MgATPase Activity is Dispensable for the Pharmacological Regulation of the Functional Effects of the KATP Channels Opening in Brain Mitochondria.

Abstract

Background: The mechanisms underlying the effects of pharmacological mitochondrial ATP-sensitive K⁺ channel (mKATP) channel openers on the functional effects of the mKATP channels opening remain disputable. Earlier we have shown that the mKATP channel activation by diazoxide (DZ) occurred at submicromolar concentrations and did not require a MgATP in liver mitochondria. This work aimed to evaluate a requirement of a MgATP for the mKATP channel opening by DZ and its blocking by glibenclamide (Glb) and 5-hydroxy decanoate (5-HD) in rat brain mitochondria and to find the effects of the mKATP channels opening on mitochondrial Ca²⁺ uptake, reactive oxygen species (ROS) production, and the mitochondrial permeability transition pore (mPTP).

Methods: The mKATP and the mPTP channels activity was assessed by the light scattering; polarography was applied to quantify K⁺ transport; Ca²⁺ transport and ROS production were monitored with fluorescent probes, chlortetracycline, and dichlorofluorescein, respectively; one-way ANOVA was used for reliability testing.

Results: ATP-sensitive K⁺ transport in native mitochondria was fully activated by DZ at <0.5 μM and blocked by Glb and 5-HD in the absence of a MgATP, however, Mg²⁺ was indispensable for the blockage of the mKATP channel by ATP. DZ increased Ca²⁺ uptake, but ROS production was regulated differently: suppressed in mitochondria respiring on glutamate, but activated on succinate. However, in the presence of rotenone, ROS production was suppressed by DZ, which indicated the involvement of reverse electron transport (RET) in the modulation of ROS production. In all cases, the mKATP channel blockers reversed the effects of DZ. The impact of DZ on the mPTP opening strongly correlated with its effects on ROS production. DZ inhibited the mPTP activity on glutamate but elevated on succinate, which was strongly suppressed by rotenone. In the presence of rotenone, the mPTP was strongly inhibited by DZ, which indicated the involvement of ROS and RET in the mechanism of mPTP regulation by DZ.

Conclusions: Brain mKATP channel exhibited high sensitivity to DZ on the low sub-micromolar scale; its regulation by DZ and Glb did not require a MgATPase activity; the impact of DZ on the mPTP activity was critically dependent on the regulation of ROS production by ATP-sensitive K⁺ transport.