**1. Introduction**

The large-conductance potassium channel activated by calcium (Ca<sup>2</sup>+) and voltage (BKCa/Slo/MaxiK) was originally cloned in *Drosophila* at the *slowpoke (slo)* locus [1–3] and addressed as *Kcnma1* in mammals. BKCa channel is ubiquitously present in the plasma membrane of all eukaryotic cells. In *Drosophila,* extensive work has been performed on *slo* mutants where BKCa was shown to carry transient Ca2+-dependent K<sup>+</sup> currents (IKCa) in muscles [2,4], and neuronal cells [5]. In addition, *slo* mutant has revealed roles of BKCa channel in neuronal functions, abnormal circadian activity, and well-characterized locomotor disorder (hence the name *slowpoke*) [3,6].

In mammals, BKCa is characterized to play similar roles in neuronal and non-neuronal cells [7]. They are the key ion channels with a large conductance, activated by gasses and lipids in addition to sensing changes in Ca2+, and voltage. In the last decade, mutations in *Kcnma1* gene have been associated

with a paroxysmal movement disorder, epilepsy, obesity, hypertension, and cancer in humans [8]. BKCa null mutant mice showed alterations in circadian rhythm, blood pressure, hearing, heart rate, bladder control, locomotion, reproductive function, neurovascular coupling, airway constriction, insulin secretion, and learning and memory [7,9]. In the absence of BKCa, the survival of mice and weight gain was hampered [10] but in contrast, the absence of Slo-1 in *Caenorhabditis elegans* was associated with slow motor aging and moderate extension of life span [11]. The majority of these functions were shown to be associated with the BKCa localized to the plasma membrane [9]. One exception to plasma membrane localization of BKCa channels is their localization to mitochondria of murine and rodent adult cardiomyocytes [8,12]. In the heart, activation of BKCa is known to play a direct role in cardioprotection from ischemia-reperfusion (IR) injury possibly via regulation of mitochondrial function [8,12–14].

Mitochondria are energy-generating organelles of the cell involved in several metabolic and signaling pathways. The inner mitochondrial membranes support the electron transport chain (ETC) tightly-coupled with membrane potential ( ψmito) that participates in the generation of ATP. Defects in ETC, ψmito, mitochondrial fusion–fission events, or ionic imbalance can cause mitochondrial permeability transition pore (mPTP) to form, and result in apoptosis [15]. One of the well-established consequences of mitochondrial dysfunction is life span [16]. Several ion channels present in the plasma membrane and intracellular organelle membranes are known to regulate mitochondrial structure as well as functional integrity [8]. Even though BKCa is shown to regulate mitochondrial function, there is no direct evidence that BKCa can directly regulate mitochondrial structural and functional integrity. Expression of BKCa in coronary arteries from old rats, as well as humans, diminishes without showing any changes in biophysical properties [17]. However, whether BKCa directly a ffects life span is not well studied. To address this question, we studied the BKCa channel mutant (*slo*) phenotypes with respect to mitochondrial functional integrity and life span using the *Drosophila* model.

In this study, we found that BKCa/Slo is present in mitochondria of *Drosophila* as a functional ion channel. The absence of BKCa results in age-related changes in mitochondrial structural and functional integrity. We also tested whether increased mitochondrial reactive oxygen species (ROS) is responsible for the early death of flies and chelating ROS could partially rescue the aging phenotype. Ablation of BKCa dramatically reduced the lifespan of *Drosophila*, while overexpression of human BKCa form surprisingly increased lifespan only in males. In agreement, our microarray data revealed various life span regulated transcripts altered in *slo* mutant flies. Taken together, our results define a novel function for BKCa channel in regulating mitochondrial structure and function and reduction in life span.

#### **2. Materials and Methods**

#### *2.1.* Drosophila *Stocks, Reagents, Dyes, and Antibodies*

All fly stocks were maintained at 25 ◦C on standard medium (jazz mix, nipagin free) unless otherwise stated. The experiments were carried out at 25 ◦C or 29 ◦C (for Gal4 efficiency) as mentioned in the results sections or figures. The Canton S strain served as the wild-type (wt) stock and is indicated as 'wt' through the manuscript. The *slo*<sup>1</sup> mutants (chemical-induced mutation, originally characterized in Elkins et al. 1986 [3]), RNAi lines, Gal4 lines, and wild type lines (Canton S and W1118) were obtained from the Bloomington Stock Center. UAS Sod2 flies were a gift from Prof. David Walker (UCLA).

#### *2.2. Immuno Cyto*/*Organelle Chemistry*

Flight muscles were dissected and fixed with 4% ( *w*/*v*) paraformaldehyde (PFA), washed and permeabilized with 0.4% (*v*/*v*) Triton-X100. Mitochondria were isolated from whole flies and loaded with mitotracker as described earlier [18]. Mitochondria and tissues were blocked with normal goa<sup>t</sup> serum (10%) and stained with primary antibodies (anti-ubiquitin 1:100 (FK2), and anti-BKCa 1:200) and secondary antibodies, followed by DAPI (for tissues) (n = 5 independent experiments).
