After HFE associated hemochromatosis mutations in SLC40A1, encoding the only known cellular iron exporter ferroportin, are the commonest cause
of genetic iron overload. Patients with mainly private SLC40A1 mutations can present
with distinct phenotypes. Loss of iron export function mutations have been associated
with the ferroportin disease phenotype whereas the hemochromatosis type 4 phenotype
is associated with gain of function mutations, where specific mutation render ferroportin
constitutively active and resistant against hepcidin mediated degradation.
However, the molecular disease mechanism of ferroportin disease and hemochromatosis
type 4 has only been reported for a subset of disease-associated mutations and limited
information on genotype-phenotype correlation exists. Recent structural information
from bacterial homologues of SLC40A1 and modelling studies suggest that the aspartate residue at position 157 is exposed
at the intracellular side of of ferroportin's iron entry channel and could therefore
be crucial for its export function. The aim of the present study was to determine
the functional consequence of D157 mutations on the iron export function and hepcidin
responsiveness of ferroportin. Residue 157 was mutated to histidine, glutamine, tyrosine
or glycine using site-directed mutagenesis and the cDNA was cloned into a mammalian
expression vector as N-terminal fusion protein with GFP and mCherry. The identity
of the constructs was confirmed by Sanger sequencing. To create an authentic disease
model of autosomal dominant SLC40A1 mutation, HEK293T cells were cotransfected with
the normal and mutant expression vectors fused to GFP and mCherry. Hepcidin responsiveness
and iron export were investigated in cells overexpressing wild type and mutant ferroportin
using flow cytometry and ferritin ELISA.
In conclusion, this overexpression model based on a dual fluorescent protein reporter
readout offers a versatile tool to study molecular disease mechanisms of autosomal
dominant transporter defects including heterozygous SLC40A1 mutations, but could also
be used for studying other autosomal dominant gastrointestinal channelopathies.
