Bharat G Reddy1, Qun Dai2, Carmel M McNicholas3, Catherine M Fuller4, John C Kappes5, Lawrence J DeLucas6. 1. Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States. 2. Dept. of Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Dept. of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States. 3. Dept. of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States. 4. Dept. of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States. 5. Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Dept. of Biochemistry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Dept. of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, AL 35233, United States. 6. Dept. of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, United States; Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address: duke2@uab.edu.
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.
The epithelial sodium channel (n class="Gene">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.
Authors: I I Ismailov; M S Awayda; B K Berdiev; J K Bubien; J E Lucas; C M Fuller; D J Benos Journal: J Biol Chem Date: 1996-01-12 Impact factor: 5.157