State-dependent Inhibition of the Mitochondrial KATP Channel by Glyburide and 5-Hydroxydecanoate

Martin Jabůrek,Vladimir Yarov-Yarovoy,Petr Paucek,Keith D Garlid

doi:10.1016/s0021-9258(19)57796-1

Martin Jabůrek, Vladimir Yarov-Yarovoy + Show 2 more

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https://doi.org/10.1016/s0021-9258(19)57796-1

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Abstract

The mitochondrial KATP channel (mitoKATP) is hypothesized to be the receptor for the cardioprotective effects of K+ channel openers (KCO) and for the blocking of cardioprotection by glyburide and 5-hydroxydecanoate (5-HD). Studies on glyburide have indicated that this drug is inactive in isolated mitochondria. No studies of the effects of 5-HD on isolated mitochondria have been reported. This paper examines the effects of glyburide and 5-HD on K+ flux in isolated, respiring mitochondria. We show that mitoKATP is completely insensitive to glyburide and 5-HD under the experimental conditions in which the open state of the channel is induced by the absence of ATP and Mg2+. On the other hand, mitoKATP became highly sensitive to glyburide and 5-HD when the open state was induced by Mg2+, ATP, and a physiological opener, such as GTP, or a pharmacological opener, such as diazoxide. In these open states, glyburide (K1/2 values 1-6 microM) and 5-HD (K1/2 values 45-75 microM) inhibited specific, mitoKATP-mediated K+ flux in both heart and liver mitochondria from rat. These results are consistent with a role for mitoKATP in cardioprotection and show that different open states of mitoKATP, although catalyzing identical K+ fluxes, exhibit very different susceptibilities to channel inhibitors.

Highlights

During steady-state respiration, Kϩ influx into mitochondria is balanced by Kϩ efflux on the Kϩ/Hϩ antiporter, and steadystate volume is maintained
Glyburide is a prototypical sulfonylurea inhibitor that acts on all KATP channels and blocks the protective effects of both Kϩ channel openers (KCO) and cardiac preconditioning [20, 21]. 5-HD, which is structurally unrelated to glyburide, has been characterized as an ischemia-selective inhibitor of KATP channels [22, 23]
The hypothesis that mitoKATP is involved in cardiac protection has been clouded by the lack of a crucial piece of evidence: a convincing demonstration that glyburide and 5-HD inhibit ATPdependent Kϩ uptake in intact mitochondria

Summary

EXPERIMENTAL PROCEDURES

Preparations—Rat liver mitochondria were prepared according to Pedersen et al [10], and rat heart mitochondria were prepared by the Glass-TeflonTM homogenization procedure according to Matlib et al [11]. We normally begin measurements at 115 milliosmolal, where the outer membrane begins to break [14] This technique is important for light scattering of rat heart mitochondria, which is only weakly sensitive to volume changes in the isosmotic domain. 115 milliosmolal assay media contained either Kϩ or TEAϩ salts of chloride (45 mM), acetate (25 mM), EGTA (0.1 mM), and TES (5 mM), pH 7.4. Assay of Kϩ Flux in Liposomes—MitoKATP was partially purified from rat liver mitochondria and reconstituted into proteoliposomes loaded with PBFI according to previously published protocols [15]. The Tris salt of ATP was titrated to pH 7.2 with Tris base

RESULTS

TABLE I

Experimental preparation

DISCUSSION