Premium
Construction of Fluorescently Tagged Proteins to Determine Potential Interactions in a Non‐native Cellular System
Author(s) -
Soska Mallory A,
Sheridan David C
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.791.18
Subject(s) - hek 293 cells , transfection , glucocorticoid receptor , depolarization , microbiology and biotechnology , voltage dependent calcium channel , receptor , green fluorescent protein , gene isoform , calcium , biology , chemistry , cell culture , biophysics , biochemistry , gene , genetics , organic chemistry
L‐type calcium channels couple membrane depolarization to muscle contraction and aid in signal conduction between cells. Cav1.3 and Cav1.2 are calcium channel isoforms that are expressed in neurons and endocrine cells. Nervous and endocrine tissues also express both Glucocorticoid (GR) and Mineralocorticoid receptors (MR) that are responsible for binding serum cortisol to induce immediate and long term changes within the cell. While neurons co‐express both GRs and MRs, GR activation occurs in response to high concentrations of cortisol. There is a direct correlation between GR activation and the amplitude of calcium currents (Champeau, 2007). To determine potential interactions between the GR and the calcium channel isoforms, we will co‐express a GFP‐tagged GR, purchased from Addgene, and a mRubyC1‐tagged L‐type calcium channel in a simplified, non‐native cellular system, Human Embryonic Kidney (HEK) cells. Specifically targeted primers were designed to clone the Cav1.3 sequence in preparation for its insertion into the mRubyC1 red fluorescent vector. The Cav1.2+mRubyC1 and Cav1.3+mRubyC1 constructs will then be separately transfected into HEK cells expressing GFP‐tagged GR. Injection of a high concentration of cortisol into the cellular environment will activate the GR receptor and fluorescence capture imaging will determine if there is an in vitro interaction between Cav1.2 and GR and / or Cav1.3 and GR. Support or Funding Information Otterbein University Student Research Fund Bert and Jane Horn Endowed Research Award This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .