Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes

Abstract

AE2 (SLC4A2) is the member of the Na þ -independent anion exchanger (AE) family putatively involved in the secretion of bicarbonate to bile. In humans, three variants of AE2 mRNA have been described: the full-length transcript AE2a (expressed from the upstream promoter in most tissues), and alternative transcripts AE2b 1 and AE2b 2 (driven from alternate promoter sequences in a tissue-restricted manner, mainly in liver and kidney). These transcripts would result in AE protein isoforms with short N-terminal differences. To ascertain their translation, functionality, and membrane sorting, we constructed expression vectors encoding each AE2 isoform fused to GFP at the C-terminus. Transfected HEK293 cells showed expression of functional GFP-tagged AE2 proteins, all three isoforms displaying comparable AE activities. Primary rat hepatocytes transfected with expression vectors and repolarized in a collagen-sandwich configuration showed a microtubule-dependent apical sorting of each AE2 isoform. This shared apical sorting is liver-cell specific, as sorting of AE2 isoforms was basolateral in control experiments on polarized kidney MDCK cells. Hepatocytic apical targeting of AE2 isoforms suggests that they all may participate in the canalicular secretion of bicarbonate to bile.

Zebrafishslc4a2/ae2anion exchanger: cDNA cloning, mapping, functional characterization, and localization

“…Nonetheless, AE2 expression in the apical membranes of human biliary canaliculi and small bile ducts has been detected with a single IgM monoclonal antibody (35) and has also been suggested by immunogold electron microscopic localization of rat AE2 to the subapical region of cholangiocytes in rat bile duct (57). More recently, apical targeting of human AE2-GFP fusion proteins in biliary canaliculus of polarized primary hepatocytes (6) was shown to contrast to their basolateral targeting in polarized Madin-Darby canine kidney cells (6,12).…”

“…Intron sizes (nt) are presented for zebrafish (Z), mouse (M, upper italicized number), and rat (R, lower number). 6,641 nt encompassing an open reading frame of 1,228 aa ( Supplementary Fig. 1).…”

Molecular physiology and genetics of Na+-independent SLC4 anion exchangers

“…AE2 is a major Cl -/HCO 3 -exchanger of the osteoclast contralacunar membrane Josephsen et al, 2009) and in the lateral membrane of dental secretory ameloblasts (Lyaruu et al, 2008). However, hepatobiliary epithelial cells exhibit apical (Aranda et al, 2004;Martinez-Anso et al, 1994) or subapical (Tietz et al, 2003) immunolocalization of AE2. Apical membrane AE2 was also detected in the ameloblast of the Ae2 a,b -/-mouse (Paine et al, 2008) in unfixed sections, but without the accompanying Ae2 -/-tissue controls included in the study detecting lateral localization in the complete Ae2 knockout mouse (Lyaruu et al, 2008).…”

Inefficient Chronic Activation of Parietal Cells in Ae2−/− Mice

“…The 5Ј diversity of these messages leads to small changes, the initial 17 amino acids of 1237 residues of Ae2a being replaced by three residues (MTQ) in Ae2b1 and by eight residues (MDFLLRPQ) in Ae2b2. The pattern of alternative transcription from intron 2 is conserved between mouse, rat, and humans, 18 although expression of variants type b in humans seems to be more tissue-specific than in rodent species. 17,19,20 In mouse and rat, but not in humans, 19 additional alternative exons 1c 1 and 1c 2 are both transcribed from overlapping sequences in intron 5 (with some differences between these species).…”