**3.5. Proteomics approach**

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

Decapitate 2 weeks old (CBA/J) mouse at the base of the foramen magnum using scalpel. Briefly rinse the head with 70% ethanol and remove the epidermis using a scalpel blade. Open the cranium along the sagittal suture using a scalpel blade bisect the head equally half and remove the forebrain, cerebellum, and brainstem using blunt dissection. Remove the temporal bones, dip them briefly in 70% ethanol, and transferred into 35 mm dish. Remove the bulla and surrounding tissue from the petrous portion of the temporal bone and identify the conch shaped cochlea and separate it from the vestibular system using forceps. Remove the calcified bony labyrinth of the cochlea carefully removed from basal region to apical end. Spiral ligament and organ of Corti is tightly attached and coiled inside the bony labyrinth. Carefully remove the organ of Corti by securing the spiral ligament at the hook region of the base using forceps and unwinding it as you move apically. Begin at the base and remove the spiral ligament from the organ of Corti using

The isolation of hair cells from mouse cochlea was described [17]. Remove the organ of Corti at the base of hook region by using two ½ cc insulin syringes with the help of U-100 28G½ needles as forceps. The organ of Corti consist of spiral limbus and sensory epithelium (outer and inner hair cells) cells starting from apex to base of organ of Corti. The sensory epithelium was separated from spiral limbus with help of insulin syringes. The sensory epithelium explant was transferred into fresh Petridis (35 mm) containing 1 ml of DMEM with 10% FBS, ampicillin (10 mg/ml), and 400 μl of each poly-L-ornithine (0.01%) and laminin

the above fresh medium then the adhesive outer and inner hair cells was started multiplica-

The mouse cochlear hair cell culture is washed with pre-incubated PBS buffer and adds 1 mL of trypsin solution then incubated 37°C for 1 min. and harvest (2.5 × 105) cells. The cells were centrifuged at 300 g for 10 min at room temperature and the supernatant removed and

**3.2. Transfection of candidate genes in mouse cochlear hair cells by nucleofector** 

. After 48 h carefully change

C- terminus of BK channel and inhibit their channel open state activity.

**3. Isolation of protein complexes from MAMs in cochlea**

*3.1.2. Micro-isolation of hair cells from sensory epithelium of organ of Corti*

(50 μg/mL). The Petri dish was incubated at 37°C with 5% CO<sup>2</sup>

**3.1. Maintenance of mouse cochlear hair cell culture**

58 Current Understanding of Apoptosis - Programmed Cell Death

*3.1.1. Isolation of the organ of Corti*

no. 55 fine forceps.

tion on the Petri dish.

**device (Lonza)**

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 control and BK transfected hair cells. The 50 μg of proteins were mixed with sample buffer and loaded in IEF gel strips. IEF will be performed using 7 cm immobilized pH gradient (IPG) gel strip, pH 3–10 (Protean IEF Cell System, Bio-Rad). Proteins were resolved by IEF in the first dimension and SDS-PAGE (12% acrylamide) in the second dimension. Precision Plus (Bio-Rad) molecular weight marker was used to determine relative mobilities. Gels were stained with silver staining and images were captured using the Molecular Imager versa doc MP Imaging System (Bio-Rad). The resolution of the scanning gel was 53 μm, and images were processed with the standard version of PDQUEST software (Bio-Rad), which is used to identify spots by pi and molecular weight with the help of standards. The BKα transfected protein gel is compared with control gel and qualitative differences of appeared (up-regulated proteins) and disappeared (down-regulated proteins) protein spots were excised and subjected to reduction, alkylation, and trypsin digestion as described previously [19]. Peptides were extracted and concentrated under vacuum centrifugation. The peptides of each sample were injected into LC–MS/MS then identification of each protein spots.

ER morphology and Ca2+ [29]. The ER morphology is still controversial even though reticulons and DP1 proteins are enriched in ER tubule rather than sheets and nuclear envelope [30]. Another protein dynamin-related membrane GTPases atlastins are involved in the control of

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

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

61

Close contact between the membrane of ER and outer mitochondrial membrane was first identified in late 1960 by several independent groups [32]. ER membranes co-purifying with mitochondrial fractions were observed under electron microscopy which, revealed that direct communication between cisternal space of ER and inner mitochondrial membrane (IMM) space [33]. The 20% of mitochondrial surface were direct contact with ER and each contact appears to vary between 10 to 25 nm in length [34]. The functional importance of these two organelles contact sites is further established by the quasi-synaptic mechanism of transmis-

The nature of ER–mitochondrial tethering has remained largely elusive. Szabadkai et al. [35] reported that IP3R is localized on membrane of ER and VDAC is localized on the OMM and both are physically attached through 75 kDa glucose-regulated proteins (GRP 75) (**Figure 2**). IP3R play a major role on the mobilization of calcium from ER to mitochondria as a function of apoptosis [36]. During steady-state transfer of Ca2+ molecules from ER to mitochondria, the mitochondrial anti-apoptotic proteins Bcl2 is drastically reduced [37]. The phosphofurin acidic cluster sorting protein-2 (PACS)-2 is also involved in the regulation of apoptosis

morphology of ER by promoting the branching of the tubules [31].

**5. Protein liaison in tethering to ER and mitochondria**

sion of Ca2+ crucial function of during apoptosis.

**Figure 2.** Tethers between ER and mitochondria.
