*2.3. Electrophysiology*

Patch-clamp experiments using mitoplasts (mitochondria without outer membranes) were performed as described previously [19,20]. Briefly, mitoplasts were prepared from mitochondria isolated from whole *D. melanogaster* placed in a hypotonic solution (5 mM HEPES, 100 μM CaCl2, pH = 7.2) to induce swelling and eventual disruption of the outer membrane. To restore the sample to an isotonic condition (150 mM KCl, 10 mM HEPES, 100 μM CaCl2, pH 7.2) a hypertonic solution (750 mM KCl, 30 mM HEPES, 100 μM CaCl2, pH 7.2) was added. The patch-clamp pipette was filled with an isotonic solution. Mitoplasts are easily recognizable due to their size, round shape, transparency, and the presence of a 'cap', characteristics that distinguish these structures from the cellular debris that is also present in the preparation. The low-calcium solution (1 μM CaCl2) contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA, and 0.752 mM CaCl2 at pH 7.2. An isotonic solution containing 100 μM CaCl2 was used as the control solution for all of the presented data. The experiments to assess the channel activity were carried out in patch-clamp inside-out mode [20]. The electrical circuit was made using Ag/AgCl electrodes and an agar salt bridge (3 M KCl) as the ground electrode. The current was recorded using a patch-clamp amplifier Axopatch 200B. The pipettes had a resistance of about 14 MΩ and were pulled using a vertical puller.

The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. The traces of the experiments were recorded in single-channel mode. The conductance of the channel was calculated from the current–voltage relationship (Figure 1I). The probability of channel opening (Po, open probability) was determined using the single-channel search mode of the Clampfit software. Data from the experiments are reported as the mean values + standard deviations (S.D.). Student's *t*-test was used for statistical analysis (n = 5 independent experiments comprising of mitochondrial isolation from 100 flies each).

**Figure 1.** Localization of dSlo in isolated mitochondria. High-resolution confocal images of isolated mitochondria from *Drosophila* (**A**–**C**, wild type, **D**–**F** *slo* mutants) loaded with mitotracker (**A**,**D** red) and labeled with an anti-Slo antibody (**B**,**E** green). Overlays are shown in (**C**,**F**). Protein proximity index for dSlo to mitotracker was 0.5 ± 0.1.

(**G**), Single-channel current-time recordings (left panel), current-voltage characteristics (middle panel) and Po analysis of single-channel events in a symmetric 150/150 mM KCl isotonic solution (100 μM Ca2+) at different voltages in mitoplast prepared from whole flies. (**H**), Effects of 1 μM Paxilline on the single-channel activity. (**I**), Single-channel current-time recordings in symmetric 150/150 mM KCl isotonic solution at control (100 μM Ca2+) and after reduction calcium concentration to 1 μM Ca2+. (**J**), Analysis opening probability in the presence of 1 and 100 μM Ca2+ at different voltages of the mitoBKCa channel in mitoplast prepared from whole flies. All data were acquired in a symmetric 150/150 mM KCl isotonic solution (n = 4). (**K**), Current–time recordings of single-channel activity in symmetric 150/150 mM KCl isotonic solution at control (100 μM Ca2+), after reduction calcium concentration to 1 μM Ca2+ and after application of 10 μM NS1619. The bar graph shows the distribution of the Po under the conditions above. \* *p* < 0.001 vs. the control. \*\* *p* < 0.001 vs. 1 μM Ca2+. The data in (**G**,**J**,**K**) are presented as the means ± S.D. The recordings were low-pass filtered at 1 kHz. "-" indicate a closed state of the channel.

#### *2.4. Reactive Oxygen Species and Quantification*
