTM. The hepatopancreas exhibited more sensitive to crowding stress than muscle. The multi-omics analysis revealed that transport stress could interfere the translation and protein folding functions of ribosomal proteins, fatty acid metabolism and degradation, physiological functions of mitochondria in CTM. This study could provide critical information to increase the understanding of the regulation mechanism of crayfish when subjected to transport stress.

Comparison between lipid and protein oxidation progress in the tail and claw muscles of signal crayfish (Pacifastacus leniusculus)

AbstractMicrobial activity and then oxidation progress are the most important freshness indicators during post‐mortem. In this study, we monitored proteomic and microbial changes, as well as biochemical degradation, in the tail and claw muscles of crayfish stored for 12 days at +4 °C. One specified protein band at 107 kDa in the claw muscle and two specified protein bands in the tail muscle at 140 and 36–40 kDa were identified. Western blotting indicated a higher amount of oxidised proteins in the tail compared to the claw muscle. Tail muscle showed higher oxidation progress and calpain activity than claw one. Both muscles were spoiled after 12 days with respect to total viable counts. In the first days, calpain activity is the main reason for protein degradation, while protein oxidation dominates for the rest of the time. Lipid–protein oxidation progress showed probably, protein oxidation started earlier than lipid oxidation in both muscles.

Developing metabolomics-based bioassessment: crayfish metabolome sensitivity to food and dissolved oxygen stress.

There is a need to develop bioassessment tools that can diagnose the effects of individual stressors that can have multiple ecological effects. Using nuclear magnetic resonance (NMR)-based metabolomics, our experiments aimed to identify the sensitivity of metabolites to changes in food availability and dissolved oxygen (DO) concentrations, and compare these results to identify metabolites that may differentiate between the effects of these two stressors. Forty-eight, laboratory-raised, red swamp crayfish (Procambarus clarkii) were randomly assigned and exposed to one of three food availability or DO treatment levels (high, normal, low). Starved crayfish had lower amounts of amino acids than fed crayfish, suggesting catabolic effects of starvation on tail muscle tissue for energy requirements. In contrast, crayfish exposed to hypoxic conditions experienced changes in abundance of metabolites primarily associated with energy metabolism. Tail muscle was the only tissue sensitive to food and DO stress, suggesting the need to select tissues for monitoring appropriately. Our evaluation of environmental metabolomics as a tool for bioassessment indicates that several identified metabolites in crayfish tail muscle may be able to diagnose food and oxygen stress. Further study is required to determine if these metabolic effects are linked with changes of individual fitness and higher levels of biological organization, such as population size.

Mechanistic understanding of low methylmercury bioaccessibility from crayfish (Procambarus clarkii) muscle tissue

Recent research indicates that dietary exposure to mercury and other metals from crayfish consumption poses a human health concern, particularly in regions with high crayfish-consuming populations. To better understand consumption risk from methylmercury (MeHg), we quantified MeHg bioaccessibility in edible tail muscle of cooked red swamp crayfish (Procambarus clarkii, collected from seven cities in China), versus cooked fillet tissue of two finfish species: yellow croaker (Larimichthys polyactis) and snakehead (Channa argus). Results indicated that digestive solubilization rate (DSR) of MeHg in crayfish (7.8±3.9% for restaurant-crayfish and 9.8±0.8% for market-crayfish) was lower than the rate in yellow croaker (25.8±2.7%) and snakehead (26.2±4.7%) tissue, suggesting that relatively low MeHg bioaccessibility in crayfish may reduce dietary exposure to humans. Three possible mechanisms for the reduced MeHg DSR in crayfish tissue were examined: MeHg-Se interactions, MeHg subcellular fractionation, and Hg-amino acid binding. Selenium concentrations were comparable among the examined species, and no significant relationship was observed between tissue Se and MeHg DSR. Similarly, observed differences in subcellular fractionation of MeHg could not explain the species-specific MeHg DSR. Therefore, MeHg-Se interactions and MeHg subcellular fractionation do not explain the relatively low MeHg bioaccessibility in crayfish. Significantly higher cysteine and arginine content was found in crayfish than in the finfish. We suspect that the lower MeHg bioaccessibility of crayfish tail muscle may be attributed to the higher cysteine concentrations, and thus, stronger MeHg-protein binding in crayfish. These results support the interpretation that bioaccessibility differences will alter risk interpretations for MeHg, especially when comparing hazard across aquatic food types.

Regulation of crayfish, Orconectes virilis, tail muscle lactate dehydrogenase (LDH) in response to anoxic conditions is associated with alterations in phosphorylation patterns.

Lactate dehydrogenase (LDH), the terminal enzyme of anaerobic glycolysis, has a crucial role in sustaining ATP production by glycolysis during periods of anoxia via regenerating NAD+ through the production of lactate. The present study examined the effects of prolonged (20h) anoxic submergence on LDH from the tail muscle of an anoxia-tolerant crayfish (Orconectes virilis). LDH was purified to homogeneity from tail muscle of both aerobic control and anoxic crayfish in a three step process. Analysis of the kinetic parameters and the stability of LDH showed that the Vmax in the pyruvate-reducing direction was significantly higher for the enzyme from anoxic crayfish whereas in the lactate-oxidizing direction the Vmax was significantly higher for the control enzyme. Differential scanning fluorimetry was used to assess thermal unfolding of crayfish LDH. The results showed that the enzyme from control muscle had a significantly higher melting temperature (greater thermal stability) than the anoxic enzyme form, suggesting that there was a structural difference between the two enzyme forms. Immunoblotting of purified LDH implicated post-translational modification as the reason for this difference; purified LDH from aerobic control crayfish showed significantly higher amounts of serine/threonine phosphorylation than did the anoxic enzyme form. This study provides evidence for anoxia-induced modifications of crayfish muscle LDH that may contribute significantly to modulating enzyme function under anoxic conditions.

Human exposure to methylmercury from crayfish (Procambarus clarkii) in China.

Methylmercury (MeHg) accumulation in aquatic food raises global concerns about human exposure to MeHg. Crayfish is the world's third largest farmed crustacean species and a favorite aquatic food in many countries. However, human health hazard due to MeHg exposure via crayfish consumption is unclear, partly because appropriate survey data are lacking. We report on mercury concentrations and speciation in edible tail muscle of crayfish collected from restaurants in 23 Chinese cities. On average, MeHg constituted 99.1% of mercury in tail muscle, and MeHg concentrations were comparable with those reported for fish in China. Variation in MeHg concentrations was not attributable to broad geographic region (i.e., provinces) or tail length. For different populations, potential health risk (characterized by hazard quotient or HQ) of MeHg exposure through crayfish consumption depended largely on crayfish consumption rates. In particular, a health hazard (HQ>1) was found for high-rate consumers (i.e., 95%ile or higher) in some cities in the middle and lower reaches of the Yangtze River (MLYR), during the peak consumption season. Our results suggest that more attention should be paid to dietary MeHg intake via crayfish consumption in China, particularly for communities with high consumption in MLYR.

Fatty acid, cholesterol and fat-soluble vitamin composition of wild and captive freshwater crayfish (Astacus leptodactylus)

The proximate analysis (dry matter, protein, fat and ash), cholesterol, fatty acid and fat-soluble vitamin compositions of the tail muscle of wild caught and captive crayfish (Astacus leptodactylus) were investigated. Captive crayfish contained higher moisture and fat content than wild crayfish. In contrast, wild crayfish contained a higher level of crude protein, ash and n-3 polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) than captive crayfish. Arachidonic acid (C20:4 n-6) was the major n-6 PUFA in wild A. leptodactylus, and linoleic acid (C18:2 n-6) was the major n-6 PUFA in captive A. leptodactylus. The percentages of total saturated fatty acids (SFA), PUFA, and n-3/n-6 ratio were higher in wild crayfish and total monounsaturated fatty acids (MUFA) were lower. Although differences existed between wild and captive crayfish in vitamins A (p < 0.001), δ-Tocopherol (p < 0.001), α-Tocopherol acetate (p < 0.05), no differences were found in vitamins D(2), D(3), α- Tocopherol and K (p > 0.05). The differences may be originated from the diet provided to captive crayfish. Since wild A. leptodactylus contained higher n-3/n-6 ratio than captive A. leptodactylus, crayfish farms can potentially produce a better quality of crayfish meat by increasing the PUFA n-3 (especially DHA and EPA) in the diets of A. leptodactylus.

Mercury in the Northern Crayfish, Orconectes virilis (Hagen), in New England, USA

Biologists and policy makers continue to seek environmental correlates of mercury bioavailability in aquatic ecosystems. In this study, we assessed the effects of drainage basin, habitat type, size class, and sex on mercury concentrations in the northern crayfish, Orconectes virilis (Hagen). Drainage basin, habitat type, and size class had significant effects on mercury concentration in crayfish tail muscle even though animals from roughly half the sites examined had mean mercury values at or below expected background levels. The low observed mercury values in crayfish tail muscle indicate a low consumptive risk. Contrary to expectations, crayfish from brooks had higher mercury concentrations than animals from other habitat types, possibly as a result of point source contamination or varying diet compositions among habitats. We suggest that crayfish represent a good indicator of mercury bioavailability in aquatic ecosystems and provide a synthesis for lower food webs. Our understanding of mercury dynamics in lower food webs has been hindered by an under appreciation of the complexity in foraging habits of macroinvertebrates. Further studies focusing on benthos with well-understood life histories and foraging behavior are essential to improve our understanding of mercury transfer and bioavailability through aquatic systems.

Icon® rice seed treatment toxicity to crayfish (Procambarus clarkii) in experimental rice paddies

Outdoor pools (2.3 x 2.3 m) were used to simulate typical rice agricultural practices in Louisiana, USA, to evaluate the toxicity of ICON (active ingredient [a.i.] fipronil) and its degradates to crayfish (Procambarus clarkii). Six paddies were planted with seed treated with ICON 6.2 FS at an exaggerated application rate of 0.05 kg a.i./ha (recommended rate, 0.042 kg a.i./ha), simulating three rice-planting scenarios. Two reference paddies were planted with untreated seed. Crayfish were exposed to tail water within 24 to 48 h after seeding, simulating standard Louisiana agricultural and water management practices. At 50 d after planting, a separate group of crayfish was caged in situ for 14 d to evaluate toxicity. An additional 50 crayfish were added to two paddies approximately 100 d after rice planting and held for 29 weeks to evaluate bioaccumulation. Residues of fipronil and its degradates in water and soil were similar to residue concentrations measured from rice fields in Louisiana. Tail water from the treated paddies was not toxic to crayfish. The fipronil 96-h median lethal concentration (LC50) for adult crayfish was 180 microg/L, which would provide at least a sixfold safety factor between the maximum fipronil concentration in tail water and the crayfish LC50. In situ exposures of crayfish also were not toxic. Concentrations of fipronil and its degradates after 29 weeks of exposure were less than 5 microg/kg in crayfish tail muscle tissue. These results demonstrate that label instructions adequately protect crayfish in a rice-crayfish cropping scenario when ICON is applied at maximum application rates as a seed treatment.

Time domain dielectric spectroscopy of crayfish tail muscle, measured in vivo

We measured the dielectric spectra of crayfish tail muscle, in vivo, in both the time and frequency domains. We inserted a linear array of four needle electrodes (denoted 1, 2, 3 and 4) into the tail muscle. For time domain dielectric spectroscopy (TDDS), we used the MacADIOS data acquisition system to apply a voltage step of amplitude V 0 and to sample the resulting voltages across electrodes 1 and 2 and also across 3 and 4 as well as the current, I, that flowed through a series resistor during polarization and depolarization. Fourier transformation of the variation of the conductance between 2 and 3 with time yielded the capacitance and conductance spectra of the muscle from about 1 kHz down to 0.1 Hz with electrode effects presumably eliminated. A Hewlett-Packard 4192A low-frequency impedance analyzer (IA) measured directly the capacitance and conductance spectra between electrodes 2 and 3 from 100 Hz to 1 MHz. The TDDS results exhibited a strong dispersion for frequencies on the order of 10 Hz that could be characterized by a KWW response ( T∼0.1 s and β∼0.8). We modeled the IA results as a Cole–Cole dispersion with n=0.5.

Effects of starvation and d- or l-alanine administration on the free d- and l-alanine levels in the muscle and hepatopancreas of the crayfish, Procambarus clarkii

Changes of d- and l-alanine and other free amino acids were examined in muscle and hepatopancreas of the crayfish Procambarus clarkii before and after a 1-month starvation in freshwater, 50% seawater, and seawater. Total free amino acids and d- and l-alanine in both tissues decreased during starvation. The largest decrease was found in the animals starved in freshwater. In 50% seawater and full-strength seawater, the ratio of d-alanine to total alanine increased in both tissues, although the level was reduced a little. A large dose of l-alanine administered into the tail muscle of freshwater crayfish was converted to d-alanine and l-alanine returned to the control level within a week. A large amount of l-alanine was transported from muscle to hepatopancreas. d-Alanine injected into freshwater crayfish was converted to l-alanine, but was not transported to the hepatopancreas. In the case of l-alanine injection into the muscle of crayfish acclimated to 50% seawater, large amounts of d- and l-alanine were maintained in the muscle during the experimental period of 4 days. These data indicate that d-alanine is metabolized actively in crayfish tissues and accumulated as an important osmolyte during osmotic stress.

Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata

From the pharyngeal baskets of the ascidians Microcosmus sulcatus and Phallusia mammilata we have purified an 85-kDa protein that is characterized as a member of the gelsolin family. These proteins from both species show the same behaviour in functional assays. The ascidian gelsolin binds two actin monomers in a highly cooperative manner. This complex formation is Ca 2+-dependent, but not completely reversible, as on removal of Ca 2+ one actin monomer dissociates leaving a 1:1 complex between gelsolin and G-actin. The properties of F-actin severing and G-actin nucleation depend on the presence of free Ca 2+ in a micromolar range, with half maximum activation at about 3×10 −6 M. The protein becomes inactivated when Ca 2+ concentrations of 0.5 mM are exceeded. Fragmentation of F-actin by the ascidian gelsolin is comparably fast to that of vertebrate gelsolin. A steady state of actin fragmentation is reached within 2–4 s. Promotion of G-actin nucleation is also comparable to that of vertebrate gelsolin. Regarding functional aspects, the ascidian gelsolin is more closely related to vertebrate gelsolin than to an arthropod gelsolin from crayfish tail muscle.

Amino Acid Sequence of C-Terminal 17 kDa CNBr-Fragment of Akazara Scallop Troponin-I1

The M(r) 52,000 subunit of Akazara scallop striated muscle troponin, which was tentatively identified as troponin I, was cleaved into two major fragments with CNBr: C-terminal 17 kDa fragment (CN17K) and N-terminal 35 kDa fragment (CN35K) [J. Biochem. 108, 519-521 (1990)]. CN17K inhibits rabbit reconstituted actomyosin Mg-ATPase activity, weakly in the absence of troponin T but strongly in its presence, together with Akazara tropomyosin. CN35K, however, hardly shows such inhibition. Thus, the amino acid sequence of the CN17K was determined by the Edman method. CN17K comprises 135 amino acid residues and its calculated molecular mass is 15,732 Da. A computer search of the SWISS-PROT data base revealed the TnIs of crayfish tail muscle, rabbit skeletal muscle, and bovine cardiac muscle to be homologous proteins with total sequence homologies of 39, 30, and 30%, respectively, to CN17K. Significantly high homology was observed among these TnIs in the regions around residues 75-95, 99-114, and 135-151 of the rabbit TnI. From these facts, we conclude that the 52K subunit is a TnI.

A gelsolin-related actin-severing protein with fully reversible actin-binding properties from the tail muscle of crayfish, Astacus leptodactylus.

A Ca(2+)-dependent actin-severing protein was purified from the tail muscle of the crayfish Astacus leptodactylus. The isolation procedure involved extraction at low ionic strength in the presence of EGTA, followed by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The purified crayfish actin modulator appeared as a single band with a molecular mass of 105 kDa on SDS/PAGE. The crustacean actin modulator revealed basic functional properties in common with vertebrate gelsolin, like the Ca(2+)-activated severing of F-actin and the nucleation of actin polymerization. However, both proteins differed in major aspects: Ca2+ activation of crayfish actin modulator started at lower threshold concentrations (0.1 microM). The effect of the modulator on shortening the nucleation phase of actin polymerization was significantly weaker at lower modulator/actin ratios. The modulator formed three distinct stoichiometric complexes with G-actin, identified as binary, ternary and quaternary. Binding of G-actin occurred in a low cooperative manner and was completely reversible by EGTA. Despite some properties being similar to those of villin, crayfish actin modulator did not cross-link actin filaments. It is regarded in principle as a gelsolin-type protein, but with characteristic functional deviations from vertebrate gelsolin.

Purification and Molecular Cloning of Arginine Kinase from Tail Muscle of Crayfish, Procambarus Clarkii.

We have purified adenosine triphosphate: arginine phosphotransferase (EC2-7-3-3, arginine kinase) and molecularly cloned the cDNA from the tail muscle of crayfish, Procambarus clarkii. An open reading frame encodes 357 amino acid residues with a calculated molecular mass of 40, 118 dalton. The predicted amino acid sequence shows 60% similarity with that of human creatine kinase B-subunit. The expression of a full-length cDNA in COS7 cells gave rise to a product of 40kDa and conferred the arginine kinase activity on mammalian cells. The amino acid sequence surrounding cys271 is well conserved as an active site for the catalytic function among a family of phosphagen kinase. We have found that chemical modification of a single cysteinyl residue of arginine kinase inhibits the enzymatic function and ATP protects the enzyme against this modification.

Amino acid sequences of the two major isoforms of troponin C from crayfish

The primary structure of the two major isoforms (alpha and gamma) of troponin C (TnC) from crayfish tail muscle has been determined by the application of manual and automated Edman degradation procedures to fragments generated by suitable chemical and proteolytic cleavages. Both amino acid sequences commence with an acetylated methionyl residue and contain 150 amino acid residues, including a single proline residue at position 29 and 2 residues of tyrosine at positions 95 and 102. No cysteine or tryptophan are present. The molecular weights calculated for alpha- and gamma-TnC are 17,157 and 16,974, respectively. The two crayfish proteins are invariable at 129 positions and conserved at 11 others. Pairwise comparisons show that the two sequences are 33-39% identical with those of seven TnCs reported so far and 39% identical with that of bovine brain calmodulin. The N-terminal end of about 10 residues, found in vertebrate TnCs, is absent in crayfish TnCs. In the latter proteins, domains I and III appear as abortive Ca2+-binding sites due to nonconservative amino acid replacements at the key Ca2+-coordinating positions in their loops. The remaining two Ca2+-binding loops (II and IV) show a remarkable similarity with the Ca2+-specific loops (I and II) found in vertebrate TnCs. These findings are consistent with the Ca2+-binding data (Wnuk, W. (1989) J. Biol. Chem. 264, 18240-18246) which indicate the presence of two Ca2+-specific sites in crayfish TnCs. These two sites display the same affinity for Ca2+ (log KCa = 4.3) on gamma-TnC but differ in their affinity (log KCa = 6.0 and 4.1) on alpha-TnC. The only structural difference between the dodecapeptide loops II and IV in both alpha- and gamma-TnC, which correlates with the existence of the high affinity (log KCa = 6.0) Ca2+-specific site on alpha-TnC, is position 11 occupied by a methionyl residue in the loop IV of alpha-TnC as opposed to negatively charged residues found in the other three loops. This suggests that the high affinity Ca2+-specific site on alpha-TnC is located in domain IV. Since the Ca2+-binding studies show that the formation of the complex of crayfish troponin I (TnI) with alpha- and gamma-TnC increases significantly the affinity of only one of their two Ca2+-specific sites and this TnI-sensitive site is not the high affinity Ca2+-specific site on alpha-TnC, we conclude that the binding of Ca2+ to site II controls the Ca2+-dependent interaction between crayfish TnCs and TnI.

Resolution and calcium-binding properties of the two major isoforms of troponin C from crayfish

Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.

L‐[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.

Amino Acid Sequence of Crayfish Troponin I

Troponin I is the actomyosin ATPase inhibitory subunit present in the thin filament regulatory complex. The complete amino acid sequence of crayfish tail muscle troponin I has been determined. The protein is composed of 201 amino acid residues and has a molecular weight of 23,547. The N terminus is blocked, likely by an acetyl group. Crayfish troponin I shows a rather low (20-25%) sequence identity with vertebrate troponin Is as compared to the 60-82% identity within the vertebrate phylum. Similar to vertebrate cardiac troponin I, crayfish troponin I contains a 30-residue-long N-terminal extension. In crayfish troponin I, this segment bears significant sequence homology with the heavy or light chains of particular myosins. The actin-binding domain of crayfish troponin I, which displays 57% sequence homology with vertebrate troponin Is, possesses 2 unusual trimethyllysine residues. The consensus sequence of this domain in five troponin Is is as follows: D-L-R-G-K-F-X-R*-P-X-L-R*-R*-V, where R+ stands for Arg/Lys, R* for Arg/trimethyllysine, and X for any amino acid residue. Troponin I possesses two Ca2+-dependent interactive sites for troponin C; one partly overlaps with the actin binding domain and is highly conserved, and the other, corresponding to the 30-residue-long segment following the N-terminal extension in vertebrate cardiac and crayfish troponin I, is poorly conserved in the different troponin Is. Troponin I also interacts with troponin T. The consensus sequence for the interacting site on troponin I is as follows: h-D- -X-D- -R+-Y-D-h-E-h, where h stands for a hydrophobic residue, D- for Asp/Glu, R+ for Arg/Lys, and X for any residue. The five troponin Is further possess one more 15-residue-long segment of high sequence identity near the C terminus. Its evolutionary conservation suggests that this domain is involved in protein-protein interaction.

The excitatory glutamate-activated channel recorded in cell-attached and excised patches from the membranes of tail, leg and stomach muscles of crayfish

Glutamate activated, excitatory single channel currents were recorded from 5 different muscles of crayfish (Austropotamobius torrentium) from abdomen, legs and stomach. Cell-attached and outside-out excised membrane patches with GΩ-seals were studied. At −70 mV membrane potential and 19 °C, single channel currents activated by 0.5 mM glutamate had an amplitude of −7.6 pA, a mean open time of 0.22 ms and a mean burst length of 0.58 ms. These values did not show significant differences in all muscles investigated. The distributions of open times and of burst durations were described by single exponentials. The distributions of closed times could be fitted only by at least two exponentials. The short component of on average 0.1 ms represented closings within bursts, a longer component of on average 0.9 ms grouping of bursts. Burst durations (but not individual open times) increased with rising glutamate concentration: the relative open time of the channel was approximately proportional to glutamate concentration between 0.1 and 5 mM. The channels described above could not be activated by the glutamate analogues kainate and NMDA, but were about 10 times more sensitive to quisqualate than to glutamate. Quisqualate elicited single channel currents of the same amplitude as those triggered by glutamate. Compared at the same concentrations, channel open times and burst durations were about 4 times longer in quisqualate than in glutamate. A model describing the kinetics of the glutamate-activated excitatory channels is discussed. In addition, a type of Ca-independent, depolarization-activated K+-channel is reported.