Cell-based homologous expression system for in-vitro characterization of
environmental effects on transmembrane peptide transport in fish.
All organisms encounter environmental changes that lead to physiological
adjustments and drive evolutionary adaptations. These, in turn, induce
behavioral, physiological and molecular changes that affect each other.
Deciphering the role of molecular adjustments in physiological changes
will help to understand how multiple levels of biological organization
are synchronized during adaptations. Transmembrane transporters are
prime targets for molecular studies of environmental effects, as they
facilitate the ability of cells to interact with the external
surrounding. Fish are subjected to fluctuations of environmental factors
of their aquatic surrounding and exhibit different coping mechanisms. To
study the molecular adjustments of fish proteins to their unique
external surrounding, suitable experimental systems must be established.
Mozambique tilapia (Oreochromis mossambicus) is an excellent model for
environmental stress studies due to its extreme osmotolerance. We
established a homologues cellular-based expression system, and an uptake
assay, that allowed us to study effects of environmental conditions on
transmembrane transport. We applied it to study the effects of
environmental conditions on the activity of PepT2, a widely studied
transporter due to its importance in absorption of dietary peptides and
drugs. We created a stable, modified fish cell-line, exogenously
expressing the tilapia PepT2 and tested the effects of temperature and
water salinity on the uptake of fluorescent di-peptide, β-Ala-Lys-AMCA.
While temperature affected the Vmax of the transport, salinity affected
both the Vmax and the Km. These assays demonstrate the importance of
suitable experimental systems for fish ecophysiology studies. The
presented tools and methods can be adapted to study other transporters