Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Direct Evaluation of Effects of Fatty-Acid Unsaturation on the Thermal Properties of Photosynthetic Activities, as Studied by Mutation and Transformation of Synechocystis PCC6803

Glycerolipids of thylakoid membranes isolated from the cyanobacteriumSynechocystis PCC6803 contained high levels of dienoic and trienoicC₁₈ fatty acids, in addition to saturated C₁₆ and monoenoicC₁₈ fatty acids. A mutant (Fadl2) of this cyanobacterium wasdefective in the desaturation of C₁₈ fatty acids at the ¹² position,and its thylakoid membranes lacked trienoic acids and containeda very reduced level of dienoic acids. A derivative strain ofFadl2 (Fadl2/desA), which had been transformed with a gene fordesaturation at the ¹² position, fully recovered the abilityto desaturate the fatty acids in the glycerolipids of thylakoidmembranes. The thermal properties of the photosynthetic activitiesof the mutant and the transformant were compared with thoseof the wild-type strain. Despite great diversity in the extentof unsaturation of fatty acids between the wild-type, Fadl2,and Fad12/desA strains, no significant differences were foundeither in the temperature dependence of photosynthesis or inthe heat stability of photosynthetic, photosystem II and photosystemI activities. These results demonstrate that the trienoic fattyacids and, probably, the dienoic acids of the lipids in thethylakoid membrane do not affect the thermal properties of theabove-mentioned activities of photosynthesis. ³Permanent address: Institute of Plant Physiology, BiologicalResearch Center of Hungarian Academy of Sciences, H-6701 Szeged,P.O. Box 521, Hungary (Received August 9, 1990; Accepted December 7, 1990)     

Activation of 20S Proteasomes from Spinach Leaves by Fatty Acids

In order to clarify the mechanism of activation of plant 20Sproteasomes by fatty acids, we examined the effects of oleic,linoleic and linolenic acids on the three peptidase activitiesof purified 20S proteasomes from spinach leaves and comparedthem with the effects of SDS, a previously characterized activatorof 20S proteasomes. The three fatty acids all activated thehydrolysis of succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide(Suc-LLVYMCA) and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide(Cbz-LLE-2NA) at low concentrations (one-third to one-sixthof that required for activation by SDS). The range of concentrationsof linolenic acid for the activation of Suc-LLVY-MCA hydrolysiswas very narrow. All the fatty acids inhibited the hydrolysisof tert-butoxycarbonyl-Leu-Arg-Arg-4-methylcoumaryl-7-amide(Boc-LRR-MCA)at extremely low concentrations (one-fifth to one-fifteenthof that required for the activation of the hydrolysis of Suc-LLVY-MCAand Cbz-LLE-2NA). In the case of hydrolysis of Suc-LLVY-MCA,SDS and the three fatty acids increased the V_max value and decreasedthe apparent K_m value to similar relative extents. In the caseof hydrolysis of Boc-LLE-MCA, SDS and the three fatty acidsalso decreased the K_m and increased the V_max. However, SDS markedlyincreased V_max. The curves representing the SDS-dependent activationwere shifted to a lower range by the addition of linoleic acid,but the maximum activity at the optimum concentration of SDSwas essentially unchanged. These results suggest that the activationby SDS and that by the fatty acids has an additive effect. Theresults imply that fatty acids, such as linolenic acid, mightact as physiological regulators in plant cells. (Received April 10, 1995; Accepted December 22, 1995)     

Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

Temperature-Induced Changes in the Fatty Acid Composition of the Cyanobacterium, Synechocystis PCC6803

Changes in glycerolipid and fatty acid composition with a change in growth temperature were studied in the cyanobacterium, Synechocystis PCC6803. Under isothermal growth conditions, temperature did not significantly affect the composition of the various classes of lipids, but a decrease in temperature altered the degree of unsaturation of C₁₈ acids at the sn-1 position, but not that of C₁₆ acids at the sn-2 position of the glycerol moiety in each class of lipids. When the growth temperature was shifted from 38°C to 22°C, the desaturation of C₁₈ acids, but not that of C₁₆ acids, was stimulated. The desaturation of fatty acids occurred only in the light and was inhibited by chloramphenicol, rifampicin and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, but not by cerulenin, an inhibitor for fatty acid synthesis. These findings suggest that desaturase activities are induced after a shift from a higher to a lower temperature, and that the desaturation of fatty acids is connected with the reactions involved in photosynthetic electron transport.     

Betaine Lipids in Lower Plants. Biosynthesis of DGTS and DGTA in Ochromonas danica (Chrysophyceae) and the Possible Role of DGTS in Lipid Metabolism

Membrane lipids and fatty acids of Ochromonas danica were analyzed.Of the two betaine lipids, the homoserine lipid DGTS mainlycontains 14:0 and 18:2 fatty acids, while the alanine lipidDGTA is enriched in 18:0, 18:2 and 22:5 fatty acids. Of thepolar moiety of DGTA, improved NMR data are presented. On incubationof cells with [3,4-¹⁴C]methionine, DGTS as well as DGTA werelabelled. With [1-¹⁴C]methionine as a substrate, the label appearedin DGTS only. If double labelled [³H](glycerol)/[¹⁴C](polarpart)DGTS was used as a precursor, radioactivity was incorporatedspecifically into DGTA in which the isotope ratio was unchangedcompared to the precursor. Thus, the glyceryltrimethylhomoserinepart of DGTS acts as the precursor of the polar group of DGTA.Labelling of cells with [1-¹⁴C]oleate in a pulse-chase mannerand subsequent analysis of the label in the fatty acids andmolecular species of different lipids including DGTS and DGTA,suggested a clearly different role of the two betaine lipids:DGTS acts as a i) primary acceptor for exogenous C₁₈ monoeneacid, ii) substrate for the desaturation of 18:1 to 18:2 acid,and iii) donor of mainly 18:2 fatty acid to be distributed amongPE and other membrane lipids. Into DGTA, in contrast, fattyacids are introduced only after elongation and desaturation.As a result, the biosynthesis of DGTA from DGTS involves a decarboxylationand recarboxylation of the polar part and a simultaneous deacylationand reacylation of the glycerol moiety. (Received January 28, 1992; Accepted March 11, 1992)     

Seasonal Changes in Activities of Enzymes Responsible for the Formation of C6-aldehydes and C6-alcohols in Tea Leaves, and the Effects of Environmental Temperatures on the Enzyme Activities

When tea leaves were homogenized and incubated, the volatileC₆-compounds hexanal, cis-3-hexenal, cis-3-hexenol and trans-2-hexenalwere formed much more by summer leaves than by winter leavesof tea plants (Camellia sinensis). The enzymes lipolytic acylhydrolase (LAH), lipoxygenase, fatty acid hydroperoxide lyase(HPO lyase) and alcohol dehydrogenase (ADH) and an isomerizationfactor were responsible for the sequential reactions of C₆-compoundformation from linoleic and linolenic acids in tea leaf lipids,and there were seasonal changes in their activities. The tealeaf enzymes were of 3 types: LAH and lipoxygenase, which hadhigh activities in summer leaves and low activities in winterleaves; ADH, which had low activity in summer leaves and highactivity in winter ones; and HPO lyase and the isomerizationfactor, which did not seem to have any effect on the rate ofC₆-compound formation throughout the year. Changes in enzymeactivities were induced by shifts in the environmental air temperaturerather than by the age of the leaves. The combined activitiesof these enzymes determined the amounts and compositions ofthe volatile C₆-compounds formed, which are the factors thatcontrol the quality of the raw leaves processed for green tea. (Received October 6, 1983; Accepted December 20, 1983)     

Inhibition of Ethylene Production by Fatty Acids in Fruit and Vegetable Tissues

Fatty acids of chain length from C₄ to C₁₂ inhibited ethyleneproduction in wounded albedo tissue of Hassaku (Citrus hassakuHort. ex Tanaka) fruit. Of the fatty acids tested, caprylicacid (C₈) and capric acid (C₁₀) were the most effective. Lauricacid (C₁₂) was less effective, and caproic acid (C₆) and butyricacid (C₄) were the least effective. Caprylic acid at 5 mM markedlyinhibited ethylene production in not only wounded albedo tissueof citrus fruit but also apple (Malus sylvestris Mill.) cortex,tomato (Lycopersicon esculentum Mill.) pericarp, cucumber (Cucumissativus L.) cortex, banana (Musa AAA group Cavendish subgroup)pulp, broccoli (Brassica oleracea L.) floret, spinach (Spinaciaoleracea L.) leaf, lettuce (Lactuca sativa L.) leaf and mungbean (Vigna radiata [L.] Wilczek) hypocotyl. Caprylic acid inhibitedethylene production at the step of conversion of l-aminocyclopropane-l-carboxylicacid to ethylene. The inhibition could be partially relievedby transferring the tissue to caprylic acid-free medium. (Received June 15, 1982; Accepted August 13, 1982)     

Lipid and Fatty-acid Composition of Diatoms

The lipids and fatty acids of two freshwater diatoms Nitzschiapalea Kutz, Navicula muralis Lewin, and one marine species,Navicula incerta Grun. have been studied. The major lipid components in all species were triglycerides,monogalactosyl, digalactosyl and sulphoquinovosyl diglycerides,phosphatidyl glycerol, phosphatidyl choline (lecithin), andphosphatidyl ethanolamine; while palmitoleic, palmitic, eicosapentaenoicand eicosate-traenoic acids were the major fatty acid constituents.The two galactolipids, monogalactosyl and digalactosyl diglyceridescontained large amounts of C₁₆ and C₂₀ polyunsaturated fattyacids. Lipids of diatoms, whether grown in the light or in the dark,were the same apart from quantitative differences. More storagelipids such as triglycerides were synthesized in the light thanin the dark.     

Acyl-CoA Synthetase in Maturing Safflower Seeds

Acyl-CoA synthetase in maturing seeds of safflower (Carthamustinctorius) was membranebound, and the highest specific activitywas associated with microsomes. Activity absolutely dependedon the concentrations of fatty acid, CoA, ATP and Mg²⁺. Theapparent K_m values were 4.2 µM for oleate, 24 µMfor CoA, and 250 µM for ATP. The optimum pH of the reactionwas 7.5. Triacsin C, a potent inhibitor of the animal and bacterialacyl-CoA synthetase, was ineffective for the safflower enzyme.The enzyme utilized C₁₆ and C₁₈ long-chain fatty acids preferentially,while medium-chain and very-long-chain fatty acids were poorsubstrates. The order of specificity for native fatty acidswas linoleate > oleate=palmitate > stearate. Althoughactivity per seed varied during seed maturation, it was enoughto account for the rate of triacylglycerol synthesis in vivo. (Received February 2, 1993; Accepted March 3, 1993)     

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C₁₈ fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.     

Unsaturated Fatty Acid Synthesis During the Development of Isolated Sunflower (Helianthus annuus L.) Seeds

The effect of temperature on unsaturated fatty acid synthesisin developing sunflower seed embryos (Helianthus annuus L.)has been studied using isolated seeds grown in culture. Variabilitybetween individual embryos in the response to temperature wasalso investigated. Oil and dry matter accumulation in cultured embryos were similarto those of embryos allowed to develop in intact plants, andthe effect of increasing temperature in lowering the amountof linoleic acid in seed oil was reproduced in cultured embryos.The isolated seed culture system, therefore, constitutes a suitablemodel system for studies of oil synthesis in developing sunflowerembryos. The decrease in linoleic acid synthesis in response to highertemperature was detectable after only 18 day-degrees incubation,and the incorporation of labelled substrates suggests that alterationsin the fatty acid composition of seed oil in response to temperatureare produced by an effect on the desaturation of newly synthesizedoleate rather than through turnover of existing lipid. Variation in fatty acid composition between individual embryosgrown at constant temperature was considerable. The detectionof embryos with high linoleic acid levels following growth athigh temperature indicates that potential may exist for theselection of cultivars for temperature-stable fatty acid compositionin sunflower oil. Key words: Fatty acid synthesis, Helianthus annuus, Sunflower seeds     

Changes in Lipid and Fatty Acid Metabolism of Vicia faba Mesophyll Protoplasts

The metabolism of the major polar and neutral lipids of Viciafaba protoplasts isolated from ¹⁴CO₂-fed leaves has been examined.The results show large losses in the radioactivity found inphosphatidylcholine and monogalactosyldiacylglycerol while thatof phosphatidylglycerol was stable. This loss was accountedfor by a rapid increase in the ¹⁴C content of the neutral lipids,particularly the triacylglycerols. Analysis of the fatty acidradioactivity in the lipids suggests that protoplast isolationinhibited fatty acid desaturation on phosphatidylcholine andpossibly on other lipids. These results also suggest a roleof phosphatidylcholine in the donation of fatty acids for triacylglycerolsynthesis in mesophyll protoplasts. The results are discussedin terms of the regulation of lipid metabolism and protoplastbiology. (Received April 20, 1984; Accepted August 27, 1984)     

Effects of inhibitors on the enzyme system producing C6-aldehydes from C18-unsaturated fatty acids in chloroplasts of Japanese silver (Farfugium japonicum) leaves

When chloroplasts isolated from Farfugium japonicum (Japanesesilver) leaves were used as an enzyme source, the activity ofthe enzyme system producing C₆-aldehydes (cis-3-hexenal andn-hexanal) from C₁₈-unsaturated fatty acids (linolenic and linoleicacids) decreased upon treatment with LAHase from potato. Thisenzyme system could not be separated from chlorophylls and lipidsby detergent treatment and was not affected by light illumination,CCCP or DCMU. The activity of the enzyme system was inhibitedby MB and NTB used as a redox reagent, SKF 525-A as an oxidaseinhibitor and DABCO as a quencher of singlet oxygen, but notby DCIP, PMS and SOD. These data suggest that; i) interactionof the enzyme system with lipids is required for maximal enzymeactivity, ii) this enzyme system may involve electron mediator(s),and iii) singlet oxygen takes part in the enzyme reaction. (Received October 28, 1977; )     

Studies on cutin-esterase I. Preparation of cutin and its fatty acid component from tomato fruit peel

Examination was made of the fatty acid component of tomato cutinvia gas-liquid chromatography and thin layer chromatography.Dihydroxyeicosanoic acid was identified as a major componentof tomato cutinic acid in contrast with the results of BAKERand MARTIN (1) who recognized 10,16-dihydroxyhexadecanoic acidas the dominant acid of cutin in all plants tested. On the thinlayer chromatograms we found more than nine kinds of fatty acidsin the cutin hydrolysate which was saponified with ethanol-potashsolution. The gas-liquid chromatogram for trimethylsilyl etherderivatives of methyl cutinate showed somewhat different results,i.e., unsaturated decanoic, t_R 1.4, unsaturated stearic, t_R4.2 and unsaturated octadecanedioic acid, t_R 16.0 as unsaturatedfatty acids. Two more than C₂₂-hydroxyfatty acids were recognizedas minor components. Beside these components, octanoic, t_R 0.9,hydroxydecanoic, t_R 7.0 and cis-epoxy-hydroxyoctadecanoic acid,t_R 18.7 were identified. The biosynthesis of cutin is positednot to be fulfilled or to be delayed due to less lipoxidaseactivity in tomato fruit. ¹Biological Laboratory, Research Department, Nihon Noyaku Co.Ltd., Kawachinagano, Osaka, Japan (Received December 8, 1969; )     

Methionine Sulfoximine Effects on C4 Plant Leaf Discs: Comparison with C3 Species

Methionine sulfoximine caused ammonia accumulation and photosyntheticrate inhibition in leaf discs from two C₄ species, Zea maysL. cv. F. M. Cross (Hybrid) and Sorghum bicolor L. Moench cv.NC-70X, as well as one C₃ plant species, Datura stramonium L.cv. stramonium. Similar results were obtained earlier with theC₃ species Spinacia oleracea L. The effect occurred in the absenceof inorganic nitrogen reduction and was light dependent. Ammoniaaccumulation rates were similar in all four species examined.The results support a role for glutamine synthetase in leafammonia recycling in both C₄ and C₃ leaves. ¹ Current address: Cetus Madison Corporation, 2208 Parview Road,Middleton, WI 53562, U.S.A. (Received November 2, 1981; Accepted April 28, 1982)     

Fat Metabolism in the West African Oil Palm (Elaeis guineensis)

During germination in the light, the endosperm, containing ahigh proportion of reserve fat (composed largely of shorter-chain(C₈ to C₁₄ saturated fatty acids), is slowly invaded by theexpanding haustorium (cotyledon). Free fatty acids accumulatein the endosperm, preferential hydrolysis of longer-chain saturatedacids (C₁₄ to C₁₈ occurring under conditions of slow growth.Lipids are absorbed by the haustorium, the process being superficiallysimilar in certain respects to intestinal fat absorption. Whencomplicating factors are removed, absorption is found to beunselective during disappearance of 75 per cent, of the endospermlipids. Amounts of lipid in the haustorium are low compared with thehigh concentration in the surrounding endosperm and, beforephotosynthesis starts, losses through respiration account fora large part of the reserves which disappear. No free fattyacids are present in the haustorium. Breakdown of fatty acids is relatively unspecific, althoughthe acids characteristic of the haustorium (C₁₆ C₁₈, oleic andlinoleic acids) are metabolized some what less rapidly thanthe shorter-chain saturated acids (C₈ to C₁₄ characteristicof the endosperm fat. Both root and shoot have a low fat content. The fatty-acid compositionof the former changes little during growth, but in the shootlinolenic acid increases proportionately during leaf expansionin the light.     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Chlorophyll Fluorescence and Organic Acid Oscillations during Transition from CAM to C3-photosynthesis inClusia minor L. (Clusiaceae)

In species of Clusia, switching from C₃-photosynthesis (C₃-PS)to crassulacean acid metabolism (CAM) may be a means of optimizingwater use, plant carbon balance and photon utilization duringperiods of stress. We ask whether, in perennial species of Clusia,the switch from CAM back to C₃-PS is also of ecophysiologicalsignificance. Our objective was to investigate the performanceof C. minor L. during a short-term shift from CAM to C₃-PS.During the transition from CAM to C₃-PS, nocturnal malate andcitrate accumulation decreased whereas CO₂uptake increased duringthe daytime. However, after 7 d, marked nocturnal accumulationof citrate and 24 h CO₂uptake occurred. In contrast to C₃-likephotosynthesis, a pronounced reduction in the effective quantumyield of photosystem II,     

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

A voltage-gated, small, persistent Na⁺ current (I_Na) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent I_Na that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on I_Na in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited I_Na. The late portion of I_Na (I_{Na late}, measured near the end of each pulse) was almost completely suppressed. I_Na returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration dependent, with IC₅₀ values of 4.0 ± 0.4 µM for I_Na peak (I_{Na peak}) and 0.9 ± 0.1 µM for I_{Na late}. EPA shifted the steady-state inactivation of I_{Na peak} by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on I_Na. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient I_Na and that n-3 PUFAs inhibit mutant I_Na. human cardiac sodium channel