Iptakalim Modulates ATP-Sensitive K⁺ Channels in Dopamine Neurons from Rat Substantia Nigra Pars Compacta

Abstract

Iptakalim, a novel cardiovascular ATP-sensitive K ϩ (K ATP ) channel opener, exerts neuroprotective effects on dopaminergic (DA) neurons against metabolic stress-induced neurotoxicity, but the mechanisms are largely unknown. Here, we examined the effects of iptakalim on functional K ATP channels in the plasma membrane (pm) and mitochondrial membrane using patch-clamp and fluorescence-imaging techniques. In identified DA neurons acutely dissociated from rat substantia nigra pars compacta (SNc), both the mitochondrial metabolic inhibitor rotenone and the sulfonylurea receptor subtype (SUR) 1-selective K ATP channel opener (KCO) diazoxide induced neuronal hyperpolarization and abolished action potential firing, but the SUR2B-selective KCO cromakalim exerted little effect, suggesting that functional K ATP channels in rat SNc DA neurons are mainly composed of SUR1. Immunocytochemical staining showed a SUR1-rather than a SUR2B-positive reaction in most dissociated DA neurons. At concentrations between 3 and 300 M, iptakalim failed to hyperpolarize DA neurons; however, 300 M iptakalim increased neuronal firing. In addition, iptakalim restored DA neuronal firing during rotenone-induced hyperpolarization and suppressed rotenone-induced outward current, suggesting that high concentrations of iptakalim close neuronal K ATP channels. Furthermore, in human embryonic kidney 293 cells, iptakalim (300 -500 M) closed diazoxide-induced Kir6.2/ SUR1 K ATP channels, which were heterologously expressed. In rhodamine-123-preloaded DA neurons, iptakalim neither depolarized mitochondrial membrane nor prevented rotenone-induced mitochondrial depolarization. These data indicate that iptakalim is not a K ATP channel opener in rat SNc DA neurons; instead, iptakalim is a pm-K ATP channel closer at high concentrations. These effects of iptakalim stimulate further pharmacological investigation and the development of possible therapeutic applications.

Mitochondrial ATP-sensitive potassium channels enhance angiotensin-induced oxidative damage and dopaminergic neuron degeneration. Relevance for aging-associated susceptibility to Parkinson’s disease

“…Doses up to 500 mmol/l have been reported to inhibit selectively mitoK(ATP) channels without affecting surface K(ATP) currents (Sato et al 2000;Hu et al 1999). However, we did not observe significant differences between the effect of 10 μM 5-HD and 100 μM 5-HD, and high doses of 5-HD may have nonspecific side effects independent of any action on mitoK(ATP), as reported for mitoK(ATP) openers, which have been observed to act as channel closers at high concentrations (Garlid 2000;Wu et al 2006). In the present study, we therefore used the lowest dose of 5-HD (10 μM), which proved effective at inhibiting the effects of angiotensin II.…”

“…The discrepancies may be related to different experimental conditions. MitoK(ATP) openers have been reported to act as channel closers at high concentrations (Wu et al 2006). It has also been suggested that opening of mitoK(ATP) may have two distinct consequences, depending on the underlying bioenergetic state (Garlid et al 2003).…”

“…5-HD is the most widely used specific inhibitor of mitoK(ATP) channels, and several studies have shown that 5-HD (10 μM) blocks the mitoK(ATP) channels without any effect on cell membrane K(ATP) channels (Costa and Garlid 2008;Garlid et al 1997;McCullough et al 1991;Zhang et al 2007). Several doses of 5-HD were tested in the present and preliminary experiments (10, 100, 500 μM; Rodriguez-Pallares et al 2009), and the lowest dose that proved effective (10 μM) was used in most of the experiments to prevent potential nonspecific side effects of high doses of 5-HD (Wu et al 2006). In addition, some cultures were treated with a second K(ATP) channel blocker (glibenclamide, 10 μM, Sigma) to confirm the involvement of K (ATP) channels in AII-induced cell death.…”

Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP‐sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors

“…Doses up to 500 mmol/L have been observed to selectively inhibit mitoKATP channels without affecting surface KATP currents (Sato et al, 1998(Sato et al, , 2000Hu et al, 1999), although 5-HD is a specific blocker of the mito-KATP channels at lower doses such as 10 mM (McCullough et al, 1991;Garlid et al, 1997;Kimura et al, 2005b;Zhang et al, 2007: Costa andGarlid, 2008). In the present study, therefore, we used the lowest dose of 5-HD (10 mM), previously found to block mitoKATP channels in DAergic neurons (Wu et al, 2006;RodriguezPallares et al, 2009), to prevent any potential nonspecific side effects of high doses of 5-HD. Furthermore, the results obtained with 5-HD were confirmed with glibenclamide, which acts as a general inhibitor of KATP channels.…”

ATP-sensitive potassium channels: novel potential roles in Parkinson’s disease

“…Mitochondrial K ATP (Mito-K ATP ) channels play the roles in controlling the mitochondrial volume, regulating the translation of metabolic status of cells, and responsing open/ close channels to injury for neurodegeneration. It is hypothesized that preconditioning of K ATP channels may render neuronal tissues resistant to neurodegenerative process [15] . To test this hypothesis , Tai and Truong et al found that, at the cellular level, activation of K ATP channels in PC12 cells confered protection against mitochondrial complex-I inhibition-induced cell death by rotenone which had been proven to be the pathogenesis of PD [16] .…”