**4. Discussion**

The presence of BKCa in mitochondria has been extensively pursued in the mitochondrial channel field in recent years [8,50]. Hence, we investigated if *Drosophila* mitochondria contain BKCa currents. Our electrophysiological studies provide clear evidence for the presence of BKCa channels in the mitochondria of *Drosophila*. The currents measured are of typical BKCa characteristics and they could be blocked by paxilline, and activated by calcium. Surprisingly, Po decreased at higher voltages which needs further characterization as this phenomenon could result from the presence or absence of additional regulatory subunit. Our immunolabelling of mitochondria also confirmed localization of BKCa to the mitochondria. These experiments indicate that the large current in *Drosophila* mitoplast is highly sensitive to changes in Ca2+ concentrations in the mitochondrial matrix, and could be blocked by highly specific BKCa inhibitor, paxilline. The large-conductance and sensitivity to NS1619, paxilline as well as Ca2+ and voltage in addition to mitochondrial immunocytochemistry indicate that *Drosophila* mitoplast possess functional BKCa proteins. These experiments for the first time establish BKCa as a mitochondrial ion channel across the species confirming an evolutionary presence. However, BKCa is located in several other membranes, for example in plasma membranes in neurons and astrocytes [7,8], along with mitochondrial membranes. It is ye<sup>t</sup> to be deciphered how mitochondrial function is controlled by BKCa with respect to its various locations.

The absence of BKCa has several consequences on mitochondria. A dramatic increase in the accumulation of ROS was observed in *slo*<sup>1</sup> mutant flies. Increased ROS can be detected in live tissues of mutant flies and also a higher amount of ROS generation was observed in isolated mitochondria. Energized *slo*<sup>1</sup> mutant mitochondria produce increased levels of ROS compared to wt mitochondria when provided with specific substrates. This indicates that the increased ROS is a consequence of dysfunctional mitochondria, although it does not rule out the contribution from NOX related enzymes that are capable of producing ROS. However, other mitochondrial readouts such as ATP and oxidative phosphorylation measurements further consolidate the hypothesis that the increased ROS observed in *slo*<sup>1</sup> mutant animals is due to mitochondria. The increase in ATP could also be due to sustained membrane potential caused by reduced potassium leak, increasing proton flux from ATP synthase. These results indicate hyper-functional mitochondria, which explain the higher level of ROS production from the mitochondria. We have recently shown that genetic activation of BKCa channels reduces ROS upon IR injury stress [13], which further supports a role for BKCa in regulating ROS.

The BKCa flies also display oxidative stress sensitivity in a ROS-based pathway [51]. When we subjected the flies to oxidative stress using PQ, the BKCa mutant flies died within 48 h compared to wt flies, which survive for more than 3 days. This indicated that BKCa flies are under high oxidative stress and any further increase in ROS could be detrimental. The converse experiments by feeding glutathione increased the survivability of *slo*<sup>1</sup> mutants indicating that ROS is at least a factor that determines the survival of *slo*<sup>1</sup> mutants.

Consistent with the functional abnormalities of mitochondria, *slo*<sup>1</sup> mutants also show several structural defects in mitochondria. Although younger flies contain mitochondria of normal appearance, occasional vacuoles and mitochondrial swirls are observed in flies even of day 1 age. As the flies age, mitochondrial structural defects are further enhanced where cristae structure is lost and mitochondrial swelling occurs. This depicts a progressive disintegration of mitochondria in an accelerated manner perhaps one of the causes leading to the early death of flies. Given that mitochondria from *slo*<sup>1</sup> mutants produced higher amounts of ATP, it is possible that the absence of BKCa results in changes in cristae as observed here which results in assembly of respiratory chain supercomplexes (RCS). RCS are quaternary supramolecular structures that allow channeling of electrons amongs<sup>t</sup> individual respiratory chain complexes facilitate selective use of RCC subsets for nicotine adenine dinucleotide (NADH)- or flavin adenine dinucleotide (FAD)-derived electrons [52]. These type of supramolecular organization is commonly found in cristae, and the mitochondrial ATP synthase is also assembled as dimers with increased ATPase activity and the dimerization is further augmented during autophagy [53]. Mitochondria are closely associated with lifespan and mitochondrial defects accumulate as the animal ages. Interestingly in *slo*<sup>1</sup> mutants, mitochondrial abnormalities can be seen on day 1 or birth. Mitochondrial swirls are occasionally seen in *slo*<sup>1</sup> mutants, a phenotype hallmark of very old/dying flies in wt situations.

Ion channels are reported to alter with age in rats and humans [17,54]. Expression of BKCa channels was shown to be reduced in aged coronary arteries possibly resulting in decreased vasodilator capacity, increased the risk of coronary spasm and myocardial ischemia in older people [17,54,55]. In mice, the absence of BKCa causes low body weight and decreased survivability in the first 10 weeks [10,56] but a complete life span analysis has not been reported. In contrast, a recent report indicated a moderate increase in life span and motor neuron activity in *C. elegans* BKCa mutants [11]. However, broad augmentation of endogenous BK currents in vivo (gain-of-function BKCa TG mice) resulted in protecting the heart from ischemia-reperfusion injury [13]. In our current study, we have discovered that *Drosophila* lacking BKCa showed a decrease in lifespan supporting mammalian observations. Flies mutant for BKCa not only die rapidly but show early and premature accumulation of aging markers. This indicates that the presence of BKCa is important in the regulation of aging. The key reason for this di fference between *C. elegans*, flies, and mammals could be attributed to the role of electron transport chain (ETC) and ROS in aging. In *C. elegans* any perturbation with ETC results in an increase in life span due to their anaerobic energy-producing capacity, which is the exact opposite to what is observed in mammals and *Drosophila* [57]. One of the best examples for this di fference is in frataxin homolog gene (frh-1), where knocking down frh-1 significantly increased the life span of *C. elegans* [58], but its ablation in mouse decreased life span [59], and recessive mutations in frataxin cause Friedreich's ataxia [60] in humans.

In agreemen<sup>t</sup> with accelerated aging, we observed the accumulation of age-related phenotypes just after the birth of flies such as intestinal perforation and polyubiquitin aggregation accompanied by motor defects in *slo*<sup>1</sup> mutant flies. This provides evidence that BKCa channel function is required from an early age, perhaps from developmental stages, for the animal to age in a wild type manner and it regulates life span. Our microarray data intriguing shows increased expression of several methuselah genes whose mutants are known to extend life span [43]. While it is not clearly shown if an increase in methuselah expression reduces the life span, it is consistent with the proposed role of methuselah where lack of it increases life span. We also observed several life span related genes altered in the *slo* mutants along with oxidative stress genes in our microarray [45–48]. These results collectively show that slo is a major regulator of oxidative stress and life span and a detailed study is required in the future to narrow down the direct role of BKCa in regulating life span. The major limitation is the contribution of mitochondrial vs. non-mitochondrial BKCa in regulation the lifespan of *Drosophila*.

Supporting our observation of ion channels regulating lifespan, it was recently shown that low temperatures activate a cold-sensitive cation channel TRPA-1, which extends a lifespan by triggering cellular signaling pathways [61]. It is also interesting that lack of BKCa only from the muscles also causes reduced lifespan similar to what is reported in earlier studies [62]. Conversely increasing BKCa by Gal4-UAS based overexpression increased the life span indicates a true role for BKCa in regulating life span. Human BKCa is 70% identical to *Drosophila* BKCa but is su fficient to rescues as well as augmen<sup>t</sup> the life span of *Drosophila* indicating the function could be conserved across species. This result is of relevance given expression of BKCa goes down with age in humans [63]. However, it is intriguing that only males show this e ffect while females do not show life span extension upon overexpression. These results are in agreemen<sup>t</sup> with increase in a life span of male flies on overexpression of specific DNA repair endonucleases [64]. DNA repair mechanisms are ATP-dependent processes, and dysfunctional mitochondria over a longer period of time could trigger apoptosis and cell death. This indicates gender-based di fferences in how BKCa regulates life span or could be involved in DNA repair mechanisms, which needs detailed study.

Taken together, our study establishes BKCa/Slo as an important player in maintaining the structure and functional integrity of mitochondria in *Drosophila*, and regulating lifespan. These findings also corroborate earlier studies that expression of BKCa reduces during aging which increases the risk of cardiovascular diseases in older people [17]. Our study also proves the existence of ion channel activity for BKCa in the *Drosophila* mitochondria. Given the dual cellular localization (intracellular membranes vs. plasma membrane) of BKCa, it is critical to evaluate its spatial specific role(s) in pathophysiology in future studies. In our findings, we have not ruled out the role of plasma membrane BKCa but introduced its new physiological role in aging. Presence of BKCa in the mitochondria and its role in modulation of ROS opens up avenues to explore antioxidant-based therapies in diseases and disorders related to these large conductance potassium channels. In the past decade, studies have indicated that pharmacological and genetic activation of BKCa results in cellular and organ protection from ischemic injuries. Despite recent successes with animal models, the translational aspect of BKCa channel openers is still lacking due to poor selectivity of these agonists. With recent advancements in gene delivery and gene therapy, our recent and current work reiterates the importance of expression of BKCa to protect organs from ischemic insult or increasing life span.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/9/945/s1, Figure S1: *slof05915* mitochondria produce increased ROS. Figure S2: *slo*<sup>1</sup> mutants result in accelerated aging when males and females are grown together. Figure S3: Increased levels of protein aggregates in *slo*<sup>1</sup> mutants. Figure S4: Heat map of oxidative stress-related genes altered in *slo*<sup>1</sup> mutants in comparison to wt flies. Figure S5: Females overexpressing human (Hs) BKCa do not show a change in life span as compared to control flies.

**Author Contributions:** Conceptualization, S.G.R. and H.S.; Methodology, S.G.R., P.B., A.T., K.S., P.K., D.P., H.N.J., S.A., B.A.S.R., and H.S.; Software, H.S.; Validation, S.G.R., E.J.V.B., A.S., D.C.W. and H.S.; Formal Analysis, S.G., P.B., A.S., S.A., and H.S.; Investigation, S.G.R., P.B., A.T., and H.S.; Resources, E.J.V.B., A.S., D.C.W., and H.S.; Data Curation, S.G.R. and H.S.; Writing—Original Draft Preparation, S.G.R.; Writing – Review & Editing, S.G.R., A.S., D.C.W., and H.S.; Visualization, S.G.R. and H.S.; Supervision, H.S.; Project Administration, H.S.; Funding Acquisition, S.G.R., D.C.W., and H.S.

**Funding:** This research was funded by the Commonwealth Universal Research Enhancement (CURE) Program Grants to S.G.R. and H.S., American Heart Association Postdoctoral Fellowship (17POST33670360) to D.P., National Institute of Health (NS021328, CA182384, and MH108592) and Department of Defense (OD10944) to D.C.W., and a gran<sup>t</sup> from the W. W. Smith Charitable Trust, American Heart Association National Scientist Development Grant (11SDG230059), American Heart Association Grant-in-Aid (16GRNT29430000), National Institute of Health (HL133050), and Drexel University College of Medicine startup funds to H.S. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537).

**Acknowledgments:** We thank Nigel Atkinson (University of Texas, Austin) for *slo* mutant flies, David W. Walker (University of California, Los Angeles) for UAS Sod2 flies, and Irwin Levitan (Thomas Je fferson University, Philadelphia) for anti-Slo antibodies.

**Conflicts of Interest:** The authors declare no conflict of interest.
