Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates.

Many neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, are characterized by abnormal accumulations of aggregated proteins. Brains in these diseases also show accumulation of autophagic vesicles in the neuronal cytoplasm, suggesting impairment of the autophagic process. As autophagy involves de novo membrane production and vesicle fusion, extensive changes in lipid molecules are necessary. However, the involvement of signaling lipid-modifying enzymes in autophagy and their roles in neurodegenerative diseases are not clear. Using specific inhibitor, we show that loss of phospholipase D1 (PLD1) activity resulted in an accumulation of microtubule-associated protein light chain 3 (LC3), p62, and polyubiquitinated proteins, signs representing malfunction in autophagic flux. Fluorescence and electron microscopic analyses demonstrated impaired fusion of autophagosomes with lysosomes, resulting in accumulation of autophagosomes. Within the cells with impaired autophagic flux, α-synuclein aggregates accumulated in autophagosomes. Knockdown of PLD1 expression using small interfering RNA also resulted in impaired autophagic flux and accumulation of α-synuclein aggregates in autophagosomes. Neuronal toxicity caused by α-synuclein accumulation was rescued by overexpression of PLD1; however, expression of activity-deficient mutant, PLD1-KRM, showed reduced rescue effects. Finally, we demonstrated that both PLD activity and expression levels were reduced in brain tissues of dementia with Lewy bodies (DLB) patients, whereas the amounts of α-synuclein and p62 were increased in the same tissue samples. Collectively, these results suggest that insufficient PLD activity, and therefore, the changes in phospholipid compositions within membranes, might be an important contributor to impaired autophagic process and protein accumulation in Lewy body diseases.

The small molecule indirubin-3'-oxime activates Wnt/ß-catenin signaling and inhibits adipocyte differentiation and obesity.

OBJECTIVES: Activation of the Wnt/ß-catenin signaling pathway inhibits adipogenesis by maintaining preadipocytes in an undifferentiated state. We investigated the effect of indirubin-3'-oxime (I3O), which was screened as an activator of the Wnt/ß-catenin signaling, on inhibiting the preadipocyte differentiation in vitro and in vivo. METHODS: 3T3L1 preadipocytes were differentiated with 0, 4 or 20 µM of I3O. The I3O effect on adipocyte differentiation was observed by Oil-red-O staining. Activation of Wnt/ß-catenin signaling in I3O-treated 3T3L1 cells was shown using immunocytochemical and immunoblotting analyses for ß-catenin. The regulation of adipogenic markers was analyzed via real-time reverse transcription-PCR (RT-PCR) and immunoblotting analyses. For the in vivo study, mice were divided into five different dietary groups: chow diet, high-fat diet (HFD), HFD supplemented with I3O at 5, 25 and 100 mg kg(-1). After 8 weeks, adipose and liver tissues were excised from the mice and subject to morphometry, real-time RT-PCR, immunoblotting and histological or immunohistochemical analyses. In addition, adipokine and insulin concentrations in serum of the mice were accessed by enzyme-linked immunosorbent assay. RESULTS: Using a cell-based approach to screen a library of pharmacologically active small molecules, we identified I3O as a Wnt/ß-catenin pathway activator. I3O inhibited the differentiation of 3T3-L1 cells into mature adipocytes and decreased the expression of adipocyte markers, CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ, at both mRNA and protein levels. In vivo, I3O inhibited the development of obesity in HFD-fed mice by attenuating HFD-induced body weight gain and visceral fat accumulation without showing any significant toxicity. Factors associated with metabolic disorders such as hyperlipidemia and hyperglycemia were also improved by treatment of I3O. CONCLUSION: Activation of the Wnt/ß-catenin signaling pathway can be used as a therapeutic strategy for the treatment of obesity and metabolic syndrome and implicates I3O as a candidate anti-obesity agent.

Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy.

Autophagy is a catabolic process in which cell components are degraded to maintain cellular homeostasis by nutrient limitations. Defects of autophagy are involved in numerous diseases, including cancer. Here, we demonstrate a new role of phospholipase D (PLD) as a regulator of autophagy. PLD inhibition enhances autophagic flux via ATG1 (ULK1), ATG5 and ATG7, which are essential autophagy gene products critical for autophagosome formation. Moreover, PLD suppresses autophagy by differentially modulating phosphorylation of ULK1 mediated by mTOR and adenosine monophosphate-activated protein kinase (AMPK), and by suppressing the interaction of Beclin 1 with vacuolar-sorting protein 34 (Vps34), indicating that PLD coordinates major players of the autophagic pathway, AMPK-mTOR-ULK1 and Vps34/Beclin 1. Ultimately, PLD inhibition significantly sensitized in vitro and in vivo cancer regression via genetic and pharmacological inhibition of autophagy, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PLD inhibition. Collectively, we show a novel role for PLD in the molecular machinery regulating autophagy.

Rebamipide abolishes Helicobacter pylori CagA-induced phospholipase D1 expression via inhibition of NFκB and suppresses invasion of gastric cancer cells.

Infection with cagA-positive Helicobacter pylori is a risk factor for the development of severe gastritis and gastric cancer (GC). CagA protein is injected into gastric epithelial cells and deregulates a variety of cellular signaling molecules. Phospholipase D (PLD) is elevated in many different types of human cancers and has been implicated as a critical factor in inflammation and carcinogenesis. In this study, we show that infection with cagA-positive H. pylori in GC cells significantly induces PLD1 expression via CagA-dependent activation of nuclear factor κB (NFκB). Interestingly, the level of PLD1 protein and IκBα phosphorylation is aberrantly upregulated in H. pylori-infected human GC tissues. Infection with cagA-positive H. pylori and expression of CagA enhanced the binding of NFκB to the PLD1 promoter, and two functional NFκB-binding sites were identified within the PLD1 promoter. Rebamipide, a mucosal-protective antiulcer agent, abolished H. pylori cagA-induced PLD1 expression via inhibition of binding of NFκB to the PLD1 promoter, and also inhibited PLD activity. Moreover, rebamipide suppressed H. pylori-induced matrix metalloproteinase-9, interleukin-8 and activation-induced cytidine deaminase expression as well as invasion of GC cells through downregulation of PLD1. Our data suggest that H. pylori cagA targets PLD1 for invasion of GC cells, and rebamipide might contribute to the antitumorigenic effect of GC cells via inhibition of the H. pylori cagA-NFκB-PLD1 signaling pathway.

Cleavage of phospholipase D1 by caspase promotes apoptosis via modulation of the p53-dependent cell death pathway.

The enzymatic activity of phospholipase D (PLD) is known to be essential for cell survival and protection from apoptosis. However, the mechanisms regulating PLD activity during apoptosis remain unknown. Here we report that cleavage of PLD1 by caspases facilitates p53-mediated apoptosis. Cleavage of PLD1 into an N-terminal fragment (NF-PLD1) and a C-terminal fragment at the amino-acid sequence, DDVD(545), led to a reduction in PLD1 activity. However, a caspase-resistant mutant form of PLD1 retained significant levels of enzymatic activity and apoptotic function as compared to wild-type PLD1. Exogenous NF-PLD1 expression induced apoptosis through a dominant-negative effect on the activity of endogenous PLD1. During apoptosis, a small fraction of PLD1 is cleaved by caspases in a p53-independent manner and NF-PLD1 amplifies apoptotic signaling through inhibition of the remaining PLD1 activity. As PLD1 suppresses the ATM-Chk2-p53 pathway, elimination of PLD1 activity through NF-PLD1 or si-RNA against PLD1 increases apoptosis in a p53-dependent manner. Taken together, our results reveal that cleavage of PLD1 by caspases promotes apoptosis via modulation of the p53-dependent cell death pathway.

Transcriptional regulation of GSDML gene by antisense-oriented HERV-H LTR element.

During the course of hominoid evolution, a new transcript variant of the GSDML (gasdermin-like protein) gene was formed by the integration of the antisense-oriented HERV-H (human endogenous retrovirus) LTR (long terminal repeat) element. To investigate regions that are critical for transcriptional regulation of the GSDML gene, we generated seven deletion mutants from a full-length clone (clone 1/630) that includes the HERV-H LTR sequence and compared their expression levels relative to the full-length parental clone using a transient transfection assay. In the transient transfection assay, deletion of the 5' flanking region (cellular origin) of the HERV-H LTR sequence led to a 4.5-fold increase in expression compared to the full-length clone, while deletion of the U5 region showed a significant decrease in transcriptional activity. Deletion of the 3' flanking region of the LTR sequence (clone 42/451) showed similar transcriptional activity to a clone missing the 5' flanking region of cellular origin (clone 42/630). Taken together, these data indicate that the HERV-H LTR sequence (viral origin) positively regulates transcriptional activity of the GSDML gene and that the 5' flanking region sequence (cellular origin) exerts negative transcriptional regulation.

NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells.

Quercetin, a flavonoid molecule ubiquitously present in nature, has multiple effects on cancer cells, including the inhibition of cell proliferation and migration. However, the responsible molecular mechanisms are not fully understood. We found that quercetin induces the expression of NAG-1 (Non-steroidal anti-inflammatory drug activated gene-1), a TGF-beta superfamily protein, during quercetin-induced apoptosis of HCT116 human colon carcinoma cells. Reporter assays using the luciferase constructs containing NAG-1 promoter region demonstrate that early growth response-1 (EGR-1) and p53 are required for quercetin-mediated activation of the NAG-1 promoter. Overexpression of NAG-1 enhanced the apoptotic effect of quercetin, but suppression of quercetin-induced NAG-1 expression by NAG-1 siRNA attenuated quercetin-induced apoptosis in HCT116 cells. Taken together, the present study demonstrates for the first time that quercetin induces apoptosis via NAG-1, providing a mechanistic basis for the apoptotic effect of quercetin in colon carcinoma cells.

Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells.

Long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) have been reported to serve as alternative promoters in functional genes. The GSDML (gasdermin-like protein) gene located on human chromosome 17q21 has been found to be an oncogenomic recombination hotspot. Here, we identified the LTR element of HERV-H with reverse orientation as an alternative promoter of the GSDML gene and analyzed its expression pattern in human tissues and cancer cells. A reporter gene assay of the promoter activity of the LTR on the GSDML gene in human cancer cell lines (HCT-116 and HeLa) and a kidney cell line (Cos7) of African green monkey indicated that the LTR promoter with reverse orientation had stronger promoter activity than forward one. The transcripts of this LTR-derived promoter were widely distributed in various human tissues and cancer cells, whereas the transcripts of the cellular promoter were found only in stomach tissues and some cancer cells (HCT116, MCF7, U937, C-33A, and PC3). These findings suggest that the LTR element on the GSDML gene was integrated into the hominoid lineage and acquired the role of transcriptional regulation of human tissues and cancer cells.

Expression and potential role of phospholipase D1 in cryoinjured cerebral cortex of rats.

The expression and potential role of phospholipase D1 (PLD1) were studied in the cerebral cortex of rats after freeze injury. Histopathologically, cryoinjury, by exposing cerebral cortex to a prechilled rod for 1 minute, produced consistent pathological lesions, specifically neuronal death, infiltration of macrophages into the center of the cryoinjury, and reactive astrogliosis at the periphery, which caused the lesion site to become encased. Western blot analysis showed that PLD1 expression in the ipsilateral cerebral cortex increased significantly during days 1 to 3 after cryoinjury and declined slightly at post-injury day 7. PLD1 immunoreactivity was very low in the brains of sham-operated control adults. After cryoinjury, there was substantial PLD1 immunostaining of numerous inflammatory cells in the ipsilateral cortex, which were identical to ED1-positive macrophages. In addition, PLD1 immunoreactivity was increased in some neurons and astrocytes at the periphery of the cryoinjury at post-injury days 3 and 7. These findings suggest that cryoinjury by means of prechilled rods induced consistent histopathological changes in the cerebral cortex. In addition, expression of a cell activation signal, PLD1, was upregulated in macrophages and astrocytes in the ipsilateral cerebral cortex after cryoinjury.

Increased expression of phospholipase D1 in the sciatic nerve of rats with experimental autoimmune neuritis.

Phospholipase D1 (PLD1) expression in the sciatic nerve was studied in induced experimental autoimmune neuritis (EAN) in Lewis rats. PLD1 immunoreactivity was seen in some Schwann cells in the sciatic nerves of normal rats. In parallel with the progression of EAN, PLD1-positive Schwann cells significantly increased in number and showed intense immunoreactivity. PLD1 was also detected in some ED1+ macrophages in EAN lesions. These results suggest that PLD1 in macrophages and Schwann cells plays an important role in the activation of these cells in the pathogenesis of EAN, an animal model of human peripheral demyelinating disease.

Differential expression of phospholipase D1 in the developing retina.

Expression patterns of phospholipase D1 (PLD1) in the developing rat retina were investigated using immunocytochemistry and Western blot analysis and compared with the expression patterns of glutamine synthetase. PLD1 immunoreactivity appeared first in a few neuroblasts in the middle of the mantle zone of the primitive retina by embryonic (E) day 13. PLD1-immunoreactive primitive ganglion cells were characterized in the ganglion cell layer by E17. Faint immunoreactivity at E17 profiled radially orientated cells and this pattern appeared up to postnatal (P) day 7. In the ganglion cell layer at P3, displaced amacrine cells and ganglion cells were classified. At P5, presumptive horizontal cells and amacrine cells were identified. By P7, a thin outermost layer of newly formed segments of the photoreceptor cells was also PLD1 immunoreactive. PLD1 immunoreactivity at P8 was limited to radial Müller cells and the outer segment layer of the photoreceptor cells, and the expression pattern was conserved to adulthood. Western blot analysis showed relatively high amounts of PLD1 protein at E17 and P3, a decrease at P7, and moderate amounts from P8 onward. Co-expression of PLD1 with glutamine synthetase in the retina appeared first after birth in differentiating neurons and in Müller cells by P8; thereafter the pattern was maintained. The expression pattern of the PLD1 during development of the retina suggests that PLD1 plays important roles in glutamate-associated differentiation of both specific neurons and radial glial cells, and in glutamate-mediated cellular signalling in Müller cells.

Effect of ethanol on spontaneous phasic contractions of cat gastric smooth muscle.

BACKGROUND: Ethanol is generally believed to inhibit extracellular Ca2+ influx, thereby inhibiting gastric muscle contraction. Recently, we observed that verapamil inhibited only the amplitude of spontaneous phasic contractions, whereas ethanol inhibited both amplitude and frequency. In our objective to investigate the mechanism of ethanol's inhibition of gastric motility, the involvement of various protein kinases in ethanol-inhibited spontaneous phasic contractions of the stomach muscle strips was tested. METHODS: Circular muscle strips (2.0 x 0.2 cm) were prepared from the corpus of cat stomach in order to measure isometric contraction in a chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 degrees C) bubbled with 5% CO2 in O2. RESULTS: Spontaneous phasic contraction was not affected by various receptor antagonists (I microM atropine, 1 microM hexamethonium, 1 microM phentolamine and 1 microM propranolol) or 1 microM tetrodotoxin. EGTA and verapamil dose-dependently inhibited only the amplitude of spontaneous phasic contractions and not the frequency. Ethanol dose-dependently inhibited both the amplitude and frequency of phasic contractions. The amplitude and frequency of spontaneous phasic contractions were significantly inhibited by protein kinase C and tyrosine kinase inhibitors. However, neither protein kinase C activator nor various phosphatase inhibitors blocked the inhibitory effect of ethanol. CONCLUSIONS: Ethanol appears to inhibit spontaneous phasic contractions by a mechanism other than the inhibition of protein kinase C or tyrosine kinase or the inhibition of extracellular Ca2+ influx.

Increased expression of phospholipase D1 in the spinal cords of rats with experimental autoimmune encephalomyelitis.

Phospholipase D1 (PLD1) expression was studied in the central nervous system (CNS) under the condition of induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. After inducing EAE, the expression of PLD1 was analyzed by Western blot and immunohistochemistry. Western blot analysis showed that expression of the isozymes PLD1 significantly increased in the spinal cord at the peak stage of EAE, and declined thereafter. Immunohistochemistry showed that PLD1-positive cells increased in number in EAE lesions, which consisted mainly of ED1-positive macrophages and glial fibrillary acidic protein-positive astrocytes. In contrast, PLD1 was only weakly expressed in some spinal cord astrocytes in control rats. These results suggest that PLD1 is increased in autoimmune CNS inflammation, and possibly involved in the activation of macrophages and astrocytes in EAE lesions.

Involvement of phospholipase D in oxidative stress-induced necrosis of vascular smooth muscle cells.

Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress-induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose- and time-dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan-1-ol, a PLD inhibitor, attenuated both pervanadate-induced necrosis and increase of intracellular Ca(2+). Removal of extracellular Ca(2+) inhibited pervanadate-induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate-induced necrosis of VSMCs, which is at least partly due to Ca(2+) toxicity.

Transient expression of phospholipase D1 in developing rat hippocampus.

We investigated the distribution of phospholipase D1 (PLD1) protein in the developing rat hippocampus using an affinity-purified peptide antibody against PLD1. Immunoreactivity for PLD1 was first seen in some scattered cells in the hippocampus at embryonic day 18. At postnatal day 1 (P1), many PLD1 immunoreactive cells were observed in the CA1 and CA3 sectors, subiculum and the hilus of the dentate gyrus. During the first postnatal week, there was an abrupt increase of immunoreactive neurons in the hippocampus, and their number and intensity peaked at P7. During the second postnatal week, there was an abrupt decrease in the number of immunoreactive hippocampal neurons. By P14, no significant labeling was found in the hippocampus. These results corresponded well with those from Western blot analysis, suggesting that PLD1 may regulate the developmental processes of hippocampal neurons.

Effects of (-)-epigallocatechin-3-gallate, the main component of green tea, on the cloned rat brain Kv1.5 potassium channels.

The interaction of (-)-epigallocatechin-3-gallate (EGCG), the main component of green tea (Camellia sinensis), with rat brain Kv1.5 channels (rKv1.5) stably expressed in Chinese hamster ovary (CHO) cells was investigated using the whole-cell patch-clamp technique. EGCG inhibited rKv1.5 currents at +50 mV in a concentration-dependent manner, with an IC50 of 101.2+/-6.2 microM. Pretreatment with protein tyrosine kinase (PTK) inhibitors (10 microM genistein, 100 microM AG1296), a tyrosine phosphatase inhibitor (500 microM sodium orthovanadate), or a protein kinase C (PKC) inhibitor (10 microM chelerythrine) did not block the inhibitory effect of EGCG on rKv1.5. The inhibition of rKv1.5 by EGCG displayed voltage-independence over the full activation voltage range positive to +10 mV. EGCG had no effect on the midpoint potential or the slope factor for steady-state activation and inactivation. EGCG did not affect the ion selectivity of rKv1.5. The activation (at +50 mV) kinetics was significantly slowed by EGCG. During repolarization (at -40 mV), EGCG also slowed the deactivation of the tail currents, resulting in a crossover phenomenon. Reversal of inhibition was detected by the application of repetitive depolarizing pulses and of identical double pulses, especially during the early part of the activating pulse, in the presence of EGCG. EGCG-induced inhibition of rKv1.5 showed identical affinity between EGCG and the multiple closed states of rKv1.5. These results suggest that EGCG interacts directly with rKv1.5 channels. Furthermore, by analyzing the kinetics of the interaction between EGCG and rKv1.5, we conclude that the inhibition of rKv1.5 channels by EGCG includes at least two effects: EGCG preferentially binds to the channel in the closed state, and blocks the channel by pore occlusion while depolarization is maintained.

Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity.

Resveratrol has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. In the present study, we determined the effect of high intracellular levels of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-gamma1 degradation and cytochrome c release during resveratrol-induced apoptosis. For this, we used U937/vector and U937/Bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with U937/vector, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 60 or 100 microM resveratrol for 24 h produced morphological features of apoptosis and DNA fragmentation in U937/vector cells, respectively. This was associated with caspase-3 activation and PLC-gamma1 degradation. In contrast, resveratrol-induced caspase-3 activation and PLC-gamma1 degradation and apoptosis were significantly inhibited in U937/Bcl-2 cells. Bcl-2 overexpressing cells exhibited less cytochrome c release and sustained expression levels of the IAP proteins during resveratrol-induced apoptosis. In addition, these findings indicate that Bcl-2 inhibits resveratrol-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase-3 that is involved in the execution of apoptosis.

Neoplastic transformation and tumorigenesis associated with overexpression of phospholipase D isozymes in cultured murine fibroblasts.

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines.

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.

Effects of norfluoxetine, the major metabolite of fluoxetine, on the cloned neuronal potassium channel Kv3.1.

The effects of fluoxetine and its major metabolite, norfluoxetine, were studied using the patch-clamp technique on the cloned neuronal rat K(+) channel Kv3.1, expressed in Chinese hamster ovary cells. In whole-cell recordings, fluoxetine and norfluoxetine inhibited Kv3.1 currents in a reversible concentration-dependent manner, with an IC(50) value and a Hill coefficient of 13.11+/-0.91 microM and 1.33+/-0.08 for fluoxetine and 0.80+/-0.06 microM and 1.65+/-0.08 for norfluoxetine at +40 mV, respectively. In inside-out patches, norfluoxetine applied to the cytoplasmic surface inhibited Kv3.1 with an IC(50) value of 0.19+/-0.01 microM. The inhibition of Kv3.1 currents by both drugs was characterized by an acceleration in the apparent rate of current decay, without modification of the activation time course and with relatively fewer effects on peak amplitude. The degree of inhibition of Kv3.1 by norfluoxetine was voltage-dependent. The inhibition increased steeply between 0 and +30 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +30 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance delta of 0.31+/-0.05. The association (k(+1)) and dissociation (k(-1)) rate constants for norfluoxetine-induced inhibition of Kv3.1 were 21.70+/-3.39 microM(-1) s(-1) and 14.68+/-3.94 s(-1), respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 0.68 microM. Norfluoxetine did not affect the ion selectivity of Kv3.1. The reversal potential under control conditions was about -85 mV and was not affected by norfluoxetine. Norfluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of norfluoxetine, were superimposed. The voltage dependence of steady-state inactivation was not changed by the drug. Norfluoxetine produced use-dependent inhibition of Kv3.1 at a frequency of 1 Hz and slowed the recovery from inactivation. It is concluded that at clinically relevant concentrations, both fluoxetine and its major metabolite norfluoxetine inhibit Kv3.1, and that norfluoxetine directly inhibits Kv3.1 as an open channel blocker.