Electrical activity encodes auditory and vestibular stimuli and sculpts peripheral and central nervous system pathways. The latter was demonstrated in the visual system, where spontaneous electrical activity regulates neural pathway formation. The underlying bases for electrical activity are pore forming proteins or ion channels inserted into the membrane of excitatory cells. In order to understand how this activity contributes to the normal and abnormal formation of the nervous system, one must answer the fundamental question of, what are the mechanisms that regulate ion channel expression during development? Potassium ion channels contribute to electrical activity in the inner ear by shaping the hair cell receptor potential and the signals transmitted to and from afferent and efferent nerve fibers, respectively. This activity can contribute to intracellular signals that regulate these cells during development. The long term goals of our research are to discover the intracellular pathways that regulate the expression of ion channels from gene to protein in the developing inner ear. These pathways are composed of protein-protein interactions that partner ion channels with transcription, cytoskeletal, chaperone, and clustering proteins, among others. Of the many K+ channels, the large conductance Ca2+-activated K+ or BK channel has a role not only in excitation, but also in intracellular signaling and metabolism. These functions are reflected in recent findings that show BK expresses, not only in the plasmalemma, but also in mitochondria and possibly the nuclear envelope. Given these different subcellular locations and the long, ligand-binding C-terminus of BK, we will examine BK interactions with BK-Associated Proteins (BKAPs) during early and late stages of mouse cochlear development. The proposed experiments focus on discovering and characterizing the role of BKAPs in promoting BK expression, function, and distribution by using proteomic and bioinformatic techniques.
The specific aims of the proposal are to (1) validate newly-discovered BKAPs from the adult cochlea, by measuring BK expression, when silencing its partners, (2) determine how specific BKAPs alter the biophysical properties of BK, (3) determine the properties of mitochondrial BK and its relation to specific BKAPs, and (4) map the BK interactome during early cochlear development. These studies will utilize proteomics, bioinformatics, siRNA, and electrophysiology.

Public Health Relevance

These studies will begin to illuminate and characterize protein-protein interactions that guide normal ion channel development. Discovering these interactions is relevant to understanding the cochlear proteome and, with it, the many signals that contribute to the development and regeneration of the auditory and vestibular systems. Moreover, these discoveries will provide insights into major intracellular pathways that regulate not only normal cochlear development but those that lead to sensorineural deafness in children.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC004295-11
Application #
8274714
Study Section
Auditory System Study Section (AUD)
Program Officer
Freeman, Nancy
Project Start
1999-12-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
11
Fiscal Year
2013
Total Cost
$325,654
Indirect Cost
$104,121
Name
University of South Florida
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Peng, Zhenling; Sakai, Yoshihisa; Kurgan, Lukasz et al. (2014) Intrinsic disorder in the BK channel and its interactome. PLoS One 9:e94331
Ding, Bo; Frisina, Robert D; Zhu, Xiaoxia et al. (2014) Direct control of Na(+)-K(+)-2Cl(-)-cotransport protein (NKCC1) expression with aldosterone. Am J Physiol Cell Physiol 306:C66-75
Darville, Lancia N F; Sokolowski, Bernd H A (2014) Bottom-up and shotgun proteomics to identify a comprehensive cochlear proteome. J Vis Exp :
Darville, Lancia N F; Sokolowski, Bernd H A (2013) In-depth proteomic analysis of mouse cochlear sensory epithelium by mass spectrometry. J Proteome Res 12:3620-30
Sokolowski, Bernd; Orchard, Sandra; Harvey, Margaret et al. (2011) Conserved BK channel-protein interactions reveal signals relevant to cell death and survival. PLoS One 6:e28532
Sakai, Yoshihisa; Harvey, Margaret; Sokolowski, Bernd (2011) Identification and quantification of full-length BK channel variants in the developing mouse cochlea. J Neurosci Res 89:1747-60
Sokolowski, Bernd; Duncan, R Keith; Chen, Stephanie et al. (2009) The large-conductance Ca(2+)-activated K(+) channel interacts with the apolipoprotein ApoA1. Biochem Biophys Res Commun 387:671-5
Kathiresan, Thandavarayan; Harvey, Margaret; Orchard, Sandra et al. (2009) A protein interaction network for the large conductance Ca(2+)-activated K(+) channel in the mouse cochlea. Mol Cell Proteomics 8:1972-87
Harvey, Margaret; Karolat, Joerg; Sakai, Yoshihisa et al. (2009) PPTX, a pentraxin domain-containing protein, interacts with the T1 domain of K v 4. J Neurosci Res 87:1841-7
Kathiresan, Thandavarayan; Harvey, Margaret C; Sokolowski, Bernd H A (2009) The use of 2-D gels to identify novel protein-protein interactions in the cochlea. Methods Mol Biol 493:269-86

Showing the most recent 10 out of 18 publications