The C. elegans mitochondrial K⁺_ATP channel: a potential target for preconditioning

Introduction

Mitochondria are essential for cellular function and are a sensitive target for damage induced by ischemia reperfusion (IR) injury, such as the overproduction of reactive oxygen species (ROS) and overload of Ca²⁺, both of which can trigger the formation of the mitochondrial permeability transition (PT) pore at reperfusion [1–3]. Mitochondrial dysfunction has significant implications for post-ischemic recovery [4; 5]. Despite being the site of such pathological events, mitochondria are also involved in the protective mechanism of ischemic preconditioning (IPC) whereby short non-lethal periods of ischemia protect the heart from subsequent prolonged ischemic insults [6]. IPC triggers an array of cell signaling pathways, many of which converge at the mitochondrion. Recent work has suggested that a primary target of IPC signaling is the mitochondrial ATP sensitive K⁺ channel (mK_ATP) [7; 8], and that this channel is essential for the mechanism of cardioprotection recruited by IPC.

The mK_ATP was first described electro-physiologically [9], then subsequently via its pharmacological sensitivity, both of which demonstrate similarities to plasma membrane and sarcoplasmic reticulum ATP sensitive K⁺ channels or “surface” K_ATP channels. For example, the sulfonylurea diazoxide’s cardioprotective activity was originally thought to be due to triggering of surface K_ATP channels, but its protective effects were later shown to relate specifically to mK_ATP channel opening [10]. Moreover, the protective effects of IPC can be mimicked by mK_ATP channel activators [11–13] and prevented by channel inhibitors [14; 15]. The protection mediated through the opening of the mK_ATP is thought to involve membrane depolarization through K⁺ influx [16], which may translate to decreased Ca²⁺ uptake and ROS generation [17]. Additionally, K⁺ entry obligates the influx of anions and water, thereby augmenting matrix volume [18] and potentially improving energy metabolism and interfering with PT pore assembly [16; 19]. Despite the central role of the mK_ATP in IPC and the well-documented molecular characterization of the surface K_ATP channels, none of the sequences or splice variants of the inward rectifying K⁺ channel (Kir) or sulfonylurea receptor (SUR) genes that comprise surface K_ATP channels [20] contain mitochondrial targeting sequences, and it is unclear whether the mK_ATP channel is derived from surface K_ATP channels [21–23] or is a unique protein entity [24].

Efforts centered on mammalian model systems have yielded no definitive answer regarding the molecular identity of the mK_ATP channels. One example of the problems faced by this field is the recent discovery that many of the antibodies used to identify SUR and Kir subunits in purified mitochondria can recognize off-target proteins unrelated to K⁺ channel function [25]. Furthermore, knockout mice for Kir and SUR genes have been generated and are viable, but the ability to test these mice in IPC experiments is confounded by the obligatory effects of knocking out surface K_ATP channel function on various cardiovascular functions. A key example is the SUR2^−/− mouse which exhibits elevated blood glucose, a known risk factor for increased IR injury [26]. These limitations may be further compounded by potential nonspecific effects of pharmacological agents used in assaying the mK_ATP channel [27–29].

One potential solution to these limitations is to employ a model organism. In this regard, IPC in the nematode worm C. elegans results in protection from ischemic death similar to “late” preconditioning seen in mammals [30; 31]. To begin testing the hypothesis that IPC in worms occurs through an evolutionarily conserved mechanism involving mK_ATP, we assayed mK_ATP activity in purified mitochondria using a K⁺ swelling assay [32–35]. Our results demonstrate mK_ATP channel activity that both pharmacologically and physiologically mimics its mammalian counterpart. The presence of the channel in this model organism provides several advantages in determining its molecular identity. The reduced cellular complexity of worms and absence of vasculature may reduce confounding systemic effects, while the use of genetics to eliminate candidate channel expression should eliminate off-target pharmacologic effects. Future determination of the identity of the mK_ATP channel in worms will represent a significant advance in the field of IR injury and protection by IPC.

Materials and Methods

All chemicals and reagents were from Sigma (St Louis MO) unless otherwise stated. Culture and growth of C. elegans N2 strain was performed essentially as described [36]. 2 × 10⁵ embryos or starved L1 worms were plated onto 2 × 150 mm plates, each seeded with 6 ml of HB101 bacteria (~50% slurry), condensed from a 500 ml culture of Terrific Broth. Plates were incubated in a humidified atmosphere at 20°C for 3–4 days, resulting in staged adult worms ready for mitochondrial isolation.

Mitochondria were isolated from adult C. elegans by a method based on that described in [37]. Worms from 2 plates were transferred at 25°C into 50 ml of M9 media (22 mM KH₂PO₄, 42 mM Na₂HPO₄, 86 mM NaCl, 1 mM MgSO₄, pH 7) in a conical tube, then incubated for 20 min. on ice resulting in precipitation / settling. Supernatant (45 ml) was removed, and the worm pellet rinsed twice with 45 ml ice-cold M9 and once with 45 ml ice-cold mitochondrial isolation medium (IM: 220 mM mannitol, 70 mM sucrose, 5 mM MOPS, 2 mM EGTA, pH 7.4, filtered) containing 0.4% BSA. Worms were settled by gravity for 10 min. between rinses, and finally by centrifugation for 1 min. at 1000 × g, and suspension of the final pellet in ~1.5 ml of IM. Worms were transferred using a cut-off pipet tip to a mortar on-ice containing 2g of washed sand per ml of worms, followed by grinding with a pestle for 1 min. Ground worms were extracted from the sand using 3 × 3 ml aliquots of IM, transferring to a 10 ml conical tube. Following 3 additional settling/wash steps in IM+BSA to remove excess sand, the worm suspension was then homogenized with a glass Dounce homogenizer in IM+BSA. The homogenate was diluted and centrifuged at 600 × g for 5 min. The supernatant was then filtered through 300 µm mesh and centrifuged at 7000 × g for 10 min. The pellet was resuspended in 1.5 ml IM, and centrifuged at 7000 × g for 5 min. Another wash/centrifugation step was performed, and the final mitochondrial pellet suspended in 150 µl IM and stored on ice until use (within 2 hrs). Protein was determined by the Folin-Phenol method [38], against a standard curve constructed with BSA. Two plates of worms typically yielded ~6 mg mitochondrial protein.

Immediately following isolation, mitochondrial viability was determined by the measuring respiratory function (O₂ consumption) using a Clark-type O₂ electrode in a stirred chamber at 25°C. Mitochondria (0.25 mg protein) were suspended in 0.25 ml respiration buffer (RB: 195 mM mannitol, 25 mM sucrose, 40 mM HEPES, 0.25% w/v fat-free BSA, pH 7.35 at 25°C). Addition of substrates (10 mM pyruvate plus 5 mM malate, or 5mM succinate) was used to initiate state 2, followed by the addition of ADP (200 µM) to initiate state 3 respiration. Oligomycin (1 µg/ml) was then added to enforce state 4 respiration.

mK_ATP channel activity was measured by the commonly used swelling/light-scatter assay [32–35] at 520 nm using a Beckman DU800 spectrophotometer. Mitochondria (0.25 mg/ml) were rapidly added to a stirred cuvette containing mK_ATP buffer (100 mM KCl, 10 mM HEPES, 2 mM succinate, 2 mM MgCl₂, 2 mM KH₂PO₄, 1 µg/ml oligomycin, pH 7.2 at 37°C). In some experiments, as indicated by figure legends, K⁺ in the buffer was replaced with Na⁺. All differences between experimental groups were determined using ANOVA; p<0.05 was considered significant.

Results

We and others have previously noted that the mK_ATP channel activity degrades rapidly in mammalian mitochondria [32; 33], thus contraindicating the use of proteases to break the C. elegans cuticle. Instead, a 2-step homogenization protocol was developed in which whole worms were first ground with sand in a pestle and mortar, followed by several passes in a glass Dounce homogenizer. This was followed by several centrifugation steps, resulting in a mitochondrial fraction. The respiratory properties of these isolated mitochondria are shown in Fig. 1., indicating that the mitochondria were moderately well coupled. Respiratory control ratio (RCR) values were not as high as those typically seen in mammalian mitochondria, however worms are poikilotherms and prolonged incubation at 37°C is lethal. Correspondingly, a lower incubation temperature was used in the respiration reactions (25°C vs. 37°C for mammalian mitochondria [33]), which slows down state 3 respiration considerably, and this may partly account for the lower RCR.

The rapid osmotic swelling of mitochondria in K⁺ based media, which results in a decrease in light scattering that is assayed spectrophotometrically, has been widely used to assay mK_ATP channel function [32–35]. As shown in Fig. 2a, we were able to recapitulate mK_ATP mediated osmotic swelling of C. elegans mitochondria, and this activity persisted for up to 3 hrs. following mitochondrial isolation. In Fig. 2b we demonstrated that the channel exhibits the classical mK_ATP pharmacologic profile as previously reported in the literature and in this laboratory [10; 15; 17; 33; 39], i.e. swelling under baseline conditions in K⁺ based media (open channel), inhibition by ATP, re-activation by diazoxide superseding the effect of ATP, and inhibition again by 5-HD over-riding the effect of diazoxide plus ATP. No effect of these agents on swelling was observed in Na⁺ based media [33], and mitochondria isolated from adult worms staged either as embryos or as starved L1 larvae behaved similarly (data not shown).

Fig. 2C shows that the C. elegans mK_ATP channel also responds to a variety of other reagents in exactly the same manner as previously observed for the mammalian mitochondrial channel [10; 15; 17; 33; 39]. The mK_ATP channel openers cromakalim, pinacidil and malonate all reversed the inhibition of swelling by ATP, and the mK_ATP channel antagonists 5-HD and glyburide both reversed the effects of cromakalim, pinacidil and malonate.

Discussion

Our results demonstrate that viable mitochondria isolated from C. elegans contain a mK_ATP channel identical to the mammalian mK_ATP channel in every aspect so far examined. This fundamental observation is the first report on the existence of a mK_ATP channel in multicellular invertebrates, suggesting that channel activity is evolutionarily conserved. Given the ability of C. elegans to undergo IPC [30], we further suggest that the mK_ATP channel is likely a fundamental component of all evolutionarily conserved preconditioning signal pathways.

Another advantage to the C. elegans model system in regard to mK_ATP channel function, is the discovery herein that the channel activity was stable in isolated mitochondria for up to 3 hours. The mK_ATP channel swelling assay has been criticized recently due to its poor reproducibility by some laboratories [40], and as discussed above a key factor governing the ability to assay the channel by this method is the freshness of isolated mitochondria [32; 33]. The issue of channel stability may account for several related discrepancies in the mK_ATP channel literature, and thus the greater stability of the C. elegans mK_ATP channel activity may permit greater consensus in the future studies on this channel.

Numerous studies have centered on unraveling the molecular mechanisms of IPC over the past two decades. Despite significant advances, the molecular identity of mK_ATP is unknown, and this impairs progress in developing therapeutics to protect the heart and other organs from IR injury, a major cause of morbidity and mortality worldwide[41]. In addition it is known that IPC is less effective in certain patient cohorts (e.g. diabetics) [42], but the mechanisms underlying this are unclear. It is possible that differences in mitochondrial K⁺ channel activity may underlie such effects, and thus identification of these channels will permit enhanced diagnosis and treatment of individuals refractory to IPC.

Despite surface K_ATP channels being well characterized at the molecular level (i.e. with well defined genes and protein structures, see [20] for review), none of the known Kir or SUR genes or their splice variants have mitochondrial targeting sequences. In addition, several antibodies used to identify SUR and Kir subunits in purified mitochondria are now known to recognize off-target unrelated proteins [25]. Thus, a competing hypothesis has emerged, that the mK_ATP channel may not be comprised of classical Kir/SUR proteins [21–23], and instead is comprised of other mitochondrial proteins [24]. Candidates for mK_ATP that have been proposed include: succinate dehydrogenase (complex II), the phosphate carrier, mitochondrial ATP-binding cassette protein-1 (mABC-1), adenine nucleotide translocator, and ATP synthase [24]. The conservation of function and direct genetic orthologies between mammalian and worm counterparts makes C. elegans an attractive model to test whether these molecules are components of or perhaps regulate mK_ATP. Our demonstration that worms possess an mK_ATP channel activity is fundamental to this process, and the future analysis of candidate mutants should allow unambiguous insight into the identity of the mK_ATP channel.

Furthermore, it has recently been discovered that, analogous to mammals, C. elegans can also be protected from ischemic / hypoxic injury by preconditioning with volatile anesthetics (so-called “anesthetic preconditioning”, APC) [31]. In addition, emerging work suggests that Ca²⁺ activated K⁺ channels in the mitochondrion (mK_Ca) may participate in APC signaling, possibly playing a similar mechanistic role to mK_ATP channels. Thus, it is possible that the application of mitochondrial isolation methods and channel activity assays outlined herein, could also be applied to the molecular identification of the mK_Ca channel and its regulators.