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Exploring an autoinhibitory domain in the electrogenic Na/HCO 3 transporter NBCe1‐B
Author(s) -
Lee SeongKi,
Boron Walter F.
Publication year - 2016
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.30.1_supplement.971.3
Subject(s) - chemistry , cotransporter , xenopus , transporter , homeostasis , biochemistry , biology , microbiology and biotechnology , gene , sodium , organic chemistry
The SoLute Carrier (SLC) superfamily of membrane transport proteins comprises over 300 genes organized into 52 families. One of the SLC groups is the SLC4 family, the members of which mainly transport HCO 3 − , or a species related to HCO 3 − (e.g., CO 3 = ), together with another “coupled” ion across the plasma membrane. Here we study a member of the SLC4 family, the electrogenic Na/HCO 3 cotransporter NBCe1 (SLC4A4). This transporter is widely expressed in cells throughout the body. NBCe1 has 5 known splice variants (A—E). A—C variants have been extensively studied. Whereas NBCe1‐A (predominantly expressed in the kidney) is very fast, NBCe1‐B (expressed throughout the body) and NBCe1‐C (mainly expressed in the brain) are very slow. The critical determinant of speed is the extreme NH 2 ‐terminus (eNt), which in NBCe1‐A comprises the first 41 amino acids (aa). In NBCe1‐B and NBCe1‐C, this cassette is replaced by a cassette of 85 aa. Truncation of the first 85 aa of NBCe1‐B/C enhances activity. Thus, it has been suggested that this 85aa cassette in NBCe1‐B/C includes an autoinhibitory domain (AID). Recently, Shcheynikov et al. (PNAS, 2015) showed that removing 23 aa between residues 40 and 62 stimulates NBCe1‐B. The deleted region contains a unique cluster (residues 40–48) of 9 consecutive positively charged residues. We hypothesize that this positively charged cluster normally inhibits NBCe1‐B. We assess NBCe1 activity by two‐electrode voltage‐clamping of Xenopus oocytes expressing NBCe1‐B constructs in which we systematically mutated or deleted elements of the cluster. We also deleted 3 to 9 aa just before the cluster. All constructs traffic well to the plasma membrane. We show that [1] removing or mutating the cluster of positive charges robustly stimulates NBCe1‐B activity (i.e., relieve autoinhibition), whereas [2] deleting aa before the cluster does not. Our findings suggest that positively charged residues between residues 40 and 48 of NBCe1‐B are a necessary component of the AID, whereas the preceding 9 residues are not a necessary part of the AID.