A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea

Abstract

The large conductance Ca 2؉ -activated K ؉ or BK channel has a role in sensory/neuronal excitation, intracellular signaling, and metabolism. In the non-mammalian cochlea, the onset of BK during development correlates with increased hearing sensitivity and underlies frequency tuning in non-mammals, whereas its role is less clear in mammalian hearing. To gain insights into BK function in mammals, coimmunoprecipitation and two-dimensional PAGE, combined with mass spectrometry, were used to reveal 174 putative BKAPs from cytoplasmic and membrane/cytoskeletal fractions of mouse cochlea. Eleven BKAPs were verified using reciprocal coimmunoprecipitation, including annexin, apolipoprotein, calmodulin, hippocalcin, and myelin P0, among others. These proteins were immunocolocalized with BK in sensory and neuronal cells. A bioinformatics approach was used to mine databases to reveal binary partners and the resultant protein network, as well as to determine previous ion channel affiliations, subcellular localization, and cellular processes. The search for binary partners using the IntAct molecular interaction database produced a putative global network of 160 nodes connected with 188 edges that contained 12 major hubs. Additional mining of databases revealed that more than 50% of primary BKAPs had prior affiliations with K ؉ and Ca 2؉ channels. Although a majority of BKAPs are found in either the cytoplasm or membrane and contribute to cellular processes that primarily involve metabolism (30.5%) and trafficking/scaffolding (23.6%), at least 20% are mitochondrial-related. Among the BKAPs are chaperonins such as calreticulin, GRP78, and HSP60 that, when reduced with siRNAs, alter BK␣ expression in CHO cells. Studies of BK␣ in mitochondria revealed compartmentalization in sensory cells, whereas heterologous expression of a BK-DEC splice variant cloned from cochlea revealed a BK mitochondrial candidate. The studies described herein provide insights into BK-related functions that include not only cell excitation, but also cell signaling and apoptosis, and involve proteins concerned with Ca 2؉ regulation, structure, and hearing loss. BK 1 channels act as sensors for membrane voltage and intracellular Ca 2ϩ , thereby linking cell excitability, metabolism, and signaling. BK channels, also known as Slo, are large conductance channels (100 -300 pS) (1) composed of four ␣-subunits that are regulated by four auxiliary ␤-subunits. The ␣-subunit of the BK channel has six to seven transmembranespanning regions (S0 -S6) where the S0 domain places the N terminus extracellularly as a binding site for the beta subunit. The transmembrane domains S1-S4 are responsible for sensing voltage changes, whereas the pore forming region, between S5-S6, conducts ions. BK has a large C-terminal region that contains target sequences for channel modulation such as a Ca 2ϩ bowl, two domains that regulate the conductance of K ϩ (RCK1 and RCK2), a tetramerization domain, leucine zipper motifs, a hemebinding motif, two phosphorylation sites, and a caveolin-targ...

mitoBK Ca is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location

“…Whether the inclusion of STREX or SV27 modifies BK Ca -DEC targeting to cardiomyocytes mitochondria is yet to be established. However, data using CHO cells indicate that SV27 does not have a major effect as a BK Ca -DEC variant with additional splice inserts including SV27 can still be observed in mitochondria of CHO cells (17).…”

“…1E and Table S1). The coding sequences of the identified peptides were localized in constitutive exons 9,12,14,17,19,21,25, and 27, and spanned exons 21 and 22. In addition, we found a peptide of a 27-aa splice insert, which we refer to as SV27.…”

Preliminary studies on differential expression of auditory functional genes in the brain after repeated blast exposures

“…Other types of molecules involved in calcium regulation, such as calreticulin and calmodulin-dependent protein kinase expression, showed significant decrease in the frontal cortex and midbrain of blast-exposed mice, respectively (Tables 3 and 4) [23,47]. Interestingly, two calcium binding proteins, calretinin and parvalbumin, that were upregulated in the cerebellum at 24 and 48 h after blast exposures by proteomic analysis were not found to be altered in cDNA microarray analysis at 6 h after blast exposures.…”

“…Molecules involved in calcium influx and calciumdependent proteins/enzymes are predominant signal transducers in auditory neurons [22][23][45][46][47][48]. The frontal cortex and midbrain of blast-exposed mice showed significant increase in the expression of calcium-dependent cysteine proteases and calpain 3 and 9, respectively (Tables 3 and 4).…”

BK Ca channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells

“…GRP78 increases in human myometrium in response to inflammation (48), is associated with the BK Ca channel in mouse cochlea (49), and is elevated in labor or preterm labor compared with nonlabor myometrium (48). We cannot rule out the possibility that GRP78 serves as a receptor for α 2 M*.…”