Molecular basis involved in the blocking effect of antidepressant metergoline on C-type inactivation of Kv1.4 channel

Bai, Hyoung-Woo,Eom, Sanung,Yeom, Hye Duck,Nguyen, Khoa V.A.,Lee, Jaeeun,Sohn, Sung-Oh,Lee, Jun-Ho Elsevier 2019 NEUROPHARMACOLOGY - Vol.146 No.-

<P><B>Abstract</B></P> <P>Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In a previous work, we have reported the regulatory effect of metergoline on Kv1.4 and Nav1.2 channel activity. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 Δ2-61) and found that it induced an outward peak and steady-state currents We also studied the modulation effect of metergoline on the activity of this Kv1.4 Δ2-61 mutant channel without having the N-terminal quick inactivation domain. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 Δ2-61 channels with acceleration current mode. The acceleration of steady-state current of deleted Kv1.4 mutant channel occurred in a concentration-dependent manner. This means that metergoline can accelerate C pattern inactivation of Kv1.4 Δ2-61 channel by acting as an open state dependent channel blocker. We also performed site-directed mutations in V561A and K532Y, also known as C-type inactivation sites. V561A, K532Y, and V561A + K532Y substitution mutants significantly attenuated the acceleration effect of metergoline on C pattern inactivation of hKv1.4 channel currents. In docking modeling study, predicted binding residues for metergoline were analyzed for six amino acids. Among them, the K532 residue known as the C-type inactivation site was analyzed to be a major site of action. Then various mutants were constructed. K532 substitution mutant significantly abolished the effect of metergoline on Kv1.4 currents among various mutants whereas other changes had slight inhibitory effects. Furthermore, we found that metergoline had specificity for Kv1.4, but not for Kv1.5 currents. In addition, the A type current in rat neuronal cell was inhibited and accelerated of inactivation. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Metergoline, ergot-derived psychoactive drug, produced inhibition on steady state current of Kv1.4 channel currents. </LI> <LI> Treatment of metergoline on Kv1.4 Δ2-61 channel currents produced significant inhibitions of the non-inactivating plateau than peak currents. </LI> <LI> Metergoline interacted predominantly with residues at channel pore region of Kv1.4 channel. </LI> <LI> Metergoline is more selective for Kv1.4 channels and is shown the effects as open channel blocker. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Role of potassium channels in chlorogenic acid-induced apoptotic volume decrease and cell cycle arrest in <i>Candida albicans</i>

Yun, JiEun,Lee, Dong Gun Elsevier 2017 Biochimica et biophysica acta, General subjects Vol.1861 No.3

<P><B>Abstract</B></P> <P><B>Background</B></P> <P>Chlorogenic acid (CRA) is an abundant phenolic compound in the human diet. CRA has a potent antifungal effect, inducing cell death in <I>Candida albicans</I>. However, there are no further studies to investigate the antifungal mechanism of CRA, associated with ion channels.</P> <P><B>Methods</B></P> <P>To evaluate the inhibitory effects on CRA-induced cell death, <I>C. albicans</I> cells were pretreated with potassium and chloride channel blockers, separately. Flow cytometry was carried out to detect several hallmarks of apoptosis, such as cell cycle arrest, caspase activation, and DNA fragmentation, after staining of the cells with SYTOX green, FITC-VAD-FMK, and TUNEL.</P> <P><B>Results</B></P> <P>CRA caused excessive potassium efflux, and an apoptotic volume decrease (AVD) was observed. This change, in turn, induced cytosolic calcium uptake and cell cycle arrest in <I>C. albicans</I>. Moreover, CRA induced caspase activation and DNA fragmentation, which are considered apoptotic markers. In contrast, the potassium efflux and proapoptotic changes were inhibited when potassium channels were blocked, whereas there was no inhibitory effect when chloride channels were blocked.</P> <P><B>Conclusions</B></P> <P>CRA induces potassium efflux, leading to AVD and G2/M cell cycle arrest in <I>C. albicans</I>. Therefore, potassium efflux via potassium channels regulates the CRA-induced apoptosis, stimulating several apoptotic processes.</P> <P><B>General significance</B></P> <P>This study improves the understanding of the antifungal mechanism of CRA and its association with ion homeostasis, thereby pointing to a role of potassium channels in CRA-induced apoptosis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Chlorogenic acid (CRA) induces apoptosis in <I>Candida albicans</I>. </LI> <LI> CRA induces potassium efflux leading to apoptotic volume decrease. </LI> <LI> Block of the potassium channels inhibits the apoptotic effect of CRA. </LI> <LI> Potassium channels play a key role in regulation of the CRA-induced apoptosis. </LI> </UL> </P>

Colonic Dysmotility in Murine Partial Colonic Obstruction Due to Functional Changes in Interstitial Cells

Qianqian Wang,Jingyu Zang,Xu Huang,Hongli Lu,Wenxie Xu,Jie Chen 대한소화기 기능성질환∙운동학회 2019 Journal of Neurogastroenterology and Motility (JNM Vol.25 No.4

Background/Aims Interstitial cells play important roles in gastrointestinal (GI) neuro-smooth muscle transmission. The underlying mechanisms of colonic dysmotility have not been well illustrated. We established a partial colon obstruction (PCO) mouse model to investigate the changes of interstitial cells and the correlation with colonic motility. Methods Western blot technique was employed to observe the protein expressions of Kit, platelet-derived growth factor receptor-α (Pdgfra), Ca2+-activated Cl− (Ano1) channels, and small conductance Ca2+- activated K+ (SK) channels. Colonic migrating motor complexes (CMMCs) and isometric force measurements were employed in control mice and PCO mice. Results PCO mice showed distended abdomen and feces excretion was significantly reduced. Anatomically, the colon above the obstructive silicone ring was obviously dilated. Kit and Ano1 proteins in the colonic smooth muscle layer of the PCO colons were significantly decreased, while the expression of Pdgfra and SK3 proteins were significantly increased. The effects of a nitric oxide synthase inhibitor (L-NAME) and an Ano1 channel inhibitor (NPPB) on CMMC and colonic spontaneous contractions were decreased in the proximal and distal colons of PCO mice. The SK agonist, CyPPA and antagonist, apamin in PCO mice showed more effect to the CMMCs and colonic smooth muscle contractions. Conclusions Colonic transit disorder may be due to the downregulation of the Kit and Ano1 channels and the upregulation of SK3 channels in platelet-derived growth factor receptor-α positive (PDGFRα+) cells. The imbalance between interstitial cells of Cajal-Ano1 and PDGFRα-SK3 distribution might be a potential reason for the colonic dysmotility. Background/Aims Interstitial cells play important roles in gastrointestinal (GI) neuro-smooth muscle transmission. The underlying mechanisms of colonic dysmotility have not been well illustrated. We established a partial colon obstruction (PCO) mouse model to investigate the changes of interstitial cells and the correlation with colonic motility. Methods Western blot technique was employed to observe the protein expressions of Kit, platelet-derived growth factor receptor-α (Pdgfra), Ca2+-activated Cl− (Ano1) channels, and small conductance Ca2+- activated K+ (SK) channels. Colonic migrating motor complexes (CMMCs) and isometric force measurements were employed in control mice and PCO mice. Results PCO mice showed distended abdomen and feces excretion was significantly reduced. Anatomically, the colon above the obstructive silicone ring was obviously dilated. Kit and Ano1 proteins in the colonic smooth muscle layer of the PCO colons were significantly decreased, while the expression of Pdgfra and SK3 proteins were significantly increased. The effects of a nitric oxide synthase inhibitor (L-NAME) and an Ano1 channel inhibitor (NPPB) on CMMC and colonic spontaneous contractions were decreased in the proximal and distal colons of PCO mice. The SK agonist, CyPPA and antagonist, apamin in PCO mice showed more effect to the CMMCs and colonic smooth muscle contractions. Conclusions Colonic transit disorder may be due to the downregulation of the Kit and Ano1 channels and the upregulation of SK3 channels in platelet-derived growth factor receptor-α positive (PDGFRα+) cells. The imbalance between interstitial cells of Cajal-Ano1 and PDGFRα-SK3 distribution might be a potential reason for the colonic dysmotility.

Regulation of human Kv1.4 channel activity by the antidepressant metergoline

Junho Lee 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na+ channels in Xenopus laevis oocytes. In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity.

Encainide, a class Ic anti-arrhythmic agent, blocks voltage-dependent potassium channels in coronary artery smooth muscle cells

Hongliang Li,Yue Zhou,Yongqi Yang,Yiwen Zha,Bingqian Ye,Seo-Yeong Mun,Wenwen Zhuang,Jingyan Liang,Won Sun Park The Korean Society of Pharmacology 2023 The Korean Journal of Physiology & Pharmacology Vol.27 No.4

Voltage-dependent K⁺ (Kv) channels are widely expressed on vascular smooth muscle cells and regulate vascular tone. Here, we explored the inhibitory effect of encainide, a class Ic anti-arrhythmic agent, on Kv channels of vascular smooth muscle from rabbit coronary arteries. Encainide inhibited Kv channels in a concentration-dependent manner with an IC₅₀ value of 8.91 ± 1.75 μM and Hill coefficient of 0.72 ± 0.06. The application of encainide shifted the activation curve toward a more positive potential without modifying the inactivation curve, suggesting that encainide inhibited Kv channels by altering the gating property of channel activation. The inhibition by encainide was not significantly affected by train pulses (1 and 2 Hz), indicating that the inhibition is not use (state)-dependent. The inhibitory effect of encainide was reduced by pretreatment with the Kv1.5 subtype inhibitor. However, pretreatment with the Kv2.1 subtype inhibitor did not alter the inhibitory effects of encainide on Kv currents. Based on these results, encainide inhibits vascular Kv channels in a concentration-dependent and use (state)-independent manner by altering the voltage sensor of the channels. Furthermore, Kv1.5 is the main Kv subtype involved in the effect of encainide.

Evidence for the Participation of ATP-sensitive Potassium Channels in the Antinociceptive Effect of Curcumin

( Marco Antonio ),( Paz Campos ),( Aracely Evangelina ),( Chavez Pina ),( Mario I Ortiz ),( Gilberto ),( Castaneda Hernandez ) 대한통증학회 2012 The Korean Journal of Pain Vol.25 No.4

Background: It has been reported that curcumin, the main active compound of Curcuma longa, also known as turmeric, exhibits antinociceptive properties. The aim of this study was to examine the participation of ATP-sensitive potassium channels (KATP channels) and, in particular, that of the L-arginine-nitric oxide-cyclic GMP-KATP channel pathway, in the antinociceptive effect of curcumin. Methods: Pain was induced by the intraplantar injection of 1% formalin in the right hind paw of Wistar rats. Formalin-induced flinching behavior was interpreted as an expression of nociception. The antinociceptive effect of oral curcumin was explored in the presence and absence of local pretreatment with L-NAME, an inhibitor of nitric oxide synthase, ODQ, an inhibitor of soluble guanylyl cyclase, and glibenclamide, a blocker of KATP channels. Results: Oral curcumin produced a dose-dependent antinociceptive effect in the 1% formalin test. Curcumin-induced antinociception was not altered by local L-NAME or ODQ, but was significantly impaired by glibenclamide. Conclusions: Our results confirm that curcumin is an effective antinociceptive agent. Curcumin-induced antinociception appears to involve the participation of KATP channels at the peripheral level, as local injection of glibenclamide prevented its effect. Activation of KATP channels, however, does not occur by activation of the L-arginine-nitric oxide-cGMP- KATP channel pathway.

Influences of Cholecystokinin on Potassium Channels in submaxillary Acinar Cells of Rat

Kim, Kyung-Nyun,Kim, Joong-Soo,Lee, Jong-Heun The Official Publication of Korean Academy of Oral 1994 International Journal of Oral Biology Vol.18 No.1

The saliva is formed by outflow of potassium due to increase of intracellular calcium activity. The principal passage of potassium ions is the calcium-activated potassium channels. This study was performed to elucidate the change in the activity of calcium-activated potassium channels during salivary secretion induced by acetylcholine (ACh) or cholecystokinin(CCK). The submaxillary acinar cells were collected from Sprague-Dawley rat (about 200 g body weight) by time-scheduled trypsin and collagenase treatment. Dispersed acinar cells were placed in 100% oxygenated HEPES-buffered Tyrode solution and perfused. Cell attached or excised inside out patch clamp method was employed. The channel activities recorded via mocropipettes (1-5 ㏁ resistance) filled with HEPES-buffered high potassium solution were evaluated with personal computer. Two distinct types of potassium channels in membranes of submaxillary acinar cells were observed. The one type had large conductance and was sensitive to pipette voltage and calcium ion activity, but the other type had small conductance and was not sensitive to pipette voltage. Addition of ACh and/or CCK increased the open probability of potassium channels without change in conductance. These results suggest that CCK could evoke salivary secretion via the same mechanism as ACh.

The large-conductance calcium-activated potassium channel holds the key to the conundrum of familial hypokalemic periodic paralysis

Kim, June-Bum,Kim, Sung-Jo,Kang, Sun-Yang,Yi, Jin Woong,Kim, Seung-Min The Korean Pediatric Society 2014 Clinical and Experimental Pediatrics (CEP) Vol.57 No.10

Purpose: Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant channelopathy characterized by episodic attacks of muscle weakness and hypokalemia. Mutations in the calcium channel gene, CACNA1S, or the sodium channel gene, SCN4A, have been found to be responsible for HOKPP; however, the mechanism that causes hypokalemia remains to be determined. The aim of this study was to improve the understanding of this mechanism by investigating the expression of calcium-activated potassium ($K_{Ca}$) channel genes in HOKPP patients. Methods: We measured the intracellular calcium concentration with fura-2-acetoxymethyl ester in skeletal muscle cells of HOKPP patients and healthy individuals. We examined the mRNA and protein expression of KCa channel genes (KCNMA1, KCNN1, KCNN2, KCNN3, and KCNN4) in both cell types. Results: Patient cells exhibited higher cytosolic calcium levels than normal cells. Quantitative reverse transcription polymerase chain reaction analysis showed that the mRNA levels of the $K_{Ca}$ channel genes did not significantly differ between patient and normal cells. However, western blot analysis showed that protein levels of the KCNMA1 gene, which encodes $K_{Ca}$1.1 channels (also called big potassium channels), were significantly lower in the membrane fraction and higher in the cytosolic fraction of patient cells than normal cells. When patient cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the altered subcellular distribution of BK channels remained unchanged. Conclusion: These findings suggest a novel mechanism for the development of hypokalemia and paralysis in HOKPP and demonstrate a connection between disease-associated mutations in calcium/sodium channels and pathogenic changes in nonmutant potassium channels.

Regulation of Antiarrhythmic Drug Propafenone Effects on the C-type KV1.4 Potassium Channel by PHo and K+

Zhiquan Wang,Jianjun Li,Xuejun Jiang,Xuejun Jiang,Neng Wang,Shimin Wang 대한의학회 2009 Journal of Korean medical science Vol.24 No.1

The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp techinique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4△N channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC50) was 121 μM/L. With high, normal, and low extracellular potassium concentrations, the changes of IC50 value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC50 values to 463 μM/L, alkalization to PH 8.0 reduced it to 58 μM/L. The results suggest that propafenone blocks the kv1.4 N channel in the open state and give some hints for an intracellular site of action.

Altered potassium ATP channel signaling in mesenteric arteries of old high salt-fed rats

Melissa A Whidden,Bilgen Basgut,Nataliya Kirichenko,Benedek Erdos,Nihal Tumer 한국운동영양학회 2016 Physical Activity and Nutrition (Phys Act Nutr) Vol.20 No.2

Purpose: Both aging and the consumption of a high salt diet are associated with clear changes in the vascular system that can lead to the development of cardiovascular disease; however the mechanisms are not clearly understood. Therefore, we examined whether aging and the consumption of excess salt alters the function of potassium ATP-dependent channel signaling in mesenteric arteries. Methods: Young (7 months) and old (29 months) Fischer 344 x Brown Norway rats were fed a control or a high salt diet (8% NaCl) for 12 days and mesenteric arteries were utilized for vascular reactivity measurements. Results: Acetylcholine-induced endothelium relaxation was significantly reduced in old arteries (81 ± 4%) when compared with young arteries (92 ± 2%). Pretreatment with the potassium-ATP channel blocker glibenclamide reduced relaxation to acetylcholine in young arteries but did not alter dilation in old arteries. On a high salt diet, endothelium dilation to acetylcholine was significantly reduced in old salt arteries (60 ± 3%) when compared with old control arteries (81 ± 4%). Glibenclamide reduced acetylcholine-induced dilation in young salt arteries but had no effect on old salt arteries. Dilation to cromakalim, a potassium-ATP channel opener, was reduced in old salt arteries when compared with old control arteries. Conclusions: These findings demonstrate that aging impairs endothelium-dependent relaxation in mesenteric arteries. Furthermore, a high salt diet alters the function of potassium-ATP-dependent channel signaling in old isolated mesenteric arteries and affects the mediation of relaxation stimuli.