Structure and Function of Potassium Chloride Cotransporter 3 Open Access

Ahn, Hyunjun (Spring 2021)

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Potassium Chloride Cotransporters (KCC) mediates electroneutral transport of one chloride ion with a potassium ion that is involved in cell volume regulation, salt reabsorption, and maintaining ion concentration across the cell membrane. As important as its physiological functions are, the exact molecular mechanism of this protein has been limited due to the lack of high-resolution 3-D structures of these proteins. To investigate the structure and the function of the protein, cryogenic electron microscopy (cryo-EM) images of the KCC3 proteins were obtained in an attempt to elucidate its structure. Here, we use negative stain and cryogenic electron microscopy to characterize the general molecular shape and the architecture of the protein. We discovered that KCC3 is in dimeric form, comparable to the previously published KCC1 structure. We also demonstrate that the cryo-EM images are much more sensitive to the type of detergents and polymers used to stabilize the membrane protein than the negative stain EM images. Furthermore, we established an optimized transient PEI-based transfection to obtain the higher yield of a membrane protein using suspension HEK293F cells. Overall, this study can serve as a groundwork for the expression and purification of other membrane proteins for structural and functional investigations. 

Table of Contents

Table of Contents

Introduction _________________________________________________________________________________ 1

     Membrane Protein Structural Studies ___________________________________________________ 3

     Traditional Detergents __________________________________________________________________ 4

     Amphipols  ______________________________________________________________________________ 5

     Nanodiscs   ______________________________________________________________________________ 6

     Fab  ______________________________________________________________________________________6

     Tag designs _____________________________________________________________________________ 7

     PEI Transfection ________________________________________________________________________ 8

Methods ___________________________________________________________________________________ 8

    Transformation __________________________________________________________________________ 8

    Plasmid Purification _____________________________________________________________________ 9

N-MBP-PPX-KCC3 Protein Expression Using Baculovirus ____________________________ 10

N-10x His tag-KCC3 Protein Expression Using PEI-based Transfection Method 1 ___ 10

N-10x His tag-KCC3 Protein Expression Using PEI-based Transfection Method 2 ___ 11

N-MBP-PPX-KCC3 Protein Purification _______________________________________________ 11

N-10x His tag-KCC3 Protein Purification _____________________________________________ 12

Nanodisc and Amphipol Reconstitution _______________________________________________ 12

Negative Stain Electron Microscopy Grid Preparation ________________________________ 13

Cryo Electron Microscopy Grid Preparation ___________________________________________ 13

Results ____________________________________________________________________________________ 14

Protein Expression and Purification of N-MBP-PPX-KCC3 ____________________________ 14

Negative Stain Images of KCC3 ________________________________________________________ 16

Cryo Grid Screening Images of KCC3 ___________________________________________________ 18

Cryogenic Electron Microscopy Image Processing ______________________________________ 23

Transformation and Plasmid purification of N-10x His tag-KCC3 ______________________ 25

PEI-based Transfection __________________________________________________________________ 26

Two Additional Studies Using Similar Techniques __________________________________________ 34

    Gel Filtration Profile and Negative Stain EM to Characterize the Protein _______________ 34

    High Resolution Structure Involving His-tag Purification and Protease Cleavage _______ 40

Discussions ___________________________________________________________________________________ 41

PEI-based transfection of N-10x his tag-KCC3 ____________________________________________ 41

N-Terminus Purificaiton Tag _______________________________________________________________ 41

General architecture and electron microscope images of N-MBP-PPX-KCC3b ___________ 42

Known Structural Insights into KCC Families ________________________________________________ 44

Structural Analysis of KCC1 ________________________________________________________________ 44

Structural Analysis of Monomeric KCC4 ____________________________________________________ 45

Structural Analysis of Dimeric KCC2, KCC3, and KCC4 ____________________________________ 46

Conclusions ___________________________________________________________________________________ 49

Supplemental Tables __________________________________________________________________________ 50

Table 1: Table Summary of Purification Tags to Different Membrane Protein for Structural Studies

Table 2: Table Summary of Buffer Conditions of Different KCC for Cryo-EM studies

Supplemental Figures __________________________________________________________________________ 55

Supplemental Figure 1: Proteins Encoded by SLC12 Gene

Supplemental Figure 2: General Workflow of Cryo-EM Image processing

Supplemental Figure 3: General Workflow of Membrane Protein Purification

Supplemental Figure 4: Comparison Between Linear and Conformational Epitope

Supplemental Figure 5: Carton Representation of Protein in Cryo-EM Grids

Supplemental Figure 6: Example Image of Particle Picking of Cryo Grid Images

Supplemental Figure 7: 2D Classifications of KCC3

Supplemental Figure 8: Gel Electrophoresis Image of the Plasmid

Supplemental Figure 9: Small Volume Expression Test

Supplemental Figure 10: Small Volume Expression Test After VPA and SB

Supplemental Figure 11: Cryo-EM Images of Potassium-Chloride Cotransporters

Supplemental Figure 12: Ion Binding Sites of KCC4

Supplemental Figure 13: N-terminal Peptide of KCC2

Supplemental Figure 14 : Hydrophobic Interactions Involved in KCC Dimerization

References _________________________________________________________________ 67




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