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Metformin restores electrophysiology of small conductance calcium-activated potassium channels in the atrium of GK diabetic rats.

ABSTRACT: BACKGROUND:Small conductance calcium-activated potassium channels (SK channels) play a critical role in action potential repolarization in cardiomyocytes. Recently, the potential anti-arrhythmic effect of metformin in diabetic patients has been recognized, yet the underlying mechanism remains elusive. METHODS:Diabetic Goto-Kakizaki (GK) rats were untreated or treated with metformin (300 mg/kg/day) for 12 weeks, and age-matched Wistar rats were used as control (n?=?6 per group). Electrocardiography, Hematoxylin-eosin staining and Masson's trichome staining were performed to assess cardiac function, histology and fibrosis. The expression levels of the SK channels in the myocardium were determined by real-time PCR and Western blotting. The electrophysiology of the SK channels in the cardiomyocytes isolated from the three groups of rats was examined by patch clamp assay, with specific blockade of the SK channels with apamin. RESULTS:Metformin treatment significantly reduced cardiac fibrosis and alleviated arrhythmia in the diabetic rats. In the atrial myocytes from control, GK and metformin-treated GK rats, the expression of KCa2.2 (SK2 channel) was down-regulated and the expression of KCa2.3 (SK3 channel) was up-regulated in the atrium of GK rats as compared with that of control rats, and metformin reversed diabetes-induced alterations in atrial SK channel expression. Moreover, patch clamp assay revealed that the SK current was markedly reduced and the action potential duration was prolonged in GK atrial myocytes, and the SK channel function was partially restored in the atrial myocytes from metformin-treated GK rats. CONCLUSIONS:Our data suggests an involvement of the SK channels in the development of arrhythmia under diabetic conditions, and supports a potential beneficial effect of metformin on atrial electrophysiology.

SUBMITTER: Fu X

PROVIDER: S-EPMC5894224 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Metformin restores electrophysiology of small conductance calcium-activated potassium channels in the atrium of GK diabetic rats.

Fu Xi X Pan Yilong Y Cao Qian Q Li Bin B Wang Shuo S Du Hongjiao H Duan Na N Li Xiaodong X

BMC cardiovascular disorders 20180410 1

<h4>Background</h4>Small conductance calcium-activated potassium channels (SK channels) play a critical role in action potential repolarization in cardiomyocytes. Recently, the potential anti-arrhythmic effect of metformin in diabetic patients has been recognized, yet the underlying mechanism remains elusive.<h4>Methods</h4>Diabetic Goto-Kakizaki (GK) rats were untreated or treated with metformin (300 mg/kg/day) for 12 weeks, and age-matched Wistar rats were used as control (n = 6 per group). El ...[more]

PMID: 29636010

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Project description:Background and purposeControlling vascular tone involves K(+) efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K(Ca)2.3 and K(Ca)3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.Experimental approachTransgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K(Ca)2.3 and K(Ca)3.1 expression by immunohistochemistry and RT-PCR. K(Ca) currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K(Ca)3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.Key resultsK(Ca)3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K(Ca)2.3 and K(Ca)3.1 mRNA expression. With 200 nM [Ca(2+)](i) , the K(Ca)2.1-2.3/K(Ca)3.1 opener NS309 increased whole-cell currents. CyPPA, a K(Ca)2.2/K(Ca)2.3 opener, was without effect. With 1 µM [Ca(2+)](i) , the K(Ca)3.1 inhibitor TRAM-34 reduced currents whereas apamin (K(Ca)2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K(Ca)3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K(Ca)3.1 and the large-conductance K(Ca) channel, K(Ca)1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.Conclusion and implicationsAstrocytes express functional K(Ca)3.1 channels, and these contribute to neurovascular coupling.

Project description:1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.

Project description:Large conductance (BK-type) calcium-activated potassium channels utilize alternative splicing and association with accessory beta subunits to tailor BK channel properties to diverse cell types. Two important modulators of BK channel gating are the neuronal-specific beta4 accessory subunit (beta4) and alternative splicing at the stress axis hormone-regulated exon (STREX). Individually, these modulators affect the gating properties of the BK channel as well as its response to phosphorylation. In this study, the combined functional consequences of STREX and the mouse beta4 subunit on mouse BK channel biophysical properties were investigated in transfected HEK 293 cells. Surprisingly, we found that the combined effects of STREX and beta4 are non-additive and even opposite for some properties. At high calcium, beta4 and the STREX individually share properties that promote BK channel opening via slowing of deactivation. However, the combined effects are a speeding of deactivation and a decreased open probability. beta4 also inhibits BK channel opening by a slowing of activation. This effect occurs across calcium concentrations in the absence of STREX, but predominates only at low calcium for STREX containing channels. BK channel responses to phosphorylation status are also altered by the combination of the beta4 subunit and STREX. beta4/STREX channels show a slowing of activation kinetics following dephosphorylation whereas beta4 channels lacking STREX do not. In contrast, beta4 confers a speeding of activation in response to cyclic AMP-dependent phosphorylation in channels lacking STREX, but not in channels containing STREX. These results indicate that the combination of the beta4 subunit and STREX confers non-additive and unique properties to BK channels. Analysis of expression in brain slices suggests that STREX and beta4 mRNA overlap expression in the dentate gyrus of the hippocampus and the cerebellar Purkinje cells, suggesting that these unique properties of BK channels may underlie BK channel gating in these cells.

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Metformin restores electrophysiology of small conductance calcium-activated potassium channels in the atrium of GK diabetic rats.

Publications

Metformin restores electrophysiology of small conductance calcium-activated potassium channels in the atrium of GK diabetic rats.

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