Expression and purification of the alpha subunit of the epithelial sodium channel, ENaC

Abstract

The epithelial sodium channel (ENaC) plays a critical role in maintaining Na(+) homeostasis in various tissues throughout the body. An understanding of the structure of the ENaC subunits has been developed from homology modeling based on the related acid sensing ion channel 1 (ASIC1) protein structure, as well as electrophysiological approaches. However, ENaC has several notable functional differences compared to ASIC1, thereby providing justification for determination of its three-dimensional structure. Unfortunately, this goal remains elusive due to several experimental challenges. Of the subunits that comprise a physiological hetero-trimeric αβγENaC, the α-subunit is unique in that it is capable of forming a homo-trimeric structure that conducts Na(+) ions. Despite functional and structural interest in αENaC, a key factor complicating structural studies has been its interaction with multiple other proteins, disrupting its homogeneity. In order to address this issue, a novel protocol was used to reduce the number of proteins that associate and co-purify with αENaC. In this study, we describe a novel expression system coupled with a two-step affinity purification approach using NiNTA, followed by a GFP antibody column as a rapid procedure to improve the purity and yield of rat αENaC.

Keywords: ENaC; Epithelial sodium channel; GFP; GFP Affinity Column; Membrane Protein Expression; Membrane Protein Purification.

Figure 1. αrENaC Construct Design and Expression

(top) The overall genetic design of the lentiviral vector is depicted. The abbreviations are as follow: LTR, long terminal repeats; ψ, Psi packaging element; RRE, Rev response element; cts, central termination sequence; ppt, polypurine tract; TRE, Tet response element; TEV, tocabo etch virus; GFP, enhanced green fluorescent protein; His, 10x histidine tag; Ubq, ubiquitin promoter; Puro, puromycin resistant gene; wpre, woodchuck hepatitis virus posttranscriptional regulatory element. (bottom) Image of doxycycline induced αrENaC expression in suspension CHO cells obtained with a Zeiss Laser Scanning 710 Confocal Microscope showing a bright field image overlaid with an eGFP image.

Figure 2. αrENaC solubilization with DDM

(a) Whole suspension CHO cell sonicated lysates were solubilized with DDM and were injected on a FSEC running a Superose 6 10/30 column (blue). Additionally, a control was run with whole suspension non-transduced CHO cell sonicated lysate (grey). Fractions corresponding to the color bars below the FSEC peaks were run on SDS PAGE and analyzed for eGFP fluorescence with the Syngene G:BOX Chemi XT4(b). The size markers on the FSEC chromatogram correspond to running the following purified protein samples to observe retention time on the column: Blue Dextran (2000kDa), Thyroglobulin (669kDa), Ferritin (440kDa), R-phycoerytherin (240kDa), Fluoroscene Fab (100kDa), and Free-GFP (28kDa). The S on the SDS PAGE represents the solubilized lysates injected into the FSEC.

Figure 3. FSEC of Differential Cell Lysis and Solubilization of αrENaC with DDM

FSEC running a Superose 6 10/30 column injected with the whole suspension CHO cell sonicated and solubilized αrENaC with DDM (blue), whole suspension CHO cell solubilized αrENaC with DDM on a stirrer (red), and isolated CHO cell membrane using nitrogen decompression and solubilized αrENaC with DDM (orange).

Figure 4. Salt Anions and αrENaC Stoichiometry

FSEC running a Superose 6 10/30 column injected with whole suspension αrENaC CHO cell sonicated and DDM solubilized lysate with DDM using a NaCl based buffer (blue) and a NaBr based buffer (purple).

Figure 5. αrENaC Purification

(a) 4–20% gradient SDS PAGE stained with Coomassie Blue after concentrating the NiNTA elutions with NaCl based buffers on the left and a 7.5% SDS PAGE stained with Coomassie Blue after concentrating the NiNTA elutions with NaBr based buffers on the right. On the NaBr gel, both αrENaC and actin were verified via mass spectrometry on the cut gel bands. The gels were imaged on a desktop scanner. (b) Concentrated NiNTA elutions were digested with PNGase, TEV, or both were run on a 7.5% SDS PAGE stained with Coomassie Blue on the left. The Coomassie stained gel was imaged with an Li-Cor Odyssey imaging system which reads the infrared signal from Coomassie and the eGFP fluorescence was imaged with a Syngene G:BOX Chemi XT4 with eGFP filters. (c) αrENaC after all purification steps including the GFP antibody column and SEC was run on a 4–20% gradient SDS PAGE and stained with Coomassie (left) or run on a western blot probed with an anti-αENaC ab using ECL on the Syngene G:BOX Chemi XT4 (right).