**3. Results**

#### *3.1. Presence of BKCa Currents in the* Drosophila *Mitochondria*

In addition to the plasma membrane, BKCa channels are known to be present in the mitochondria of rodent neurons [24] and endothelial cells [19]. In adult cardiomyocytes, they are exclusively present in the mitochondria [12,25] but not in the plasma membrane [12,26]. In *Drosophila*, BKCa has been well-characterized in the plasma membrane at the biophysical and physiological levels [7,27], however, it is not known whether it is present or active in the mitochondria. In order to test for the presence of BKCa in mitochondria, we loaded isolated mitochondria with mitotracker [18] and labeled with anti-BKCa antibodies (Figure 1A,B,D,E). Mitochondria isolated from the whole wild-type but not *slo*<sup>1</sup> mutant flies [1,3] showed the presence of a BKCa-specific signal (Figure 1A–F). Protein proximity index (PPI) analysis [12] to estimate colocalization of BKCa to mitotracker-loaded mitochondria showed a value of ~0.5 ± 0.1 (n = 6), indicating ~50% of BKCa signal colocalized with mitochondria.

BKCa has been recorded from cardiac and endothelial mitoplasts [8,19,25], but not in *Drosophila* mitoplast (inner membrane of mitochondria). To examine whether BKCa is active in *Drosophila* mitoplast (n = 5 independent experiments, mitochondria isolated from 100 flies each), we isolated mitoplast from wild type flies and carried out patch-clamp analysis [19]. Approximately 80% of the currents detected in the mitoplasts were attributed to BKCa-specific channels. We recorded channel activity (Figure 1G) in the presence of 100 μM Ca2+ in the bath pipette at holding potentials ranging from +60 mV to −60 mV in a symmetrical solution (150 mM KCl, 10 mM HEPES, 100 μM Ca2+, pH 7.2). The current (I) vs. voltage (V) curve (Figure 1G) calculated from the single-channel currents showed a conductance of 382 ± 8 pS (n = 5) for mitoBKCa. Surprisingly, the open probability of single-channel current increases from ~0.6 at +60 mV to ~1.0 at −60 mV holding potentials (Figure 1G). On addition of paxilline (BKCa antagonist), the large channel conductance was completely blocked (Figure 1H), confirming that the large currents were originated from paxilline-sensitive BKCa.

Since BKCa is a Ca2<sup>+</sup>-sensitive channel, we also changed the Ca2+ concentration of bath solution from 100 μM to 1 μM. Single-channel recordings showed a decrease in open probability (Po) at holding potentials ranging from −40 mV to 40 mV (Figure 1I). Po vs. V plot shows increase in Po at 100 μM as compared to 1 μM Ca2+ (Figure 1J). Large conductance channels were not observed at 1 μM Ca2+. However, on the addition of 1 μM NS1619 (BKCa agonist), the large-conductance channel reappeared with a high Po (Figure 1K). Our immuno-organelle chemistry data indicate the presence of BKCa channels in isolated mitochondria. In addition, our electrophysiological approach demonstrates the presence of BKCa in *Drosophila* mitochondria corroborating the immuno-organelle chemistry data.

#### *3.2. Mitochondrial Functional Aberrations in BKCa Mutants*

Given the presence of BKCa in the mitochondria, we sought to investigate if BKCa plays a direct role in its functional integrity using the BKCa (*slo*1) mutant [1,28].

We tested if ROS, the major byproduct of mitochondria, is altered in *slo*<sup>1</sup> mutants. The *slo*<sup>1</sup> mutant showed higher levels of DHE staining (a detector of ROS) in indirect flight muscles indicating significantly (*p* < 0.05, n = 5) increased production and accumulation of ROS (Figure 2A vs. 2B, quantified in 2C). We examined ETC function where ROS is generated and found that *slo*<sup>1</sup> mutant mitochondria showed a significant increase in ROS production (Figure 2D–G). The increase was significant when pyruvate was used as a substrate (Figure 2D,G, *p* < 0.01). To dissect which complex is generating this ROS, we used specific substrates for complex I and complex II of ETC. With glutamate/malate (substrate for complex I), we did not see any significant difference in ROS generation (Figure 2E,G). However, succinate (substrate for complex II) showed a much higher level of ROS generation (Figure 2F,G) in *slo*<sup>1</sup> mutants, indicating that increased ROS produced could be due to complex III and or backflow of electrons to complex I [29]. Another mutant for dSlo, *slo[f05915]* [30] also showed the elevated rate as well as the amount of ROS production by complex III (Supplementary Figure S1).

**Figure 2.** Mitochondrial functional defects in *slo*<sup>1</sup> mutants. (**A**) (wt) and (**B**) (*slo*1) show indirect flight muscles stained with DHE to detect ROS. (**C**) Quantification of ROS fluorescence in (**A**) and (**B**) (wt-black, *slo*1-grey). The graphs in (**D**–**F**) show ROS generation in isolated wt (black) and *slo*<sup>1</sup> mutant mitochondria (gray) in the presence of pyruvate (**D**), glutamate/malate (**E**), or succinate (**F**) as substrates. Succinate and pyruvate, but not glutamate/malate show an increase in ROS as detected by the amplex red dye, compared to wt mitochondria. (**G**) Quantification of (**D**,**E**), and (**F**,**H**) shows ATP levels increased in *slo*<sup>1</sup> mutants (wt-black, *slo*1-grey). (**I**) Quantification of oxygen flux from enriched mitochondria from 40 thoraces of wt (black) and *slo*<sup>1</sup> mutants (gray) flies. Basal, complex I, complex II, and ROX (non-mitochondrial residual oxygen consumption rate) oxygen consumption rates do not significantly vary between wt and *slo*<sup>1</sup> mutants but combined complex I and complex II and maximum ETC consumption are significantly higher in *slo*<sup>1</sup> mutants.

During oxidative phosphorylation, the energy released from oxidation/reduction reactions drives the synthesis of ATP. Mitochondrial disintegration is often associated with a decrease in ATP-generation [31]. We tested ATP-generation by mitochondria from two-week-old wt and *slo*<sup>1</sup> mutant flies. Surprisingly, *slo*<sup>1</sup> mutant flies showed a significant increase (*p* < 0.001, n = 5) in ATP-generation compared to wt flies (Figure 2H). We also measured the activity of complexes from both wt and *slo*<sup>1</sup> mutant flies by measuring substrate driven oxygen consumption rates. In comparison to wt, *slo*<sup>1</sup> mutant flies had similar basal rates and higher but not significant complex I and complex II oxygen consumption rates (Figure 2I). However, upon substrate saturation of both complex I and II combined, the oxygen consumption rate was highly significant (*p* < 0.05, n = 5, Figure 2I). The maximum electron transport system (ETS) capacity was also increased in *slo*<sup>1</sup> mutants suggesting a higher index of mitochondrial uncoupling in these mutant mitochondria (Figure 2I). We did not observe a sex-based difference between in *slo*<sup>1</sup> mutants.

#### *3.3. Absence of BKCa Renders Flies Susceptible to Oxidative Stress*

Our findings indicate abnormally hyper-functional mitochondria, which explain the higher level of ROS production from the mitochondria. To analyze if increased ROS renders *slo*<sup>1</sup> mutant flies sensitive to oxidative stress, we fed them with paraquat (PQ), a compound known to induce oxidative stress [32]. We found that *slo*<sup>1</sup> mutants (Figure 3A) are highly sensitive to PQ feeding. Flies (2–3 days old) maintained on starvation media for 2 h followed by exposure to 5% (*w*/*v*) sucrose combined with 20 mM PQ showed 50% death of *slo*<sup>1</sup> flies within 15 h whereas the 50% wt survived up to 25 h (Figure 3A,B). Hypersensitivity of *slo*<sup>1</sup> mutants to PQ was highly intriguing indicating that ROS plays a detrimental role on the survivability of *slo*<sup>1</sup> mutants. We tested hypersensitivity to ROS by feeding the flies with reduced glutathione (GSH) to see if glutathione feeding helps them survive in oxidative stress. We observed improved survival of *slo*<sup>1</sup> mutants similar to wild type in PQ treatment upon feeding of GSH (Figure 3C,D). These results show that increased ROS in *slo*<sup>1</sup> mutants is responsible for oxidative damage and perhaps influences the survival of flies.

**Figure 3.** Oxidative stress on fly survival. (**A**) Survival of *slo*<sup>1</sup> mutants is significantly lower compared to wt flies fed on 20 mM PQ in 5% (*w*/*v*) sucrose. (**B**) Histogram shows 50% survival for and *slo*<sup>1</sup> and wt flies. (**C**) Survival of *slo*<sup>1</sup> mutants while PQ feeding with or without glutathione. (**D**) Histogram shows 50% survival for and *slo*<sup>1</sup> mutants with or without reduced glutathione (GSH). GSH increased the survival of wild-type and *slo*<sup>1</sup> mutants.

#### *3.4. Mitochondrial Structural Abnormalities in BKCa Mutants*

In order to study the structure of mitochondria in *slo*<sup>1</sup> mutant flies, we analyzed the ultrastructure of mitochondria in wt and *slo*<sup>1</sup> flies (Figure 4, n = 5). Electron microscopic analysis revealed major differences in the ultrastructure of mitochondria (Figure 4B vs. E). We studied day 1 and day 30 time points based on the differences observed in our initial experiments. The number of mitochondria in *slo*<sup>1</sup> mutant flies was less compared to wt from older flies (day 30). The mitochondria of older *slo*<sup>1</sup> mutants showed severe defects in terms of cristae arrangemen<sup>t</sup> (Figure 4E). The size of mitochondria in *slo*<sup>1</sup> mutant older flies was also increased as compared to the young flies, which could be attributed to their swollen appearance and loss of continuous inner mitochondrial membrane (Figure 4D,E,G). No major differences were observed between young (day 1) vs. older wt flies (day 30, Figure 4A,B,G).

**Figure 4.** Mitochondrial structural defects in *slo*<sup>1</sup> mutants. (**A**) Wt mitochondria from indirect flight muscles at age 1 day show normal cristae organization. The *slo<sup>1</sup>* mutant mitochondria also show normal structure but there are increased numbers of vacuoles in the muscles (**D**). (**B**) Wt mitochondria at the age of day 30 also show a normal cristae organization. However, the *slo<sup>1</sup>* mutant mitochondria show disorganized cristae and swollen mitochondria (**E**). (**C**) Mitochondria of very old flies (at day 60) in wt show swirling of cristae, a phenotype characteristic of old age but occasionally young (day 1) *slo<sup>1</sup>* flies also show such swirls (**F**), indicated by white arrows. (**G**) The Average area indicated by histogram showed no difference in day 1 mitochondria in between wt (black) and *slo<sup>1</sup>* (gray) but significant (\* *p* < 0.05) difference at day 30.

Mitochondrial swirls are known to represent early events of deterioration. Unusually close packing of cristae in an onion peel arrangemen<sup>t</sup> in the flight muscle mitochondria makes it feasible to detect it by electron micrograph [33]. We observed sporadic mitochondrial swirls in very old wt flies (≥60 days, Figure 4C) but *slo*<sup>1</sup> mutant showed mitochondrial swirls from day 1 in the flight muscle (Figure 4F, one to two occurrences per field). We have also observed the appearance of vacuoles in young *slo*<sup>1</sup> mutant flies (Figure 4D) whereas they were not seen in the wt counterparts. Taken together these analyses indicate major disorganization in mitochondrial structure in *slo*<sup>1</sup> mutant flies, some of them being hallmarks of the early aging phenotype. Mitochondrial structural disintegration, as well as the appearance of swirls, indicated possible oxidative damage to mitochondria consistent with our earlier results. Age-related abnormalities in mitochondria from flight muscles and other tissues of *Drosophila* are well-documented [34,35]. Older flies show severe mitochondrial deterioration; including loss of cristae, increase in size (swelling) and loss of arrangemen<sup>t</sup> in muscle fibers [33]. This prompted us to investigate if there are differences in the lifespan of *slo*<sup>1</sup> mutant flies.

#### *3.5.* slo*<sup>1</sup> Mutants Show Reduced Lifespan*

Mitochondria are energy-generating organelles of the cell involved in several metabolic and signaling pathways [15] such as lifespan. Our results showed mitochondrial structural and functional defects in BKCa mutants. Hence, we further investigated the consequence of absence of BKCa in lifespan.

We compared the lifespan of *slo*<sup>1</sup> mutants with wt flies. Even though flies were cultured in the optimal nutritional conditions and temperature (25 ◦C), *slo<sup>1</sup>* mutants surprisingly died within 45 ± 3 days (Figure 5A, B, and Supplementary Figure S2) whereas wt flies survived up to 85 ± 5 days, showing that the *slo*<sup>1</sup> mutant has only ~50% of lifespan compared to wt flies. There was no significant difference between females (Figure 5A) and males (Figure 5B) *slo*<sup>1</sup> mutants as they both showed decreased lifespan by ~50%. Reductions in the lifespan of female *Drosophila* are also associated with mating [36]. To test whether BKCa has any role in 'cost of mating', we performed a parallel study where males and females were housed together. We did not detect any significant differences in the observed lifespan of flies cultured separately or together (Supplementary Figure S2).

**Figure 5.** *slo*<sup>1</sup> mutants reveal accelerated aging. *Drosophila* BKCa (*slo*1) mutants show significantly reduced lifespan of females (**A**) and males (**B**) by approximately 50% compared to wild-type (wt) flies. The inset shows 50% survival for wt (black) and *slo*<sup>1</sup> (gray), which was reduced significantly for *slo*<sup>1</sup> mutants. (**C**) Negative geotaxis assay for wt and slo flies at young (day 3) and older flies (day 30) shows reduced ability of *slo*<sup>1</sup> mutants to climb the marked distance in a given time in vials compared to their controls. (**D**) Increased polyubiquitination staining is observed in *slo*<sup>1</sup> mutants (red) as compared to wt in both young and older ages and quantification is provided in (**E**). (**F**) *slo*<sup>1</sup> mutants show increased intestinal perforations as determined by the leakage of fluorescein dye (green) from the gu<sup>t</sup> unlike control flies, which only show the dye in their gu<sup>t</sup> at both young and older ages. (**G**) Quantification of fluorescein signal from (**F**). (**H**) Microarray data showing differential expression of life span-related genes in wt and *slo<sup>1</sup>* mutants.

Age-related locomotor impairments including negative geotaxis [23] are well-documented in *Drosophila* [37]. *Drosophila slo*<sup>1</sup> mutants are known to have locomotor impairments [38] which were also observed here in both males and females (Figure 5C). No significant changes in negative geotaxis were observed in wt flies in between 3 days and 30 days old in both genders. Wild type flies survive up to ~90 days and our geotaxis assays were performed on comparatively younger wild type flies. However, with age *slo*<sup>1</sup> mutants showed a dramatic reduction in locomotion (Figure 5C, n = 3, 5 flies each in each trial). Reduction in lifespan is directly associated with increased proteotoxicity [39,40]. We characterized *slo*<sup>1</sup> mutants at young and old age along with wt flies to study the age-related deposition of protein aggregates in a flight muscle by immunofluorescence (Figure 5D). As shown earlier [39,40] anti-Ubiquitin (Ubq) antibody labels' protein aggregates in indirect flight muscle in old flies, we also observed a significant increase in protein aggregates (Figure 5D,E) in *slo*<sup>1</sup> mutants. Surprisingly, the *slo*<sup>1</sup> mutant showed a higher amount of aggregates from a young age which increased with old age (Figure 5D). Integrated fluorescence of protein aggregates showed a significant increase in Poly Ubq fluorescence with age in both wt and *slo*<sup>1</sup> mutants (Figure 5E) (n = 5). We also observed similar results with western blot studies where poly-Ubq streak was increased in *slo*<sup>1</sup> mutants. In corroboration, we observed an increase in the levels of refractory to Sigma P, Ref(2)P, a Drosophila orthologue of mammalian p62, which is a major component of protein aggregates in flies [41] (Supplementary Figure S3). Age-dependent intestinal-perforations are utilized as markers of aging and physiological changes associated with aging [42]. We tested age-related intestinal perforation by feeding fluorescein dye to young and old flies from wt as well as *slo*<sup>1</sup> mutant groups (Figure 5F). Surprisingly, the mutant flies showed fluorescent dye leakage through the intestinal perforations from a young age (3 days) indicating the premature or accelerated aging phenotype (Figure 5F). Taken together, our results sugges<sup>t</sup> that *slo*<sup>1</sup> mutants not only show shortened lifespan but several accelerated aging phenotypes (n = 5).

We conducted microarray studies using 3-week old wild type and *slo* mutants (n = 3) to investigate if life span related genes are di fferentially regulated in the mutants. We found several genes implicated in life span regulation altered in the *slo* mutants (Figure 5G). Methuselah mutants are well known to expand the life span of *Drosophila* [43]. We indeed found overexpression of two Methuselah genes explaining the converse phenotype of shortened life span in the *slo* mutants. Overexpression of methionine sulfoxide reductase A (Epi71CD) is shown to increase life span, whereas in our arrays we found a decrease of this enzyme, along with the mitochondrial antioxidant peroxiredoxin 3 [44]. Several other life span related genes were altered such as Thor, NLaz, Hsp22, and Daxx [45–48] suggesting the absence of BKCa channel having an important role in regulating life span. In line with the observed mitochondria-related oxidative stress in slo mutants, we also found 63 oxidative stress-related genes altered (Supplementary Figure S4).

We further wanted to investigate if overexpression of BKCa in flies has a converse e ffect on life span compared to the *slo* mutants. We created full-length BKCa pUAST plasmids and injected into flies. Consistent with our previous results in Figure 5A,B flies overexpressing human BKCa at 29 ◦C, at which Gal4 e fficiency is maximum, resulted in an increase in a life span of male flies (Figure 6A). The e ffect was not seen in female flies where they had a similar life span compared to wild type flies (Supplementary Figure S5). This showed that BKCa has a definitive role in regulating life span and the function is genetically conserved.

**Figure 6.** *slo*<sup>1</sup> expression modulates survival. ( **A**) Males overexpressing human (Hs) BKCa increase life span. Inset shows quantification of 50% survival of control and Hs BKCa overexpressing flies. (**B**) Lifespan of control, slo RNAi under 24B Gal4 and *slo*<sup>1</sup> mutants at 29 ◦C. ( **C**) *slo*<sup>1</sup> mutants are partially rescued by the overexpression of Daughterless Gal4-UAS; Sod2. Inset histograms represent the 50% survivability of the mutants. Histograms represent the 50% survivability of the mutants in both (**B**) and ( **C**).

Using RNAis against BKCa we also narrowed down that the reduction in lifespan is at least partly through its action in the muscles. We tested global (Daughterless Gal4), neuronal (Elav Gal4), and muscle (24B Gal4) knockdown of BKCa and found that muscle knockdown of BKCa showed a reduction in lifespan compared to control flies (Figure 6B). We found that *24BGal4*>*slo* RNAi decreased the lifespan (Figure 6B) from 52 ± 4 days to around 42 ± 3 days at 29 ◦C, at which Gal4 efficiency is maximum. The 50% survivability bar graphs show a significant decrease in the lifespan of *24BGal4*>*slo RNAi* (Figure 6B). The reduction in locomotor activity with age could also be associated with loss of BKCa in the muscles where mitochondria play an important role [49].

As ROS generation was elevated in *slo<sup>1</sup>* mutants, we attempted to rescue the reduction in lifespan of BKCa mutants by chelating ROS. We overexpressed SOD2 using UAS-SOD2 in *slo*<sup>1</sup> mutants using a ubiquitous daughterless-Gal4 driver and cultured them to study their lifespan. As shown in Figure 6C, both wt, and *slo<sup>1</sup>* mutants showed a modest but significant increase in lifespan on overexpression of SOD2 at 25 ◦C (we observed similar results at 29 ◦C as well). We further calculated the time at which 50% of flies survived. Overexpression of SOD2 increased 50% survivability by 10% but for *slo<sup>1</sup>* mutants, we observed ~36 ± 8% increase. These results partially implicate ROS in the reduction of the life span of *slo<sup>1</sup>* mutants and chelating ROS rescued the lifespan of *slo*<sup>1</sup> mutant flies. This suggests, in addition to ROS, other mitochondrial abnormalities observed in *slo*<sup>1</sup> mutants could be contributing to the reduction of lifespan.
