Efficacy of ajwain (Trachyspermum ammi L.) seed at graded levels of dietary threonine on growth performance, serum metabolites, intestinal morphology and microbial population in broiler chickens.

The present study investigated the influence of dietary threonine (Thr) levels and ajwain seed (AS) on the growth performance, immunity, blood metabolites, ileal microflora and jejunum morphology of broiler chickens. A total of 600-day-old male broiler chickens (Ross 308) were randomly allocated among five replicates of eight dietary treatments according to a 4 × 2 factorial arrangement of treatments, including four different levels of Thr (100, 105, 110 and 115% of requirements) and two inclusion rates of AS (0 or 10 g/kg of the diet). Body weight gain and FCR improved in broiler chickens fed 105% Thr supplement during the starter, finisher and whole periods of the experiment (p < .05). The broiler chickens fed AS supplemented diet had a greater weight gain (25-42 days) and better FCR (1-10 and 25-42 days) than those without AS (p < .05). A significant interaction was observed between Thr and AS supplementation for lymphocyte, heterophil/lymphocyte (H/L), bursa and spleen (p < .05). The liver enzyme activity of aspartate aminotransferase (AST) was higher in the chicks that received diet without AS supplement than those fed AS diet (p < .05). Inclusion of 105% dietary Thr and 10 g/kg AS increased the viable cell counts of Lactobacilli and decreased the population of the Escherichia coli in broilers (p < .05). In jejunum morphology, the broiler chickens fed 105% Thr diet had a greater villus length, width and crypt depth (p < .05). Also, the villus length was longer in broilers that received AS supplemented diet (p < .05). It is concluded that the dietary 105% of Thr and AS supplement individually improved growth performance, ileal microbial population and jejunum morphology of broiler chickens.

Manganese threonine chelate-a new enteric contrast agent for MRI: a pilot study on rats.

Enteric contrast agents are important in gastrointestinal MRI. However, no currently available agent is well established as the standard of care. In this study, in vitro relaxivities of manganese threonine chelate (Mn-Thr), a common nutritional food supplement, were measured at 1.5 T and 3 T with further investigation of its efficacy and safety in vivo as an enteric contrast agent. According to the calculated relaxivities, T1 W and T2 W TSE sequences of Mn-Thr solutions at different concentrations were acquired, and the optimal concentration for dark lumen imaging on both T1 W and T2 W images was determined in vitro. To validate the optimal concentration in vivo, eight Sprague-Dawley rats were randomly divided into two groups. Each group received rectal injection of either 2.00 g/L (about 3.80 mM) Mn-Thr or saline as an enteric contrast agent and underwent MRI. After a time interval of one week, the same procedures were repeated with the alternative contrast agent. Animals were sacrificed after the second MRI. Tissue manganese quantification and histopathological examination were obtained. Qualitative MR image quality assessments were performed and compared between Mn-Thr and saline. Measured T1 and T2 relaxivities of Mn-Thr were significantly higher than those of MnCl2 in vitro (p < 0.05). At the concentration of 2.00 g/L (about 3.80 mM), Mn-Thr produced a dark lumen on T1 W and T2 W images both in vitro and in vivo. Compared with saline, Mn-Thr showed significantly more homogenous luminal signal and increased bowel wall conspicuity in image quality assessments. Tissue manganese concentrations were not significantly different between two groups. Histopathological examinations were normal in both groups. Our data suggest that Mn-Thr possesses favorable paramagnetic properties and can create a homogenous dark lumen on T1 W and T2 W images without obvious side effects in healthy rats. As a commercially available nutritional food supplement, Mn-Thr appears to be a promising enteric contrast agent for MRI.

Antidepressant-Like Effect of ß-Lactolin, a Glycine-Threonine-Tryptophan-Tyrosine Peptide.

The number of patients with mental illnesses, including depression, is rapidly increasing, and daily lifestyle is closely associated with the development of symptoms. Consequently, corrective measures, such as diet-based treatment for diseases, are receiving great attention. We previously showed that ß-lactolin, a ß-lactopeptide of glycine-threonine-tryptophan-tyrosine peptide, inhibits monoamine oxidase and improves memory impairment in mice, but the effects on depression have not been investigated. Here we showed that ß-lactolin improved depression-like behavior via dopamine-D1-like receptor. Orally administered ß-lactolin reduced immobility time in tail suspension test (TST). Pretreatment with SCH23390, dopamine D1-like receptor antagonist, attenuated the reduction in TST by ß-lactolin. These effects were observed by the treatment with whey digest rich in ß-lactolin. In addition, ß-lactolin increased the levels of dopamine in the frontal cortex associated with the depression-like behavior. The present study suggests that supplements or nutraceutical compounds in whey digests (such as ß-lactolin) show antidepressant-like effect.

A Versatile Boc Solid Phase Synthesis of Daptomycin and Analogues Using Site Specific, On-Resin Ozonolysis to Install the Kynurenine Residue.

A de novo solid-phase synthesis of the cyclic lipodepsipeptide daptomycin via Boc chemistry was achieved. The challenging ester bond formation between the nonproteinogenic amino acid kynurenine was achieved by esterification of a threonine residue with a protected tryptophan. Subsequent late-stage on-resin ozonolysis, inspired by the biomimetic pathway, afforded the kynurenine residue directly. Synthetic daptomycin possessed potent antimicrobial activity (MIC100 =1.0 µg mL-1 ) against S. aureus, while five other daptomycin analogues containing (2R,3R)-3-methylglutamic acid, (2S,4S)-4-methylglutamic acid or canonical glutamic acid at position twelve prepared using this new methodology were all inactive, clearly establishing that the (2S,3R)-3-methylglutamic acid plays a key role in the antimicrobial activity of daptomycin.

Cytotoxic activity of broussochalcone a against colon and liver cancer cells by promoting destruction complex-independent ß-catenin degradation.

Aberrant activation of ß-catenin-response transcription (CRT) is a well-recognized characteristic of colorectal and liver cancers and thus a potential therapeutic target for these malignancies. Broussonetia papyrifera (paper mulberry) has been used as a herbal medicine to treat various diseases. Using a sensitive cell-based screening system, we identified broussochalcone A (BCA), a prenylated chalcone isolated from Broussonetia papyrifera, as an antagonist of CRT. BCA accelerated the turnover of intracellular ß-catenin that was accompanied by its N-terminal phosphorylation at Ser33/37/Thr41 residues, marking it for ubiquitin-dependent proteasomal degradation. Pharmacological inhibition of glycogen synthase kinase-3ß could not abrogate BCA-mediated degradation of ß-catenin. BCA decreased the intracellular ß-catenin levels in colon and liver cancer cells with mutations in ß-catenin, adenomatous polyposis coli, and Axin. BCA repressed the expressions of cyclin D1, c-Myc, and Axin2, which are ß-catenin/T-cell factor-dependent genes, and thus decreased the viability of colon and liver cancer cell. Moreover, apoptosis was elicited by BCA, as indicated by the increase in the population of Annexin V-FITC positive cells and caspase-3/7 activities in colon and liver cancer cells. These findings indicate that BCA exerts its cytotoxic effects by promoting phosphorylation/ubiquitin-dependent degradation of ß-catenin and may potentially serve as a chemopreventive agent for colonrectal and liver cancers.

A new actinomycin Z analogue with an additional oxygen bridge between chromophore and ß-depsipentapeptide from Streptomyces sp. KIB-H714.

Actinomycin Z6 (1), a new member of the actinomycin family, along with three congeners of the Z-type (Z1, Z3, Z5) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the ß-ring of the new compound actinomycin Z6 includes an additional ring linked between the actinoyl chromophore and ß-peptidolactone. In Z3 and Z5, the L-threonine in ß-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.

Depsidomycins B and C: New Cyclic Peptides from a Ginseng Farm Soil-Derived Actinomycete.

LC/MS-based chemical profiling of a ginseng farm soil-derived actinomycete strain, Streptomyces sp. BYK1371, enabled the discovery of two new cyclic heptapeptides, depsidomycins B and C (1 and 2), each containing two piperazic acid units and a formyl group at their N-terminus. The structures of 1 and 2 were elucidated by a combination of spectroscopic and chemical analyses. These new compounds were determined to possess d-leucine, d-threonine, d-valine, and S-piperazic acid based on the advanced Marfey's method and a GITC (2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate) derivatization of their hydrolysates, followed by LC/MS analysis. Depsidomycins B and C displayed significant antimetastatic activities against metastatic breast cancer cells (MDA-MB-231).

Novel activity-based probes for N-acylethanolamine acid amidase.

The cysteine hydrolase, N-acylethanolamine acid amidase (NAAA) is a promising target for analgesic and anti-inflammatory drugs. Here, we describe the development of two unprecedented NAAA-reactive activity-based probes as research tools for application in the discovery of new inhibitors and for the in-depth characterization of NAAA in its cellular environment.

The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3ß-dependent threonine-286 phosphorylation in human colorectal cancer cells.

Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3ß by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells.

The involvement of cyclin D1 degradation through GSK3ß-mediated threonine-286 phosphorylation-dependent nuclear export in anti-cancer activity of mulberry root bark extracts.

BACKGROUND: Mulberry root bark was shown to induce cyclin D1 proteasomal degradation in the human colorectal cancer cells. Still, the molecular mechanisms whereby mulberry root bark induces cyclin D1 proteasomal degradation remain largely unknown. PURPOSE: In this study, the inhibitory effect of mulberry root bark (MRB) on the proliferation of human colorectal cancer cells and the mechanism of action were examined to evaluate its anti-cancer activity. METHODS: Anti-proliferative effect was determined by MTT assay. RT-PCR and Western blotting were used to assess the mRNA and protein expression of related proteins. RESULTS: MRB inhibited markedly the proliferation of human colorectal cancer cells (HCT116, SW480 and LoVo). In addition, the proliferation of human breast cancer cells (MDA-MB-231 and MCF-7) was suppressed by MRB treatment. However, MRB did not affect the growth of HepG-2 cells as a human hepatocellular carcinoma cell line. MRB effectively decreased cyclin D1 protein level in human colorectal cancer cells and breast cancer cells, but not in hepatocellular carcinoma cells. Contrast to protein levels, cyclin D1 mRNA level did not be changed by MRB treatment. Inhibition of proteasomal degradation by MG132 attenuated MRB-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, MRB increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated MRB-mediated cyclin D1 degradation. Inhibition of GSK3ß by LiCl suppressed cyclin D1 phosphorylation and downregulation by MRB. MRB decreased the nuclear level of cyclin D1 and the inhibition of nuclear export by LMB attenuated MRB-mediated cyclin D1 degradation. CONCLUSION: MRB has anti-cancer activity by inducing cyclin D1 proteasomal degradation through cyclin D1 nuclear export via GSK3ß-dependent threonine-286 phosphorylation. These findings suggest that possibly its extract could be used for treating colorectal cancer.

Glassin, a histidine-rich protein from the siliceous skeletal system of the marine sponge Euplectella, directs silica polycondensation.

The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.

The inhibition of human farnesyl pyrophosphate synthase by nitrogen-containing bisphosphonates. Elucidating the role of active site threonine 201 and tyrosine 204 residues using enzyme mutants.

Farnesyl pyrophosphate synthase (FPPS) is the major molecular target of nitrogen-containing bisphosphonates (N-BPs), used clinically as bone resorption inhibitors. We investigated the role of threonine 201 (Thr201) and tyrosine 204 (Tyr204) residues in substrate binding, catalysis and inhibition by N-BPs, employing kinetic and crystallographic studies of mutated FPPS proteins. Mutants of Thr201 illustrated the importance of the methyl group in aiding the formation of the Isopentenyl pyrophosphate (IPP) binding site, while Tyr204 mutations revealed the unknown role of this residue in both catalysis and IPP binding. The interaction between Thr201 and the side chain nitrogen of N-BP was shown to be important for tight binding inhibition by zoledronate (ZOL) and risedronate (RIS), although RIS was also still capable of interacting with the main-chain carbonyl of Lys200. The interaction of RIS with the phenyl ring of Tyr204 proved essential for the maintenance of the isomerized enzyme-inhibitor complex. Studies with conformationally restricted analogues of RIS reaffirmed the importance of Thr201 in the formation of hydrogen bonds with N-BPs. In conclusion we have identified new features of FPPS inhibition by N-BPs and revealed unknown roles of the active site residues in catalysis and substrate binding.

A norepinephrine coated magnetic molecularly imprinted polymer for simultaneous multiple chiral recognition.

A newly designed molecularly imprinted polymer (MIP) material was developed and successfully used as recognition element for enantioselective recognition by microchip electrophoresis. In this work, molecularly imprinted polymers were facilely prepared employing Fe3O4 nanoparticles (NPs) as the supporting substrate and norepinephrine as the functional monomer in the presence of template molecule in a weak alkaline solution. After extracting the embedded template molecules, the produced imprinted Fe3O4@polynorepinephrine (MIP-Fe3O4@PNE) NPs have cavities complementary to three dimensional shape of template molecules favoring high binding capacity and magnetism property for easy manipulation. The MIP-Fe3O4@PNE NPs prepared with l-tryptophan, l-valine, l-threonine, Gly-l-Phe, S-(-)-ofloxacin or S-(-)-binaphthol as template molecules were packed in the polydimethylsiloxane microchannel via magnetic field as novel stationary phase to successful enantioseparation of corresponding target analysts. The MIP-Fe3O4@PNE NPs-based microchip electrophoresis system exhibited strong recognition ability, excellent high-performance, admirable reproducibility and stability, which provided a powerful protocol for separation enantiomers within a short analytical time and opened up an avenue for multiplex chiral compound assay in various systems.

Phosphorylation site prediction in plants.

Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community.

Molecular determinants of orexin receptor-arrestin-ubiquitin complex formation.

BACKGROUND AND PURPOSE: The orexin system regulates a multitude of key physiological processes, particularly involving maintenance of metabolic homeostasis. Consequently, there is considerable potential for pharmaceutical development for the treatment of disorders from narcolepsy to metabolic syndrome. It acts through the hormonal activity of two endogenous peptides, orexin A binding to orexin receptors 1 and 2 (OX1 and OX2) with similar affinity, and orexin B binding to OX2 with higher affinity than OX1 receptors. We have previously revealed data differentiating orexin receptor subtypes with respect to their relative stability in forming orexin receptor-arrestin-ubiquitin complexes measured by BRET. Recycling and cellular signalling distinctions were also observed. Here, we have investigated, using BRET, the molecular determinants involved in providing OX2 receptors with greater ß-arrestin-ubiquitin complex stability. EXPERIMENTAL APPROACH: The contribution of the C-terminal tail of the OX receptors was investigated by bulk substitution and site-specific mutagenesis using BRET and inositol phosphate assays. KEY RESULTS: Replacement of the OX1 receptor C-terminus with that of the OX2 receptor did not result in the expected gain of function, indicating a role for intracellular domain configuration in addition to primary structure. Furthermore, two out of the three putative serine/threonine clusters in the C-terminus were found to be involved in OX2 receptor-ß-arrestin-ubiquitin complex formation. CONCLUSIONS AND IMPLICATIONS: This study provides fundamental insights into the molecular elements that influence receptor-arrestin-ubiquitin complex formation. Understanding how and why the orexin receptors can be functionally differentiated brings us closer to exploiting these receptors as drug targets.

An enzymatic route to selenazolines.

Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines.

Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine ß-synthase.

Human cystathionine ß-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

Subunit-specific inhibition of glycine receptors by curcumol.

Emerging evidence has suggested that inhibitory glycine receptors (GlyRs) are an important molecular target in the treatment of numerous neurological disorders. Rhizoma curcumae is a medicinal plant with positive neurological effects. In this study, we showed that curcumol, a major bioactive component of R. curcumae, reversibly and concentration-dependently inhibited the glycine-activated current (IGly) in cultured rat hippocampal neurons. The inhibitory effect was neither voltage- nor agonist concentration-dependent. Moreover, curcumol selectively inhibited homomeric α2-containing, but not α1- or α3-containing, GlyRs. The addition of ß subunit conferred the curcumol sensitivity of α3-containing, but not α1-containing, GlyRs. Site-directed mutagenesis analysis revealed that a threonine at position 59 of the α2 subunit is critical for the susceptibility of GlyRs to curcumol-mediated inhibition. Furthermore, paralleling a decline of α2 subunit expression during spinal cord development, the degree of IGly inhibition by curcumol decreased with prolonged culture of rat spinal dorsal horn neurons. Taken together, our results suggest that the GlyRs are novel molecular targets of curcumol, which may underlie its pharmaceutical effects in the central nervous system.

The effects of added sulphur amino acids, threonine and an ideal amino acid ratio on nitrogen metabolism in mature, overweight dogs.

The objectives of this study were to investigate the effects of added essential amino acids in conjunction with a dietary lysine/MJ of 0.72 on nitrogen (N) metabolism in dogs. Treatments were; a control diet, a diet that provided an ideal amino acid profile (IAA), a diet with added total sulphur amino acids (TSAA), and a diet with added TSAA and threonine (TT). Diets were fed to eight overweight, mature, female hounds using a replicated 4 x 4 Latin Square design. Food intake was similar across treatments, however, food N intake was higher (p < 0.001) for TSAA than control, IAA or TT. Nitrogen absorbed was higher (p < 0.01) for TSAA than IAA and control. Urea N excretion was greater for control than TT (p < 0.05). Urine N excretion did not differ between diets. There were no differences in digestibility or N retention of diets. There were no differences in protein turnover, synthesis, or degradation. Blood metabolites were within normal ranges and did not differ due to dietary treatment. Based on the measurements made in this study, there is no benefit for added TSAA, TT or additional EAA in diets for mature dogs formulated to provide a 0.72 g lysine/MJ ME ratio.

Unconventional myristoylation of large-conductance Ca²âº-activated K⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression.

Protein myristoylation is a means by which cells anchor proteins into membranes. The most common type of myristoylation occurs at an N-terminal glycine. However, myristoylation rarely occurs at an internal amino acid residue. Here we tested whether the α-subunit of the human large-conductance voltage- and Ca(2+)-activated K(+) channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [(3)H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [(3)H]myristoyl-hSlo1 with alkaline NH(2)OH (which cleaves hydroxyesters) but not neutral NH(2)OH (which cleaves thioesters) completely removed [(3)H]myristate from hSlo1, suggesting the involvement of a hydroxyester bond between hSlo1's hydroxyl-bearing serine, threonine, and/or tyrosine residues and myristic acid; this type of esterification was further confirmed by its resistance to alkaline Tris·HCl. Treatment of cells expressing hSlo1 with 100 µM myristic acid caused alteration of hSlo1 activation kinetics and a 40% decrease in hSlo1 current density from 20 to 12 nA*MΩ. Immunocytochemistry confirmed a decrease in hSlo1 plasmalemma localization by myristic acid. Replacement of the six serines or the seven threonines (but not of the single tyrosine) of hSlo1 intracellular loops 1 and 3 with alanines decreased hSlo1 direct myristoylation by 40-44%, whereas in combination decreased myristoylation by nearly 90% and abolished the myristic acid-induced change in current density. Our data demonstrate that an ion channel, hSlo1, is internally and posttranslationally myristoylated. Myristoylation occurs mainly at hSlo1 intracellular loop 1 or 3, and is an additional mechanism for channel surface expression regulation.