Molecular mapping of the conductance activity linked to tAE1 expressed in Xenopus oocyte

Abstract

It was previously shown that expressed in Xenopus oocyte the trout (tAE1) and the mouse (mAE1) anion exchangers behave differently: both elicit anion exchange activity but only tAE1 induces a transport of organic solutes correlated with an anion conductance. In order to identify the structural domains involved in the induction of tAE1 channel activity, chimeras have been prepared between mouse and trout AE1. As some constructs were not expressed at the plasma membrane, skate exchanger (skAE1) was used instead of mouse exchanger to complete the structure-function analysis. The present paper shows that skAE1, highly similar to mAE1, does not induce a chloride conductance when expressed in Xenopus oocyte. Construct expression analysis showed that only tAE1 transmembrane domain is linked to the anion conductance. More precisely, we identified two regions composed of helices 6, 7 and 8 and putative helices 12 and 13 which are required for this function.

Consequences of point mutations in trout anion exchanger 1 (tAE1) transmembrane domains: Evidence that tAE1 can behave as a chloride channel

“…This Western blot is representative of different examples. The two bands correspond to the glycosylated form of tAE1 (≈ 120 kDa) and the unprocessed form (core‐glycosylated), which has a theoretical molecular weight of 100 kDa, as shown by deglycosylation experiments (Borgese et al, 2004).…”

“…The aim of this work was to identify amino acids involved in the conductance activity of tAE1. In previous works, we have shown that mouse AE1 (mAE1) and skate AE1 (skAE1) expressed in Xenopus oocyte were exclusively electroneutral exchangers and that conductive properties of tAE1 are confined to the second half transmembrane domain of the exchanger (TM6 to the carboxy terminal end) (Borgese et al, 2004; Guizouarn et al, 2005). Since these proteins are highly similar, an alignment between mAE1, skAE1, and tAE1 amino acid sequences was performed in order to characterize specific tAE1 amino acids not shared by the other two sequences, which might be involved in the conductive property of tAE1 (Fig.…”

The role of disease-linked residue glutamine-913 in support of the structure and function of the human electrogenic sodium/bicarbonate cotransporter NBCe1-A

“…These data indicate that it is the specific introduction of a bulky and/or positively-charged residue at 913 that causes the exhibition of the leak, although we cannot determine which of those two factors are critical due to the limitations of naturally-occurring amino-acid residues with which to substitute. Thus is seems unlikely that the leak pathway is a cryptic feature of WT that could be unveiled in response to pathophysiological stimuli such as cell swelling, as has been described for a Cl − leak that is native to trout AE1 22 , 23 .…”

A conductive pathway generated from fragments of the human red cell anion exchanger AE1

“…Intact tAE1, which retains the putative TM(6: 7) region that we removed from human AE1 to make AE1Δ(6: 7), is ∼51% identical at the amino‐acid level to intact human AE1. Expression of chimeric constructs of tAE1 and mouse AE1, which does not display the large anion conductance, shows that the conductive pathway requires trout‐specific sequences in the third extracellular loop (just before TM6) together with several regions between this loop and the C‐terminus of the molecule (Fievet et al 1995; Borgese et al 2004). In the present work, we show that the physical removal of (6: 7) from human AE1 unmasks a similar pathway, raising the possibility that a conformation change involving (6: 7) in tAE1 enables the appearance of a conductive pathway.…”

“…The tAE1 conductive pathway becomes constitutively active only when tAE1 is heterologously expressed in oocytes (Fievet et al 1995). skAE1 plays a similar role in RVD (reviewed by Perlman & Goldstein, 2004), although the conductive pathway is not manifest as a macroscopic anionic current with expression of skAE1 in Xenopus oocytes (Borgese et al 2004). Macroscopic currents are not a usual feature of human AE1, but a small, partially DIDS‐sensitive conductive pathway detected in red cells has been attributed to the anion exchanger (Knauf et al 1977; Kaplan et al 1983).…”