Isolation of the Cytosolic C-Terminus Segment of Mammalian Kca Channel a Subunit

<p>The last 20 years of research into ion channels has unraveled unexpected features of the BK_ca channel as a rheostast for cell activity in excitable tissues and homeostatically fine tuning many biological activation processes in the cell. Great attention has been dedicated to the allosteric regulatory mecanisms by which the BK_ca channel is activated or inhibited. Most of these mechanisms point to the involvment of cytoplasmic, C-terminal, ‘tail’ domain of the channel. Many laboratories have cloned portions of the ‘tail’ domain in an attempt to to study specific regulatory sites. Very little is known about the BK ‘tail’ structure, and functional studies have been the leading approach into understanding the diversity of modulation present in the ‘tail’ domain. Our lab was interested in isolating a natively expressed BK_cafrom a GH3 cells. Immunoblots using lystes of GH3 cells unexpectedly revealed a 70 kDa BK ‘tail’ like protein because it reacted with anti-BK antibodies. We determined that the high-speed supernatant fraction of these GH2 cell lysates was enriched with this 70 kDa protein. We then endeavored to isolate this protein using immunoprecipitation and affinity chromatography. Results show that our affinity column using the dye Cibacron blue and elution NAD buffer at low pH was able to isolate this 70 kDa protein. Further investigation is needed to determine the pH dependence of this affinity column approach and the sequence of this protein. If proven to be the ‘tail’ of the BK_ca channel, purification of this protein will open doors for subsequent biological experiments to determine how different ligands such as diatomic gases may interact with the channel, and elucidate mechanisms regarding the channel’s sensitivity to gaseous ligands, amongst others. xiv</p>

Name thesis:degree_name

Doctor of Science (DSc)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physiology

Year

2010

Author dc:creator

• Remigio, Wilton

Contributors dc:contributor

• David Hessinger
• Everett Lohman
• Lee Berk
• Ernie Schwab

• Physiology
• Potassium Channels – physiology; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Ion Channel Gating – physiology; Antineoplastic Agents, Phytogenic – pharmacology; Muscle, Smooth, Vascular -- drug effects; Coronary Vessels – physiology; Electric Stimulation; Molecular Sequence Data.

Statement dc:rights

• This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/696

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-1818