Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C026

Oral Communications

Trafficking, localization and degradation of the Na+, HCO3- co-transporter NBCn1 in breast cancer and kidney epithelial cells

C. W. Olesen1, J. Vogensen1, I. Axholm1, M. Severin1, I. S. Pedersen1, J. von Stemann1, J. M. Schroeder1, D. P. Christensen1, S. F. Pedersen1

1. Department of Biology, University of Copenhagen, Copenhagen O, Denmark.

The Na+, HCO3- co-transporter NBCn1 (SLC4A7) is a major physiological regulator of intracellular pH (1). Consequently, NBCn1 dysfunction is linked to human diseases such as cardiovascular disease and cancer. Specifically, it has been reported that some human breast cancers exhibit increased NBCn1 plasma membrane expression and NBCn1 knockout reduces breast tumor development in mice. NBCn1 trafficking and turnover, however, are essentially unstudied. The aim of this work was to delineate mechanisms of NBCn1 trafficking in epithelial cells. Polarized or non-polarized epithelial MCF-7 and MDCK-II cells were cultured under standard cell culture conditions (5% CO2, 95% humidity and 37°C). By confocal microscopy, endogenous NBCn1 and exogenous GFP-NBCn1 (N-terminally GFP-coupled rat NBCn1-D) were consistently found in the basolateral membrane (n=3-5) (2). In congruence with this, endogenous NBCn1 co-immunoprecipitated with the cell polarity protein lethal giant larvae (LLGL1). GFP-NBCn1 membrane localization was abolished by deletion of the full NBCn1 C-terminal tail (C-tail) but unchanged following mutation or deletion of a putative C-terminal PDZ-binding motif in the most distal part of the C-tail (n=3). Glutathione-S-Transferase-pulldown of the C-tail followed by mass spectrometry analysis revealed putative interactions with multiple sorting-, degradation- and retention factors, including the scaffolding protein RACK1 (2). Pulldown of FLAG-tagged deletion constructs mapped the RACK1 interaction to the proximal portion of the NBCn1 C-tail (n=3). Native NBCn1 and RACK1 interaction was confirmed in a cellular context by Proximity Ligation Assay and co-immunoprecipitation assays (n=4). RACK1 knockdown reduced NBCn1 membrane localization without affecting total NBCn1 expression (p<0.05, n=3) arguing for a role of RACK1 in NBCn1 trafficking. NBCn1-RACK1 plasma membrane co-localization (p<0.05, n=3) was only present in non-confluent cells, consistent with the notion that RACK1 regulates the trafficking of NBCn1 to the membrane (2). One-third of surface NBCn1 was constitutively endocytosed from the basolateral membrane within 60 min (p<0.05, n=3) while total NBCn1 degradation, predominantly by a lysosomal mechanism, was slow (half-life >24h; n=3). Together these data suggest that a fraction of NBCn1 exhibits recycling between the basolateral membrane and intracellular compartment(s). In support of this idea, preliminary data show NBCn1 co-immunoprecipitation with the recycling regulating transport protein Rab4 and that NBCn1 endocytosis partly depends on activity of the vesicle scission GTPase dynamin. In conclusion our findings shed new light on the trafficking, localization and degradation of NBCn1 in epithelial cells and contribute to a broader understanding of NBCN1 regulation in human physiology.

Where applicable, experiments conform with Society ethical requirements