Premium
Comparing Lipid and Detergent Environments as Models for Studying Renal Transmembrane Proteins
Author(s) -
Pavkov Isabela,
Strickland Kerry,
Cottrill Kirsten,
Imhoff Barry R.,
McCarty Nael A.
Publication year - 2020
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.2020.34.s1.09662
Subject(s) - popc , nanodisc , transmembrane protein , membrane protein , lipid bilayer , chemistry , biochemistry , atpase , biophysics , membrane , biology , receptor , enzyme
Background Mutations in transmembrane proteins cause kidney diseases such as Dent’s disease, Bartter syndrome, and polycystic kidney disease. 1 Understanding the structure and function of renal membrane proteins reveals how they contribute to kidney function as well as kidney disease. Recent studies have shown the importance of generating a native lipid environment when studying membrane proteins. 2 However, membrane proteins are often studied in the presence of detergent, which may modify their natural structure and lead to decreased function. Nanodiscs, a novel lipid bilayer system, provide a more native environment for protein reconstitution. Methods In this study we used two proteins found in the kidney, the Na + /K + ‐ATPase pump and CFTR ion channel, to determine whether nanodiscs or detergent micelles serve as the better model for studying renal transmembrane proteins. To do this, we expressed and purified our own Membrane Scaffold Protein (MSP), a base protein for nanodiscs, in Escherichia coli. We generated empty nanodiscs with three different lipid types: 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC), and 1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE). Results We have and are continuing to reconstitute CFTR and Na + /K + ‐ATPase into nanodiscs with different lipid compositions. We evaluate membrane protein stability using ATPase activity in detergent and different lipid nanodisc environments. Preliminary data show improved ATPase activity for CFTR in nanodiscs versus detergent. Higher ATPase activity of proteins reconstituted in nanodiscs would indicate that detergent structurally modifies these proteins and consequently decreases their function. Nanodiscs provide a more stable environment in which to study these and other proteins found in the kidneys, allowing for more accurate assessment of structure and function.1 Kuo IY , Ehrlich BE : Ion Channels in Renal Disease . Chemical Reviews 112 : 6353 – 6372 , 20122 Taylor NMI , Manolaridis I , Jackson SM , Kowal J , Stahlberg H , Locher KP : Structure of the human multidrug transporter ABCG2 . Nature 546 : 504 – 509 , 2017