**6. The role of bk channel role in mitochondria and ER interactions**

Earlier, the molecular mechanism of BK channel function in mouse cochlea we studied, through the system biology approach aided by the sensitivity of coimmunoprecipitation, shotgun mass spectrometry methods to identify 174 BK channel associated proteins (BKAPs). Based on BKAPs developed transient BKα interaction networks are enriched with functional attributes of metabolism, trafficking and scaffolding, development and differentiation, signal transduction, and transport [2]. We, for the first time, employed a large number of ion channel associating proteins in the cochlea. The BKAPs were analyzed through subcellular localization. The majority of the BKAPs nearly 30 and 15% were localized in mitochondria (both membrane and matrix) and endoplasmic reticulum. Comprehensive understanding of the BK channel role in mitochondria and endoplasmic reticulum function has remained unclear. The past decade has concealed an number of unexpected proteinprotein interactions that fundamentally changed our view of the localization and functional interactions of proteins inside cells. The functional role of BK channel in mitochondria is no exception.

One of our recent bioinformatics studies has revealed that the proteomics data (BKAPs) with functional attributes remarkably identified nearly 20 and 10% of novel mitochondrial and endoplasmic reticulum BKAPs involved in pro-apoptotic and anti-apoptotic properties which have not been looked in to, so far. Based on these results the preliminary experiments of tunnel assay for the BK overexpressed CHO cells that confirmed 65% cell death when compared with control. Till now, the exact mechanism behind the mitoBK and ER-BK channel role in pro-apoptosis is not known. It is expected that proposed project will give a new dimension in the biomedical field, which in turn may be useful for understanding the mechanism of hearing loss and noise-induced hearing loss (NIHL).

The Ca2+ activated potassium channel is playing a predominant role in mitochondria and endoplasmic reticulum-associated proteins. These proteins were contributing either K<sup>+</sup> influx or K<sup>+</sup> efflux of cells through the Ca2+ ion binds with Ca2+ bowl of c-terminus region [2]. Superoxide dismutase, glutathione S transferase μ, GAPDH, VDAC, and peroxidase are involved in ROS pathway as well as the candidate for BK channel associated proteins. The endoplasmic reticulum proteins calreticulin, GRP78, inositol triphosphate receptor (InsP3R), protein SET, VCP, HSP70, and protein disulfide isomerase are involved in the regulation of calcium-induced calcium release (CICR), protein folding and clearance. These proteins are known to BK channel associated proteins [2]. These two groups of proteins are involved in the mitochondrial associated-ER membrane (MAMs) to regulate calcium signaling of CICR and cell death. However, we are only beginning to understand the spatial organization and interorganellar signaling in between ER and mitochondria and their functional regulations.

Mitochondria-associated ER membranes (MAMs) control Ca2+ influx between ER and mitochondrial subunit. We found that BKα subunits [2] localized in the inner mitochondrial membrane directly interact with other BKAPs like IP3R, calreticulin at the ER face of the MAMs. The molecular chaperone, glucose-regulated-protein 78 (grp78), is linked with inositol 1,4,5 triphosphate receptor-1 (InsP3R-1) and voltage-dependent anion channel (VDAC-1) of the outer mitochondrial membrane (**Figure 2**) [42]. Both Ca2+ overload and depletion of the ER Ca2+ pool can result in changes of two signaling pathways of unfolding protein response (UPR) and ER overload response (EOR) [43]. Latter induces mitochondrial membrane permeabilization (MMP), opening of the permeability transition pore (PTP), and release of cytochrome c and subsequent engagement of the mitochondrial apoptotic pathway [44]. The Ca2+-sensitive dehydrogenases of the Krebs cycle [45] are stimulated as increased mitochondrial Ca2+ boosts ATP production. Increasing ATP production leads to more leakage of free electrons, causing the formation of superoxides. The resulting oxygen ions, free radicals, and peroxides are collectively called reactive oxygen species (ROS), which are effective to damage DNA and apoptosis.

ER membrane BK channels play a predominant role in ER retention, retrieval and normal trafficking [46]. InsP3R and BK channels are localized in the same hotspot region of glioma cells of lipid raft however; they are not directly interacting with each other [47]. The InsP3R are tightly linked with BK channel through continuous releasing of Ca2+ molecules in organelles and they are bind with EF-hand motif of Ca2+ bowl and activate BK channel to release the K<sup>+</sup> ion (K<sup>+</sup> efflux).

The cellular Ca2+ ion are transported through the plasma membrane by receptor and voltagesensitive channels. Once inside the cell, Ca2+ can either interact with Ca2+ binding proteins or sequestered into the ER or mitochondria. Both Ca2+ overload and depletion of the ER Ca2+ pool can result in changes of protein folding and an increase of ER stress by either unfolding protein response (UPR) or ER overload response (EOR) [42]. From these two responses, both incorrectly folded and accumulated proteins are eliminated through apoptosis [48]. Ca2+ depleted from the ER is then taken up by the mitochondria, leading to Ca2+ overload in this organelle. The latter induces mitochondrial membrane permeabilization (MMP), opening of the permeability transition pore (PTP), and release of cytochrome c and subsequent engagement of the mitochondrial apoptotic pathway [44]. The increasing mitochondrial Ca2+ boots ATP production leading to more leakage of free electrons, which results in the formation of superoxides. These oxygen ions, free radicals, and peroxides, collectively called ROS, damage DNA and leads to apoptosis. Reactive oxygen species (ROS) play a key role in mechanism for induction of cochlear damage under various patho-physiological conditions [2]. Caspase-12 is localized in ER and activated by ER stress, including the accumulation of excess proteins that leads to cell death.
