A Novel Mitochondrial K _ATP Channel Assay

Abstract

Key Words: mitochondrial ATP-sensitive potassium channel Ⅲ ischemia/reperfusion Ⅲ ischemic preconditioning Ⅲ fluoxetine T he mitochondrial ATP-sensitive potassium channel (mK ATP ) is thought to be essential for cardioprotection recruited by ischemic preconditioning (IPC), 1,2 but despite intense research the molecular identity of this channel remains unclear. The simplest thesis is that mK ATP channels are derivative of surface K ATP channels, and thus composed of inward rectifying K ϩ channels (K IR ) and sulfonylurea receptors (SURs). The cardiomyocyte surface K ATP channel is comprised of K IR 6.2 and SUR2A isoforms, 3 but efforts to conclusively assign these proteins to the cardiac mK ATP have been unsuccessful to date.Neither K IR 6 nor SUR genes contain mitochondrial target sequences, and K IR 6/SUR proteins are not found in mitochondrial proteome databases or prediction engines. 4,5 Furthermore, immune-based methods to detect K IR /SUR subunits in mitochondria are plagued by issues of antibody specificity 6 and mitochondrial purity/contamination. Several of the key pharmacological reagents used to study mK ATP channels (eg, the agonist diazoxide and antagonist 5-hydroxydecanoate) are also known to exhibit off-target effects. 7,8 Targeted gene deletion in mice to identify the mK ATP channel involved in IPC has proven futile, because of the confounding cardiovascular effects of knocking out K IR 6 and SUR genes (Kcnj8, Kcnj11, Abcc8, and Abcc9) on surface K ATP channel function. In general, K IR and SUR knockouts exhibit profound defects in glucose/insulin handling, 9 -12 which impacts the response to IPC. 13 A recent study using custom-made antibodies and SUR knockout mice identified short-form splice variants of SUR2 in mitochondria. 14 Furthermore, recent pharmacological evidence suggests that complex II of the respiratory chain (succinate dehydrogenase) may be a regulatory component of the mK ATP channel. 15,16 However, both these findings leave the identity of the K ϩ channel-forming subunit of mK ATP unknown. In this regard, mK ATP is similar to other mitochondrial ion channels which exist at a phenomenological level but have not been molecularly identified (eg, the mitochondrial Ca 2ϩ uniporter). A major obstacle in studying the mK ATP channel has been the availability of a reliable assay. Most studies to date have used an isolated mitochondrial rapid swelling assay, in which Original received October 20, 2009; resubmission received December 18, 2009; revised resubmission received January 28, 2010; accepted February 12, 2010. From K ϩ uptake into mitochondria is followed by osmotically-obliged water, leading to mild swelling that is assayed as light scattering in a spectrophotometer. 17,18 This assay has been criticized as irreproducible by some laboratories, 19 with the precise timing of mitochondrial isolation appearing to be a critical factor. 20 Studying the literature on surface K ATP channels, two key biochemical properties that appeared to have been overlooked in the mK ATP channel field...

Cardiac Slo2.1 Is Required for Volatile Anesthetic Stimulation of K+ Transport and Anesthetic Preconditioning

“…22,34 All four genotypes were studied at the isolated mitochondrial level, but due to our initial results (vide supra) indicating that Slick but not Slack is involved in APC, studies in cardiomyocytes were limited to WT and Slo2.1 −/− .…”

The Slo(w) path to identifying the mitochondrial channels responsible for ischemic protection

“…K + entry into mitochondria is accompanied by osmotically obliged water, resulting in swelling and a decreased refractive index [67], making mitochondrial volume (easily measured spectrophotometrically as the scattering of light by isolated mitochondrial suspensions) a useful surrogate measure of K + uptake [68–70]. Energetically driven swelling in K + -containing buffers was initially attributed to an inherent K + permeability of the mitochondrial membrane [71,72], and subsequent studies identified an electrically neutral KHE supporting the existence of a mitochondrial K + cycle [73] (Figure 2).…”

A Highly Selective Mitochondria‐Targeting Fluorescent K+ Sensor

“…[3] Recent research found that KCh is a potential pharmacological target in cancer, autoimmune disease, cardioprotection, and diabetes. [4] Typical research tools for KCh study include: 1) patch-clamp technique, [5] 2) fluxOR ™ assay method using Tl + ion and corresponding fluorescent probes, [6] and 3) Rb + ion method. [7] These methods are well-developed in high throughput screening of drugs with certain type of KCh, but have limitations in understanding the relationship in a multi-factor cellular singaling pathway.…”