**2. Structure and functions of BK channel**

BKα channels are involved in regulating a diversity of physiological processes such as metabolism, signaling, phosphorylation, neurotransmitter release, and modulation of smooth muscle contractions [7]. The BKα channels are activated by the cooperative effects of two distinct stimuli, membrane depolarization, and elevation of free cytoplasmic Ca2+ concentration. BK channels are assembled in membrane as tetramers of pore-forming α-subunits enclosing two regions, transmembrane spanning region containing two domains. They are voltage-sensing domain (VSD), which senses membrane potential and pore-gated domain (PGD) which opens and closes to control the permeability of K+ ions. The other region, the cytoplasmic C-terminus region comprises many protein phosphorylation sites [8] such as RCK1, RCK2, leucine zipper, heme and caveolin-binding motif and Ca2+ bowl that regulates PGD and permeability of K<sup>+</sup> ions (**Figure 1**).

The pore-forming and the C-terminus domain of the BKα subunits contain several protein kinases (cAMP-dependent PKA, PKC, cGMP-dependent PKG, c-Src) and phosphatase (**Figure 1**) binding motifs which are mainly associated with a number of interacting partners to regulate the channel gating and signaling pathways. They activate the BK channel by increasing sensitivity to intracellular Ca2+. The Ca2+ ions are bound to the electron dense of Ca2+ bowl The Role of Calcium-activated Potassium Channel in Mitochondria-Associated ER Membrane… http://dx.doi.org/10.5772/intechopen.77329 57

**Figure 1.** Significant phosphorylation sites in BKα subunit.

known to be involved in noise-induced hearing loss (NIHL) [1] through activation of Ca2+ induced Ca2+ release and ROS pathway by association of BKAPs like SOD, peroxidase, catalase and GSTμ, [2]. In addition, BK channel is known to be associated with deafness proteins like γ-actin and methylthioadenosine phosphorylase (MTAP) [3]. The molecular mechanisms that regulate the BK channel and their role in NIHL and deafness remain unclear. Therefore, understanding mechanisms of BK channel regulation and its associated proteins (BKAPs) will

Mitochondria-associated ER membranes (MAMs) control Ca2+ influx between ER and mitochondria. We found that BKα subunits [2] are localized in the inner mitochondrial membrane and interacted directly with other BKAPs like, IP3R1, calreticulin at the ER face of the MAMs, and the molecular chaperone grp78, which bridges the IP3R-1 with voltage-dependent anion channel (VDAC-1) of the outer mitochondrial membrane (OMM) [4]. The BK channel is associated with all other proteins having a contribution in mitochondria-associated ER membranes. Therefore, the functional regulation of BK channel and its role in MAMs remains unclear.

The novel concept of mechanism of apoptosis is in addition to molecular genes, ionic homeo-

linked with apoptotic cell shrinkage. Inhibition of BK channel with iberiotoxin dramatically

tor not only for early apoptotic cell shrinkage but also for downstream of caspase-3 activation

BKα channels are involved in regulating a diversity of physiological processes such as metabolism, signaling, phosphorylation, neurotransmitter release, and modulation of smooth muscle contractions [7]. The BKα channels are activated by the cooperative effects of two distinct stimuli, membrane depolarization, and elevation of free cytoplasmic Ca2+ concentration. BK channels are assembled in membrane as tetramers of pore-forming α-subunits enclosing two regions, transmembrane spanning region containing two domains. They are voltage-sensing domain (VSD), which senses membrane potential and pore-gated domain (PGD) which opens

region comprises many protein phosphorylation sites [8] such as RCK1, RCK2, leucine zipper, heme and caveolin-binding motif and Ca2+ bowl that regulates PGD and permeability of K<sup>+</sup>

The pore-forming and the C-terminus domain of the BKα subunits contain several protein kinases (cAMP-dependent PKA, PKC, cGMP-dependent PKG, c-Src) and phosphatase (**Figure 1**) binding motifs which are mainly associated with a number of interacting partners to regulate the channel gating and signaling pathways. They activate the BK channel by increasing sensitivity to intracellular Ca2+. The Ca2+ ions are bound to the electron dense of Ca2+ bowl

early steps in apoptosis [6]. The BK channels play a critical role in mediating the K+

in cell [5]. The ionic mechanism of apoptosis associ-

ions. The other region, the cytoplasmic C-terminus

efflux resulting in the

efflux is an essential media-

efflux

provide insights in understanding the problems in deafness and NIHL.

ates the accumulation of intracellular Ca2+ leading to uncontrolled K+

efflux and prevents apoptosis. Therefore, enhanced K<sup>+</sup>

stasis also induces apoptosis especially K+

56 Current Understanding of Apoptosis - Programmed Cell Death

**2. Structure and functions of BK channel**

and closes to control the permeability of K+

reduced K+

ions (**Figure 1**).

and DNA fragmentation.

and activate cytosolic domain. The cytosolic domains are connected with transmembrane spanning region, S6 by 17 amino acid peptide chain, called linker peptide. A cytosolic domain through linker peptide opens and activates the gate PGD domain. Recently the cryo-EM study illustrates the BK channel structure and gating pore size is 1.7–2.0 ηm resolution [9].

The leucine zipper (LZ) motif is originally described as DNA binding proteins and reported to play an important role in both assemblies of ion channels and interactions of protein kinase and protein phosphatase. The LZ motifs serve to anchor a number of different BK channel associated proteins [10]. The LZ and EF-hand motif containing proteins regulate the mitochondrial swelling leading to apoptosis [11]. Therefore, it can be concluded that BK and other interacting proteins are regulating apoptosis through post-translational modification of phosphorylation or palmitoylation (**Figure 1**) [10].

BKα channels are sensitive to Ca2+ regulation through phosphorylation by serine-threonine and tyrosine kinases [12, 13]. Thirty putative phosphorylation sites were identified from seven different BKα splice variants [8]. Among them, the BK-DEC variant has an additional 60 amino acids at the extreme end of the C-terminus which contains 11 serine/threonine and tyrosine residues. The BK channels are directly involved in tyrosine phosphorylation in the presence of c-Src kinase domain in C-terminus of channel. The vital role for c-Src kinase mediating signal transduction on G-protein coupled and integrin receptor activation leads to the regulation of membrane ion channels [12]. The α5β1 integrin activation leads to increasing activity of BK channel. The BK channel phosphorylation of α5β1 integrin at Tyr-766 through intracellular signaling pathway involving c-Src kinase [14].

The PKC phosphorylation site (S1076) is lying on c-terminus of human BKα channel that influences the regulation of protein kinase on BKα channel activity which may subsequently alter pulmonary smooth muscle tone functions [15, 16]. This reveals the dual role of PKC on BK channel on tracheal smooth muscle. They are phosphorylation of S695 by PKC on BK channel which is located in between the conductance of two regulators (RCK1 and RCK2) and inhibits the channel open state probability. The second phosphorylation of S1151 by PKC on C- terminus of BK channel and inhibit their channel open state activity.

appropriate nucleofector solution (containing 2 μg of plasmid vector or 100 ηM of SiRNA and 100 μL of P4 primary cell 4D-nucleofector X solution) added into the nucleocuvette. Gently tap the nucleocuvette vessels to make sure the samples were premixed and the cover bottom of the cuvette. Place the nucleocuvette vessels and close the lid into retainer of the 4D-nucleofector X unit and select the appropriate program [18]. After completion of the run carefully remove the nucleocuvette vessels and resuspended cells with pre-warmed culture medium. The gene expression or down-regulation will be observed after 4 h transfection to

The Role of Calcium-activated Potassium Channel in Mitochondria-Associated ER Membrane…

http://dx.doi.org/10.5772/intechopen.77329

59

**3.3. Mitochondria and endoplasmic reticulum isolation from mouse hair cell culture**

get mitochondria and endoplasmic reticulum proteins.

**3.4. Transmission electron microscopic studies**

pathways in mouse cochlear cells.

**3.5. Proteomics approach**

The mitochondria were isolated from mouse cochlear hair cell cultures using a kit per manufacturer's instructions (Qproteome TM Qiagen). The cells were washed with PBS buffer and harvest these cells with 1 ml of disruption buffer containing protease inhibitor cocktail and incubated 10 min at RT. After 10 min the cells were centrifuged at 6000×g for 10 min and collect the pellet and discard the supernatant. The pellet was resuspended in purification buffer followed by spun at 20,800×g for 15 min. Mitochondria and ER were layered on the surface of a density gradient centrifugation. Both mitochondria and endoplasmic reticulum were removed from the respective gradient and diluted in storage buffer, and spun at 8000×g for 10 min. The pellet consisting of purified mitochondria and endoplasmic reticulum were either resuspended in storage buffer and store in −80C or resuspended in protein lysis buffer

The BKα gene cloned in pCDNA3.1 mammalian expression vector and transfected in mouse cochlear hair cell cultures by using Nucleofector device. After transfection, both control and BK transfected cells were harvested and the cells were fixed with glutaraldehyde. The fixed cells were transferred in to wire gauge. The morphological changes of hair cells with respective of apoptosis such as plasma membrane dissolution; mitochondrial bulging, ER, and nuclear fragmentation were observed under electron microscopy with different concentration of BK transfection in the absence and presence of curcumin loaded silica nanoparticles. The synthesis of silica nanoparticles and encapsulation of curcumin will be carried out using a published procedure. One of the Co-PI is familiar with the synthesis and characterization of silica nanoparticles. The silica nanoparticles will be coated with polymers (polyethylene glycol) (PEG) or polyethylenimine (PEI) to enhance the biocompatibility of the nanoparticles. Initially, the amount of BK with appropriate time intervals is evaluated to activate apoptotic

The appropriate BKα gene was transfected with mouse cochlear hair cell cultures. After 48–72 h transfection, the mitochondria were harvested from the control and BK transfected mouse cochlear hair cell cultures. The proteins from mitochondria were isolated from both

4 days.
