Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
India El - Norfolk State University
Co-Author(s): Brittney K. Burton, Norfolk State University, Norfolk, VA
Calcium ions (Ca2+) and Protein Kinase C enzymes are necessary for the release of insulin. The calcium ions stimulate or fuel the release of insulin into the bloodstream. In previous works it has been discovered that Beta (β) subunit blocks insulin secretion and the mechanism is not known. It is hypothesized that Beta (β) subunit interferes with the interaction between the calcium channel Alpha 1 (α1) subunit and the PKC enzymes. The purpose of this research is to understand this interaction between the subunit proteins and determine the basis of the inhibition of insulin secretion by β subunit. Cav Alpha 1 (α1) and Beta (β) subunit proteins were synthesized in the lab. Concentrations of β3 proteins were obtained; Sample 1 (β3 insert +PFN21K) 1.206mg/mL, Sample 2 (β3 insert +PFN21K) 1.159mg/mL, Sample 3 (β3 insert +PFN21K) 1.3735mg/mL, Sample 4 (PFN21K-Control) 1.3335mg/mL. Protein concentrations have been obtained which will be analyzed through the SDS-PAGE and further analyzed through Western Blotting. SDS-PAGE and Western Blotting will be used to reassure that we have the appropriate protein size. Obtaining the proteins from the DNA will allow us to further the research by combining the α1 and β in-vitro and studying their interaction. This combination of the proteins will expectantly yield the location of the blockage Beta creates on the Alpha 1 subunit.
References: Catterall WA. Voltage-gated calcium channels. Cold Spring Harb Perspect Biol. 2011 Aug 1;3(8):a003947. doi: 10.1101/cshperspect.a003947. Review. PubMed PMID: 21746798.
Kamatchi GL, Chan CK, Snutch T, Durieux ME, Lynch C 3rd. Volatile anesthetic inhibition of neuronal Ca channel currents expressed in Xenopus oocytes. Brain Res. 1999 Jun 12;831(1-2):85-96. PubMed PMID: 10411986.
Kamatchi GL, Tiwari SN, Durieux ME, Lynch C 3rd. Effects of volatile anesthetics on the direct and indirect protein kinase C-mediated enhancement of alpha1E-type Ca(2+) current in Xenopus oocytes. J Pharmacol Exp Ther. 2000 May;293(2):360-9. PubMed PMID: 10773003.
Rajagopal S, Fang H, Lynch C 3rd, Sando JJ, Kamatchi GL. Effects of isoflurane on the expressed Cav2.2 currents in Xenopus oocytes depend on the activation of protein kinase Cδ and its phosphorylation sites in the Cav2.2α1 subunits. Neuroscience. 2011 May 19;182:232-40. Epub 2011 Mar 21. PubMed PMID: 21402126.
Materon, L. A. (1992). Polymerase Chain Reaction – Xeroxing DNA. Retrieved June 16, 2016, from http://faculty.utpa.edu/lmateron/3401/pcr.html
Muench, D. (n.d.). Background Information. Retrieved June 30, 2016, from http://www.odec.ca/projects/2006/sidh6h2/bg.html
Funder Acknowledgement(s): I thank Ganesan Kamatchi and Brittney K. Burton for the guidance with this experiment. I also thank Zenora Spellman for the opportunity to participate in the research experience. Funding was provided by NSU/HBCU-UP.
Faculty Advisor: Ganesan Kamatchi, firstname.lastname@example.org
Role: I did each part of the experiment which includes PCR Reaction, electrophoresis gel analysis, gel extraction, bacterial transformation(Eschericia coli or E.Coli), transfections (HEK-293 human cells), lysis of mammalian cells, and protein estimations.