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Gβγ-dependent modulation of ion channels

Gβγ-dependent modulation of ion channels
Bak, Da Som
DGIST Authors
Bak, Da Som; Suh, Byung Chang; Moon, Sang Jun
Suh, Byung Chang
Moon, Sang Jun
Issue Date
Available Date
Degree Date
2014. 2
Calcium ionpotassium ionGβγ proteinregulationelectrophysiology
Ion channels selectively allow ions to pass in or out of cells and are exquisitely sensitive to modulation by specific G protein-coupled receptors. This gives excitable cells the ability to undergo and regulate dynamic alterations in membrane potential to perform critical biological functions. Specialized voltagegated calcium channels, CaV2.2 (N-type) channels and G protein-coupled potassium 2A channels (GIRK2A) are both regulated by the direct binding of Gβγ protein dissociated from the Gi/o-protein-coupled receptors (Gi/oPCRs). The focus of this study lies within this fast, voltage-dependent, and membrane delimited mechanism of ion channel modulation. In the first section, the possible existence of this pathway following the activation of a GqPCR is explored. Previous studies have demonstrate the distinctpathway of inhibition following GqPCR activation to be mostly due to a slow, voltage-independent, lipiddependent pathway. However the exclusivity of this traditionally believed mechanism is questioned with the possibility of Gβγ involvement. The results clearly defend this hypothesis. A significant reduction in CaVβ2a current inhibition was recorded with cells pre-incubated with PTX and expressing. PTX is a Gi/oPCR and thus Gβγ blocker which suggests Gβγ also has a role in the GqPCR mediated inhibition of CaV2.2 channels. In the second section, the significance of two electrostatic residues recently found to be present within the binding interface of GIRK2A subunits and Gβγ, but not GIRK1 subunits is explored. Using molecular biology techniques, site-directed mutagenesis has been performed to produce two mutations of the GIRK2 channel, E350K (negative to positive) and E358A (negative to hydrophobic). Both these mutations resemble the amino acids present in GIRK1 which have been shown to contribute to the binding interface between the two proteins. This is particularly interesting as GIRK channels are the only channels known to be activated upon Gβγ binding among the large group of potassium ion channels. The reason for this difference is still not well understood. The results illustrate a significant decrease in this GIRK2A mutant current activation which indicates the significance of the conserved residues. Furthermore, GIRK1 is a crucial subunit in physiology, as GIRK channels exist more abundantly as heterotetramers of GIRK1/2 in the brain and GIRK1/4 in the heart. Exploring the detailed mechanism of its activation through the binding of Gβγ is therefore a relevant subject of interest. The results present a distinct decrease in activation extent of the GIRK2 channel mutants relative to that of the wild-type however, change in the activation kinetics was not observed. ⓒ 2014 DGIST
Table Of Contents
Chapter1 -- 1 INTRODUCTION 1 -- 2 MATERIALS AND METHODS 4 -- 2.1 Expression of calcium channels in mammalian cell lines 4 -- 2.1.1 Mammalian cell lines 4 -- 2.1.2 Passage of tsA201 cell Line 4 -- 2.1.3 Lipofectamine transfection 5 -- 2.1.4 Transfer of transfected tsA201 cells to Poly-L-Lysine coated chips 5 -- 2.2 Electrophysiological Recordings 6 -- 2.2.1 Solutions and Materials 6 -- 2.2.2 Whole cell recording 6 -- 2.2.3 Data analysis 8 -- 3 RESULTS AND DISCUSSION 9 -- 3.1 Gi/o and GqPCR inhibition of CaV2.2(β2a) channels 9 -- 3.2 The lipid-dependent component of GPCR mediated inhibition 10 -- 3.3 Effects of PTX on the Gi/oPCR induced inhibition of CaV2.2(β2a) 11 -- 3.4 Effects of PTX on the GqPCR induced inhibition of CaV2.2(β2a) 11 -- 4 FIGURE LEGENDS 13 -- 5 FIGURES 15 -- Chapter2 -- 1 INTRODUCTION 20 -- 2 MATERIALS AND METHODS 23 -- 2.1 Expression of GIRK channels in mammalian cell lines 23 -- 2.1.1 Mammalian cell lines 23 -- 2.1.2 Passage of tsA201 cell Line 23 -- 2.1.3 Lipofectamine transfection 23 -- 2.1.4 Transfer of transfected tsA201 cells to Poly-L-Lysine coated chips 24 -- 2.2 Electrophysiological Recordings 24 -- 2.2.1 Solutions and Materials 24 -- 2.2.2 Whole cell recording 24 -- 2.2.3 Data analysis 25 -- 2.3 Site-directed mutagenesis on wild type GIRK2A channels 25 -- 2.3.1 All-around Polymerase Chain Reaction using Megaprimers 26 -- 2.3.2 Quantification of Nucleic Acids, Gel Electrophoresis, DNA Digest, Purification 26 -- 2.3.3 Heatshock transformation, inoculation and extraction of DNA 27 -- 3 RESULTS AND DISCUSSION 28 -- 3.1 GIRK2A channels activation following M2R activation 28 -- 3.2 GIRK2A channels measured in the presence of PTX 29 -- 3.3 Gi/oPCR activation of GIRK2A mutants 30 -- 4 FIGURE LEGENDS 32 -- 5 FIGURES 34 -- REFERENCES 38 -- ABSTRACT IN KOREAN 41 -- CURRICULUM VITAE 42
Brain Science
Brain and Cognitive SciencesThesesMaster

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