Inhibition of astroglial Kir4.1 channels by selective serotonin reuptake inhibitors

The inward rectifier potassium (Kir) channel Kir4.1 plays essential roles in modulation of neurotransmission and renal sodium transport and may represent a novel drug target for temporal lobe epilepsy and hypertension. The molecular pharmacology of Kir4.1 is limited to neurological drugs, such as fluoxetine (Prozac ª ), exhibiting weak and nonspecific activity toward the channel. The development of potent and selective small-molecule probes would provide critically needed tools for exploring the integrative physiology and therapeutic potential of Kir4.1. A fluorescence-based thallium (Tl + ) flux assay that utilizes a tetracycline-inducible T-Rex-HEK293-Kir4.1 cell line to enable high-throughput screening (HTS) of small-molecule libraries was developed. The assay is dimethyl sulfoxide tolerant and exhibits robust screening statistics (Z 0 = 0.75 -0.06). A pilot screen of 3,655 small molecules and lipids revealed 16 Kir4.1 inhibitors (0.4% hit rate). 3,3-Diphenyl-N-(1-phenylethyl)propan-1-amine, termed VU717, inhibits Kir4.1-mediated thallium flux with an IC 50 of *6 lM. An automated patch clamp assay using the IonFlux HT workbench was developed to facilitate compound characterization. Leak-subtracted ensemble ''loose patch'' recordings revealed robust tetracycline-inducible and Kir4.1 currents that were inhibited by fluoxetine (IC 50 = 10 lM), VU717 (IC 50 = 6 lM), and structurally related calcium channel blocker prenylamine (IC 50 = 6 lM). Finally, we demonstrate that VU717 inhibits Kir4.1 channel activity in cultured rat astrocytes, providing proof-of-concept that the Tl + flux and IonFlux HT assays can enable the discovery of antagonists that are active against native Kir4.1 channels.

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“…1C), as reported by Kurachi and colleagues. 28,29 The Figure 1D. Fluoxetine also inhibited the endogenous outward current in HEK cells (Fig.…”

Section: Cell Line Characterizationmentioning

confidence: 82%

Development and Validation of Fluorescence-Based and Automated Patch Clamp–Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1

RaphemotRene

KadakiaRishin

Olsen

et al. 2013

ASSAY and Drug Development Technologies

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“…For example, fluvoxamineinhibitedtheKir4.1channelinhumanembryonic kidney (HEK) 293T cells 24) and G protein-activated inwardly rectifying K + (GIRK) in Xenopus oocytes. 25) Fluvoxaminealso directly blocked the Kv1.5 current in Chinese hamster ovary (CHO) cells 10) and the human ether-a-go-go related (HERG) K + channel in HEK 293 cells.…”

Section: Discussionmentioning

confidence: 99%

The Effects of the Selective Serotonin Reuptake Inhibitor Fluvoxamine on Voltage-Dependent K<sup>+</sup> Channels in Rabbit Coronary Arterial Smooth Muscle Cells

Hong

Kim

et al. 2015

Biological & Pharmaceutical Bulletin

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We demonstrated the inhibitory effect of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Fluvoxamine reduced the amplitude of Kv currents in a concentration-dependent manner with an IC 50 value of 3.71 1.09 µM and a Hill coefficient of 0.62 0.14. Although fluvoxamine did not significantly affect the steady-state activation curve, it shifted the steady-state inactivation curve toward a more negative potential. Pretreatment with another SSRI, paroxetine, did not affect the basal Kv current and did not alter the inhibitory effect of fluvoxamine on Kv channels. We concluded that fluvoxamine inhibits the Kv current in a concentration-dependent manner and in a closed (inactivated) state of the Kv channels independent of serotonin reuptake inhibition.Key words fluvoxamine;voltage-dependentK + channel; coronary artery; smooth muscle; selective serotonin reuptake inhibitorFluvoxamine is a selective serotonin reuptake inhibitor (SSRI) that has been used for the treatment of major depression, anxiety, and panic disorder since 1983 in many countries, including those in Europe and Japan.1,2) The antidepressant activity of fluvoxamine is derived from inhibition of serotonin reuptake in the central nervous system with negligible effects on other neurotransmitter reuptake systems.3,4) A recent study indicated that fluvoxamine showed high affinity for the σ1 receptor, which has a neuromodulatory role on the neurotransmitter system and an antidepressant-like effect. 5-7)Although fluvoxamine has beneficial effects in the treatment of depression, its administration is associated with common side effects, including nausea, anorexia, palpitations, headache, and dizziness. 8)Fluvoxamine also affects ion channels, such as the human ether-a-go-go related (hERG) K + and Kv1.5 channels. 9,10) However, these studies were performed primarily in cultured cell lines rather than in native cells. Therefore, questionsremainabouttheeffectoffluvoxamineonionchannels from freshly isolated cells, particularly vascular smooth muscle cells. 11,12) Our previous reports suggested that Kv channels participate in the regulation of resting membrane potential and thus resting tone in some arteries. 13,14) In addition, pathological states such as hypertension, diabetes, and hypoxia, are closely related with alteration of Kv channel activity and expression in the vasculature.15) Therefore, the adverse effects of drugs on Kv channels should be clearly determined to correctly interpret experimental data regarding vascular disorders.Considering the usefulness of fluvoxamine as a SSRI and the physiological importance of Kv channels, the effects of fluvoxamine on vascular Kv channel should be clearly examined to avoid toxic effect on the vasculature when applying fluvoxamineasantidepressantdrug.In the present study, we investigated the effects of fluvoxamine on Kv channels using freshly isolated coronary ...

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“…Despite the intense molecular and physiological studies on the Kir channels, pharmacological studies are limited (Spassova and Lu, 1998;Yamada et al, 1998;Kobayashi et al, 2004;Kikuta et al, 2006;Liu et al, 2007;Ohno et al, 2007;Su et al, 2007;Rodríguez-Menchaca et al, 2008). In particular, little is known about the interaction of small organic chemicals with Kir channels (Rodríguez-Menchaca et al, 2008).…”

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confidence: 99%

Mutational and In Silico Analyses for Antidepressant Block of Astroglial Inward-Rectifier Kir4.1 Channel

Furutani¹,

Ohno²,

Inanobe³

et al. 2009

Mol Pharmacol

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Drug interaction with target proteins including ion channels is essential for pharmacological control of various cellular functions, but the majority of its molecular mechanisms is still elusive. We recently found that a series of antidepressants preferentially block astroglial K ϩ -buffering inwardly rectifying potassium channel (Kir) 4.1 channels over Kir1.1 channels. Here, using electrophysiological analyses of drug action on mutated Kir4.1 channel as well as computational analyses of three-dimensional (3D) arrangements of the ligands (i.e., bidirectional analyses), we examined the underlying mechanism for the antidepressant-Kir4.1 channel interaction. First, the effects of the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant nortriptyline on chimeric and site-directed mutants of Kir4.1 expressed in Xenopus laevis oocytes were examined using the two-electrode voltage-clamp technique. Two amino acids, Thr128 and Glu158, on pore and transmembrane domain 2 helices were critical for the drug inhibition of the current. The closed and open conformation models of the Kir4.1 pore suggested that both residues faced the central cavity, and they were positioned within a geometrical range capable of interacting with the drugs. Second, to represent molecular properties of active ligands in geometric terms, a 3D quantitative structure-activity relationship model of antidepressants was generated, which suggested that they share common features bearing a hydrogen bond acceptor and a positively charged moiety. 3D structures and physicochemical features of receptor and ligand were fitted together. Our results strongly suggest that antidepressants interact with Kir4

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Inhibition of astroglial Kir4.1 channels by selective serotonin reuptake inhibitors

Cited by 102 publications

References 36 publications

Development and Validation of Fluorescence-Based and Automated Patch Clamp–Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1

Development and Validation of Fluorescence-Based and Automated Patch Clamp–Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1

The Effects of the Selective Serotonin Reuptake Inhibitor Fluvoxamine on Voltage-Dependent K<sup>+</sup> Channels in Rabbit Coronary Arterial Smooth Muscle Cells

Mutational and In Silico Analyses for Antidepressant Block of Astroglial Inward-Rectifier Kir4.1 Channel

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