KCNJ11 gene mutation in 3 cases with neonatal diabetes mellitus / 中华内分泌代谢杂志

KCNJ11 gene mutation was searched in 3 families with neonatal diabetes. A KCNJ11 175 G>A (V59M) mutation was found in one child, while no KCNJ11 gene mutation was found in his parents. No mutation was found in the other two families. The result indicated that KCNJ11 gene mutation might lead to the onset of neonatal diabetes mellitus in Chinese.

Detection of Mitochondrial ATP-Sensitive Potassium Channels in Rat Cardiomyocytes

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while mitoKATP channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of mitoKATP channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands (~4, ~6, and ~0 kDa) were detected, and a specific ~6-kDa immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of mitoKATP channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.

Nitric Oxide-cGMP-Protein Kinase G Pathway Contributes to Cardioprotective Effects of ATP-Sensitive K+ Channels in Rat Hearts

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium (KATP) channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that KATP channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide (100muM), SNAP (300muM) and 8- (4-Chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (8-pCPT-cGMP) (100muM) followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide (50muM), 5-hydroxydecanoate (5-HD) (100muM) and 8- (4-Chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) (100muM). These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of KATP channels in rat ventricular myocytes.

Identification of ATP-sensitive K+ Conductances in Male Rat Major Pelvic Ganglion Neurons

Major pelvic ganglia (MPG) neurons are classified into sympathetic and parasympathetic neurons according to the electrophysiological properties; membrane capacitance (Cm), expression of T-type Ca2+ channels, and the firing patterns during depolarization. In the present study, function and molecular expression of ATP-sensitive K+ (K(ATP)) channels was investigated in MPG neurons of male rats. Only in parasympathetic MPG neurons showing phasic firing patterns, hyperpolarizing changes were elicited by the application of diazoxide, an activator of K(ATP) channels. Glibenclamide (10microM), a K(ATP) channel blocker, completely abolished the diazoxide-induced hyperpolarization. Diazoxide increased inward currents at high K+ (90 mM) external solution, which was also blocked by glibenclamide. The metabolic inhibition by the treatment with mitochondrial respiratory chain inhibitors (rotenone and antimycin) hyperpolarized the resting membrane potential of parasympathetic neurons, which was not observed in sympathetic neurons. The hyperpolarizing response to metabolic inhibition was partially blocked by glibenclamide. RT-PCR analysis revealed that MPG neurons mainly expressed the K(ATP) channel subunits of Kir6.2 and SUR1. Our results suggest that MPG neurons have K(ATP) channels, mainly formed by Kir6.2 and SUR1, with phenotype-specificity, and that the conductance through this channel in parasympathetic neurons may contribute to the changes in excitability during hypoxia and/or metabolic inhibition.

Block of ATP-sensitive K+ channels expressed in Xenopus oocytes by dimethyl sulfoxide

The effects of dimethyl sulfoxide (DMSO) were studied in two groups of Xenopus oocytes, one expressing ATP sensitive K+ (KATP) channel comprised of sulfonylurea receptor SUR1 and inwardly rectifying K+ channel subunit Kir6.2, and the other expressing renal KATP channel ROMK2. At concentrations of 0.3~10% (vol/vol) DMSO inhibited whole cell Kir6.2/SUR1 currents elicited by bath application of sodium azide (3 mM) in a concentration-dependent manner. The inhibition constant and Hill coefficient were 2.93% and 1.62, respectively. ROMK2 currents, however, was not affected significantly by DMSO. The results support the idea that DMSO inhibits KATP channel expressed in Xenopus oocyte through a protein-specific mechanism(s) that remains to be further elucidated.

Activation of the Cardiac ATP-Sensitive K+Channel by KR-30816,Newly Synthesized Potassium Channel Opener

BACKGROUND: The effects of a newly synthesized potassium channel opener, KR-30816((-)(nitro-2-hydroxymethyl-2-methy-2H-1-benzopyran-4-y1)pyridine oxide) on the action potential of papillary muscles of guinea pigs and the ATP-sensitive potassium channel current(IKATP) of single ventricular muscle cells of rats were examined to make clear its action mechanism of the KATPchannel. METHODS: We used the conventional microelectrode and the excised inside-out patch configuration. RESULTS: KR-30816 caused a shortening of the action potential duration in dose-dependent manner, which was inhibited by glibenclamide(3microM). Before run-down of the K+channel, KR-30816 activated the cardiac ATP-sensitive K+ channel only in the presence of ATP and shifted the dose-response relation curve between [ATP]i and the channel activity to the right in parallel. After run-down of the KATP channel, KR-30816 did not after the channel opening either in the absence or in the presence of UDP. CONCLUSION: These results suggest that KR-30816 antagonizes the inhibitory effect of ATP on the KATPchannel in a competitive manner, thereby enhancing the channel openings.

Effects of vanadate on vascular contractility and membrane potential in the rabbit aorta

Isolated rabbit aortic ring with intact endothelial cell preparations precontracted with NE (10(-7) M) were relaxed by vanadate in a dose dependent manner (from 0.2 to 2 mM). Application of vanadate and ACh during the tonic phase of high K+(100 mM)-induced contraction showed a slight relaxation in contrast to that in NE-induced contraction, but sodium nitroprusside (10 microM) more effectively relaxed the aortic ring preparations in high K+ contraction than that of vanadate. Vanadate-induced relaxation in NE-contracted aortic rings was reversed by application of BaCl2 (50 microM) or glibenclamide (10 microM). Furthermore, Vanadate hyperpolarized membrane potential of smooth muscle cells in endothelium-intact aortic strips and this effect was abolished by application of glibenclamide. The above results suggest that vanadate release EDHF (Endothelium-Derived Hyperpolarizing Factor), in addition to EDRF (Endothelium-Derived Relaxing Factor) from endothelial cell. This EDHF hyperpolarize the smooth muscle cell membrane potential via opening of the ATP-sensitive K+ channel and close a voltage dependent Ca++ channel. So it is suggested that the vanadate-induced relaxation of rabbit thoracic aortic rings may be due to the combined effects of EDRF and EDHF.