A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line

D C Soler,A Engel,T S Mccormick,T Kowatz,R Stepanyan,A Vahedi-Faridi,K D Cooper,A E Sloan

doi:10.1038/s41598-021-93117-8

D C Soler, A Engel + Show 6 more

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https://doi.org/10.1038/s41598-021-93117-8

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Abstract

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194–213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

Highlights

Aquaporins are proteins composed of six transmembrane α-helices that form a single water-permeable channel
Each C-terminally attached to Aquaporin 3-turbo GFP (tGFP) Reporter (AGR), we identified the amino acid sequence PARSFGPAVIVGKFAVHWIF as the one responsible for endoplasmic reticulum (ER) retention (Fig. 4B–E)
The aquaglyceroporin subtype Aquaporin 6 (AQP6) has been known for quite some time to be very difficult to express in heterologous s ystems[18]

Summary

Introduction

Aquaporins are proteins composed of six transmembrane α-helices that form a single water-permeable channel. A single amino acid substitution of asparagine by glycine at position 60 (N60G) eliminates AQP6 anion permeability completely when expressed in Xenopus oocytes[11]. This occurs with a concomitant increase of water permeability, similar to AQP2, which is not inhibited by H gCl2. Similar N60G mutations in other aquaporins, such as AQP0, AQP1 and AQP2 result instead in a failure of the protein to traffic to the plasma membrane This strongly suggests that the interaction of Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106‐4965, USA. Heterologous expression of AQP6 would ideally be carried out in a system which has the ability for post-translational modifications

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