-
Catharina G Faber,
Giuseppe Lauria,
Ingemar S J Merkies,
Xiaoyang Cheng,
Chongyang Han, Hye-Sook Ahn,
Anna-Karin Persson,
Janneke G J Hoeijmakers,
Monique M Gerrits,
Tiziana Pierro,
Raffaella Lombardi,
Dimos Kapetis,
Sulayman D Dib-Hajj,
Stephen G Waxman
[show abstract]
[hide abstract]
ABSTRACT: Painful peripheral neuropathy often occurs without apparent underlying cause. Gain-of-function variants of sodium channel Na(v)1.7 have recently been found in ∼30% of cases of idiopathic painful small-fiber neuropathy. Here, we describe mutations in Na(v)1.8, another sodium channel that is specifically expressed in dorsal root ganglion (DRG) neurons and peripheral nerve axons, in patients with painful neuropathy. Seven Na(v)1.8 mutations were identified in 9 subjects within a series of 104 patients with painful predominantly small-fiber neuropathy. Three mutations met criteria for potential pathogenicity based on predictive algorithms and were assessed by voltage and current clamp. Functional profiling showed that two of these three Na(v)1.8 mutations enhance the channel's response to depolarization and produce hyperexcitability in DRG neurons. These observations suggest that mutations of Na(v)1.8 contribute to painful peripheral neuropathy.
Proceedings of the National Academy of Sciences 10/2012; · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A vast diversity of salient cues is sensed by numerous classes of primary sensory neurons, defined by specific neuropeptides, ion channels, or cytoskeletal proteins. Recent evidence has demonstrated a correlation between the expression of some of these molecular markers and transmission of signals related to distinct sensory modalities (eg, heat, cold, pressure). Voltage-gated sodium channel Na(v)1.8 has been reported to be preferentially expressed in small-diameter unmyelinated sensory afferents specialized for the detection of noxious stimuli (nociceptors), and Na(v)1.8-Cre mice have been widely used to investigate gene function in nociceptors. However, the identity of neurons in which Cre-mediated recombination occurs in these animals has not been resolved, and whether expression of Na(v)1.8 in these neurons is dynamic during development is not known, rendering interpretation of conditional knockout mouse phenotypes problematic. Here, we used genetics, immunohistochemistry, electrophysiology, and calcium imaging to precisely characterize the expression of Na(v)1.8 in the peripheral nervous system. We demonstrate that 75% of dorsal root ganglion (DRG) neurons express Na(v)1.8-Cre, including >90% of neurons expressing markers of nociceptors and, unexpectedly, a large population (∼40%) of neurons with myelinated A fibers. Furthermore, analysis of DRG neurons' central and peripheral projections revealed that Na(v)1.8-Cre is not restricted to nociceptors but is also expressed by at least 2 types of low-threshold mechanoreceptors essential for touch sensation, including those with C and Aβ fibers. Our results indicate that Na(v)1.8 underlies electrical activity of sensory neurons subserving multiple functional modalities, and call for cautious interpretation of the phenotypes of Na(v)1.8-Cre-driven conditional knockout mice.
Pain 06/2012; 153(10):2017-30. · 5.78 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of ranolazine, an antianginal drug, on potassium channel Kv4.3 were examined by using the whole-cell patch-clamp technique. Ranolazine inhibited the peak amplitude of Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 128.31 μM. The activation kinetics were not significantly affected by ranolazine at concentrations up to 100 μM. Applications of 10 and 30 μM ranolazine had no effect on the fast and slow inactivation of Kv4.3. However, at concentrations of 100 and 300 μM ranolazine caused a significant decrease in the rate of fast inactivation, and at a concentration of 300 μM it caused a significant decrease in the rate of slow inactivation, resulting in a crossover of the current traces during depolarization. The Kv4.3 inhibition by ranolazine increased steeply between -20 and +20 mV. In the full activation voltage range, however, no voltage-dependent inhibition was found. Ranolazine shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant (K(i)) for ranolazine for interacting with the inactivated state of Kv4.3 was calculated to be 0.32 μM. Ranolazine produced little use-dependent inhibition at frequencies of 1 and 2 Hz. Ranolazine did not affect the time course of recovery from the inactivation of Kv4.3. The results indicated that ranolazine inhibited Kv4.3 and exhibited a low affinity for Kv4.3 channels in the closed state but a much higher affinity for Kv4.3 channels in the inactivated state.
Journal of Pharmacology and Experimental Therapeutics 09/2011; 339(3):952-8. · 3.83 Impact Factor
-
Catharina G Faber,
Janneke G J Hoeijmakers, Hye-Sook Ahn,
Xiaoyang Cheng,
Chongyang Han,
Jin-Sung Choi,
Mark Estacion,
Giuseppe Lauria,
Els K Vanhoutte,
Monique M Gerrits,
Sulayman Dib-Hajj,
Joost P H Drenth,
Stephen G Waxman,
Ingemar S J Merkies
[show abstract]
[hide abstract]
ABSTRACT: Small nerve fiber neuropathy (SFN) often occurs without apparent cause, but no systematic genetic studies have been performed in patients with idiopathic SFN (I-SFN). We sought to identify a genetic basis for I-SFN by screening patients with biopsy-confirmed idiopathic SFN for mutations in the SCN9A gene, encoding voltage-gated sodium channel Na(V)1.7, which is preferentially expressed in small diameter peripheral axons.
Patients referred with possible I-SFN, who met the criteria of ≥2 SFN-related symptoms, normal strength, tendon reflexes, vibration sense, and nerve conduction studies, and reduced intraepidermal nerve fiber density (IENFD) plus abnormal quantitative sensory testing (QST) and no underlying etiology for SFN, were assessed clinically and by screening of SCN9A for mutations and functional analyses.
Twenty-eight patients who met stringent criteria for I-SFN including abnormal IENFD and QST underwent SCN9A gene analyses. Of these 28 patients with biopsy-confirmed I-SFN, 8 were found to carry novel mutations in SCN9A. Functional analysis revealed multiple gain of function changes in the mutant channels; each of the mutations rendered dorsal root ganglion neurons hyperexcitable.
We show for the first time that gain of function mutations in sodium channel Na(V)1.7, which render dorsal root ganglion neurons hyperexcitable, are present in a substantial proportion (28.6%; 8 of 28) of patients meeting strict criteria for I-SFN. These results point to a broader role of Na(V)1.7 mutations in neurological disease than previously considered from studies on rare genetic syndromes, and suggest an etiological basis for I-SFN, whereby expression of gain of function mutant sodium channels in small diameter peripheral axons may cause these fibers to degenerate.
Annals of Neurology 05/2011; 71(1):26-39. · 11.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons).
We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs.
We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system.
Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.
Molecular Pain 01/2011; 7:32. · 3.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K(+) (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The K(i) for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.
AJP Cell Physiology 12/2010; 300(3):C567-75. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Dominant gain-of-function mutations that hyperpolarize activation of the Na(v)1.7 sodium channel have been linked to inherited erythromelalgia (IEM), a disorder characterized by severe pain and redness in the feet and hands in response to mild warmth. Pharmacotherapy remains largely ineffective for IEM patients with cooling and avoidance of triggers being the most reliable methods to relieve pain. We now report a 5 year old patient with pain precipitated by warmth, together with redness in her hands and feet. Her pain episodes were first reported at 12 months, and by the age of 15-16 months were triggered by sitting as well as heat. Pain has been severe, inducing self-mutilation, with limited relief from drug treatment. Our analysis of the patient's genomic DNA identified a novel Na(v)1.7 mutation which replaces isoleucine 234 by threonine (I234T) within domain I/S4-S5 linker. Whole-cell voltage-clamp analysis shows a I234T-induced shift of -18 mV in the voltage-dependence of activation, accelerated time-to-peak, slowed deactivation and enhanced responses to slow ramp depolarizations, together with a -21 mV shift in the voltage-dependence of slow-inactivation. Our data show that I234T induces the largest activation shift for Na(v)1.7 mutations reported thus far. Although enhanced slow-inactivation may attenuate the gain-of-function of the I234T mutation, the shift in activation appears to be dominant, and is consistent with the severe pain symptoms reported in this patient.
European journal of pain (London, England) 04/2010; 14(9):944-50. · 3.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Na(v)1.7 sodium channels can amplify weak stimuli in neurons and act as threshold channels for firing action potentials. Neurotrophic factors and pro-nociceptive cytokines that are released during development and under pathological conditions activate mitogen-activated protein kinases (MAPKs). Previous studies have shown that MAPKs can transduce developmental or pathological signals by regulating transcription factors that initiate a gene expression response, a long-term effect, and directly modulate neuronal ion channels including sodium channels, thus acutely regulating dorsal root ganglion (DRG) neuron excitability. For example, neurotrophic growth factor activates (phosphorylates) ERK1/2 MAPK (pERK1/2) in DRG neurons, an effect that has been implicated in injury-induced hyperalgesia. However, the acute effects of pERK1/2 on sodium channels are not known. We have shown previously that activated p38 MAPK (pp38) directly phosphorylates Na(v)1.6 and Na(v)1.8 sodium channels and regulates their current densities without altering their gating properties. We now report that acute inhibition of pERK1/2 regulates resting membrane potential and firing properties of DRG neurons. We also show that pERK1 phosphorylates specific residues within L1 of Na(v)1.7, inhibition of pERK1/2 causes a depolarizing shift of activation and fast inactivation of Na(v)1.7 without altering current density, and mutation of these L1 phosphoacceptor sites abrogates the effect of pERK1/2 on this channel. Together, these data are consistent with direct phosphorylation and modulation of Na(v)1.7 by pERK1/2, which unlike the modulation of Na(v)1.6 and Na(v)1.8 by pp38, regulates gating properties of this channel but not its current density and contributes to the effects of MAPKs on DRG neuron excitability.
Journal of Neuroscience 02/2010; 30(5):1637-47. · 7.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The action of fluoxetine, a serotonin reuptake inhibitor, on the cloned neuronal rat Kv3.1 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Fluoxetine reduced Kv3.1 whole-cell currents in a reversible, concentration-dependent manner, with an IC(50) value and a Hill coefficient of 13.4 muM and 1.4, respectively. Fluoxetine accelerated the decay rate of inactivation of Kv3.1 currents without modifying the kinetics of current activation. 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 0.38. The binding (k(+1)) and dissociation (k(-1)) rate constants for fluoxetine-induced block of Kv3.1 were 5.7 microM(-1)s(-1) and 53.5 s(-1), respectively. The theoretical K(D) value derived by k(-1)/k(+1) yielded 9.3 microM. Fluoxetine did not affect the ion selectivity of Kv3.1. Fluoxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of fluoxetine, were superimposed. Inhibition of Kv3.1 by fluoxetine was use-dependent. The present results suggest that fluoxetine acts on Kv3.1 currents as an open-channel blocker.
Journal of Pharmacological Sciences 02/2008; 106(1):38-45. · 2.08 Impact Factor
-
Hye Sook Ahn,
Sung Eun Kim,
Bok Hee Choi,
Jin-Sung Choi,
Myung-Jun Kim,
Duck-Joo Rhie,
Shin Hee Yoon,
Yang-Hyeok Jo,
Myung-Suk Kim,
Ki-Wug Sung,
Oh-Joo Kwon,
Sang June Hahn
[show abstract]
[hide abstract]
ABSTRACT: The interaction of FK-506 with K(V)1.3, stably expressed in Chinese hamster ovary cells, was investigated with the whole cell patch-clamp technique. FK-506 inhibited K(V)1.3 in a reversible, concentration-dependent manner with an IC(50) of 5.6 microM. Rapamycin, another immunosuppressant, produced effects that were similar to those of FK-506 (IC(50) = 6.7 microM). Other calcineurin inhibitors (cypermethrin or calcineurin autoinhibitory peptide) alone had no effect on the amplitude or kinetics of K(V)1.3. In addition, the inhibitory action of FK-506 continued, even after the inhibition of calcineurin activity. The inhibition produced by FK-506 was voltage dependent, increasing in the voltage range for channel activation. At potentials positive to 0 mV (where maximal conductance is reached), however, no voltage-dependent inhibition was found. FK-506 exhibited a strong use-dependent inhibition of K(V)1.3. FK-506 shifted the steady-state inactivation curves of K(V)1.3 in the hyperpolarizing direction in a concentration-dependent manner. The apparent dissociation constant for FK-506 to inhibit K(V)1.3 in the inactivated state was estimated from the concentration-dependent shift in the steady-state inactivation curve and was calculated to be 0.37 microM. Moreover, the rate of recovery from inactivation of K(V)1.3 was decreased. In inside-out patches, FK-506 not only reduced the current amplitude but also accelerated the rate of inactivation during depolarization. FK-506 also inhibited K(V)1.5 and K(V)4.3 in a concentration-dependent manner with IC(50) of 4.6 and 53.9 microM, respectively. The present results indicate that FK-506 inhibits K(V)1.3 directly and that this effect is not mediated via the inhibition of the phosphatase activity of calcineurin.
AJP Cell Physiology 06/2007; 292(5):C1714-22. · 3.54 Impact Factor
-
Sung Eun Kim, Hye Sook Ahn,
Bok Hee Choi,
Hyun-Jong Jang,
Myung-Jun Kim,
Duck-Joo Rhie,
Shin-Hee Yoon,
Yang-Hyeok Jo,
Myung-Suk Kim,
Ki-Wug Sung,
Sang June Hahn
[show abstract]
[hide abstract]
ABSTRACT: The effects of sibutramine on voltage-gated K+ channel (Kv)4.3, Kv1.3, and Kv3.1, stably expressed in Chinese hamster ovary cells, were investigated using the whole-cell patch-clamp technique. Sibutramine did not significantly decrease the peak Kv4.3 currents, but it accelerated the rate of decay of current inactivation in a concentration-dependent manner. This phenomenon was effectively characterized by integrating the total current over the duration of a depolarizing pulse to +40 mV. The IC50 value for the sibutramine block of Kv4.3 was 17.3 microM. Under control conditions, the inactivation of Kv4.3 currents could be fit to a biexponential function, and the time constants for the fast and slow components were significantly decreased after the application of sibutramine. The association (k+1) and dissociation (k-1) rate constants for the sibutramine block of Kv 4.3 were 1.51 microM-1s-1 and 27.35 s-1, respectively. The theoretical KD value, derived from k-1/k+1, yielded a value of 18.11 microM. The block of Kv4.3 by sibutramine displayed a weak voltage dependence, increasing at more positive potentials, and it was use-dependent at 2 Hz. Sibutramine did not affect the time course for the deactivating tail currents. Neither steady-state activation and inactivation nor the recovery from inactivation was affected by sibutramine. Sibutramine caused the concentration-dependent block of the Kv1.3 and Kv3.1 currents with an IC50 value of 3.7 and 32.7 microM, respectively. In addition, sibutramine reduced the tail current amplitude and slowed the deactivation of the tail currents of Kv1.3 and Kv3.1, resulting in a crossover phenomenon. These results indicate that sibutramine acts on Kv4.3, Kv1.3, and Kv3.1 as an open channel blocker.
Journal of Pharmacology and Experimental Therapeutics 06/2007; 321(2):753-62. · 3.83 Impact Factor
-
Hye Sook Ahn,
Sung Eun Kim,
Hyun-Jong Jang,
Myung-Jun Kim,
Duck-Joo Rhie,
Shin-Hee Yoon,
Yang-Hyeok Jo,
Myung-Suk Kim,
Ki-Wug Sung,
Seong Yun Kim,
Sang June Hahn
[show abstract]
[hide abstract]
ABSTRACT: The effects of rosiglitazone and troglitazone were examined on cloned Kv1.3 channels stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Rosiglitazone decreased the Kv1.3 currents and accelerated the decay rate of current inactivation in a concentration-dependent manner with an IC(50) of 18.6 microM. These effects were reversible after washout of the drug. Troglitazone caused the block of Kv1.3 with a similar pattern but was five times more potent than rosiglitazone with an IC(50) of 3.5 microM. The block of Kv1.3 by rosiglitazone and troglitazone was voltage-dependent at a membrane potential coinciding with the activation of the channels. Both drugs decreased the tail current amplitude and slowed the deactivation process of Kv1.3, resulting in a tail crossover phenomenon. These results indicate that rosiglitazone and troglitazone block the open state of Kv1.3 channels, suggesting that it is an important pharmacological target for rosiglitazone as a potent blocker of Kv1.3 channels.
Archiv für Experimentelle Pathologie und Pharmakologie 02/2007; 374(4):305-9. · 2.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effect of riluzole on Kv4.3 was examined using the whole-cell patch-clamp technique. Riluzole inhibited the peak amplitude of Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 115.6 microM. Under control conditions, a good fit for the inactivation of Kv4.3 currents to a double exponential function, with the time constants of the fast component (tau(f)) and the slow component (tau(s)), was obtained. tau(f) was not altered by riluzole at concentrations up to 100 microM, but tau(s) became slower with increasing riluzole concentration, resulting in the crossover of the currents. The inhibition increased steeply with increasing channel activation at more positive potentials. In the full activation voltage range positive to (+)30 mV, however, no voltage-dependent inhibition was found. Riluzole shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. However, the slope factor was not affected by riluzole. The K(i) for riluzole for interacting with the inactivated state of Kv4.3 was estimated from the concentration-dependent shift in the steady-state inactivation curve and was determined to be 1.2 muM. Under control conditions, closed state inactivation was fitted to a single exponential function. Riluzole caused a substantial acceleration in the closed state inactivation. In the presence of riluzole, the recovery from inactivation was slower than under control conditions. Riluzole induced a significant use-dependent inhibition of Kv4.3. These results suggest that riluzole inhibits Kv4.3 by binding to the closed inactivated state of the channels and that the unbinding of riluzole occurs from the closed state during depolarization.
Journal of Pharmacology and Experimental Therapeutics 11/2006; 319(1):323-31. · 3.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.
Brain Research 09/2004; 1018(2):201-7. · 2.73 Impact Factor
-
Jin-Sung Choi,
Bok Hee Choi, Hye Sook Ahn,
Myung-Jun Kim,
Duck-Joo Rhie,
Shin Hee Yoon,
Do Sik Min,
Yang-Hyeok Jo,
Myung-Suk Kim,
Ki-Wug Sung,
Sang June Hahn
[show abstract]
[hide abstract]
ABSTRACT: The effect of fluoxetine (Prozac) on 5-hydroxytryptamine(3) (5-HT(3))-mediated currents in NCB-20 neuroblastoma cells was examined using the whole-cell patch-clamp technique. Fluoxetine produced a significant reduction of peak amplitude without altering the activation time course of 5-HT(3)-mediated currents. These effects were concentration-dependent, with an IC(50) value of 4.15 microM. No voltage dependence was evident in fluoxetine's block of 5-HT(3)-mediated currents over the entire voltage range tested. The extent of block by pre-application of fluoxetine was significantly greater than that by co-application. Fluoxetine also increased the apparent rate of current desensitization to 5-HT application. Using a first-order kinetics analysis, the open-channel blocking rate constants were 0.06 microM(-1)s(-1) (k(+1), association rate constant) and 0.05 s(-1) (k(-1), dissociation rate constant), with an apparent K(d) (=k(-1)/k(+1)) of 0.83 microM. This value is close to an IC(50) of 1.11 microM obtained from the reduction in tau, the time constant of desensitization. Intracellular application of fluoxetine for long durations had no effect on the amplitude or kinetics of 5-HT(3)-mediated currents. Similarly, norfluoxetine, the major metabolite of fluoxetine, reduced the peak current, and enhanced the rate of current desensitization in a concentration-dependent manner with an IC(50) of 2.66 microM, indicating that norfluoxetine is more potent than fluoxetine in blocking 5-HT(3)-mediated currents. These results indicate that, at clinically relevant concentrations, fluoxetine and its metabolite, norfluoxetine, block 5-HT(3)-mediated currents in NCB-20 neuroblastoma cells.
Biochemical Pharmacology 01/2004; 66(11):2125-32. · 4.70 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The effects of fluoxetine were studied on cloned K+ channel Kv1.4 stably expressed in Chinese hamster ovary (CHO) cells using the whole-cell configuration of the patch-clamp technique. Extracellular application of various concentrations of fluoxetine inhibited the amplitude of the peak current of Kv1.4 and accelerated its inactivation time course in a concentration-dependent manner. Thus, fluoxetine decreased Kv1.4 (the integral of the outward current) in a concentration-dependent manner; the IC50 was 33.1 +/- 2.5 microM. The inhibitory effect of fluoxetine was time-dependent. The apparent association (k) and dissociation (l) rate constants measured at +40 mV were 3.5 +/- 0.7 microM-1s-1 and 132.5 +/- 13.3 s-1, respectively. The Kd (= l/k) was 37.9 microM, which was close to the value obtained from the concentration-response curve. The block produced by fluoxetine increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. The fluoxetine block was constant at more depolarized potentials, suggesting that the block by fluoxetine was not voltage dependent. Our data indicate that fluoxetine blocks Kv1.4 channels by preferentially binding to open state.
Neuroreport 01/2004; 14(18):2451-5. · 1.66 Impact Factor
