Biomedical Engineering Reference
In-Depth Information
choline, cimetidine, lidocaine, N-methylnicotinamide, and verapamil. Significant dif-
ferences were seen in
K
m
values, suggesting that residue 503 influences substrate
affinity. These studies nicely illustrate how detailed functional analysis of mem-
brane transporter variants can provide important information regarding transporter
biology.
In some cases, substrate selectivity of genetic variants is assessed indirectly by in-
hibition studies. Four OCT2 variants were analyzed in
Xenopus laevis
oocytes using
the model substrate MPP
+
, and only the Lys432Gln variant had altered transport.
36
In-
terestingly, tetramethylammonium, tetraethylammonium, and tetrapropylammonium
each inhibited the reference OCT2 and the Met165Ile, Ala270Ser, Arg400Cys, and
Lys432Gln variants to a similar degree. In contrast, tetrabutylammonium was a more
potent inhibitor of the Arg400Cys and Lys432Gln variants and a weaker inhibitor of
the Ala270Ser variant than was the reference.
21.3.2. ABC Transporters
Amino acid variants of multiple ABC transporters have been characterized function-
ally in vitro, and many of the results from these studies are summarized in Table 21.1.
The most widely studied ABC transporter is P-glycoprotein (Pgp), which is encoded
by
ABCB1
. There are dozens of polymorphic sites in
ABCB1
that have been iden-
tified through multiple SNP discovery efforts.
8
-
10
Functional analyses of all of the
nonsynonymous variants are slowly accumulating but the most frequently studied
SNP is the triallelic variant at amino acid position 893 that is located in the sixth
intracellular loop near the C-terminus. The reference Ala can change into a com-
mon Ser or a lower frequency Thr.
9
,
10
The Ala-to-Ser variant was first seen in the
drug-resistant cell line MCF-7/Adr, which is a breast cancer cell line that overex-
presses ABCB1.
79
ABCB1 was cloned from MCF-7/Adr and transfected into differ-
ent drug-sensitive cell types. The cells acquired the drug resistance phenotype but no
comparison was made with the reference Ala at codon 893. The functional effects of
the Ala893Ser polymorphism were first examined in a specialized mouse fibroblast
cell line (NIH3T3-GP+E86).
9
In this study the Ser893 Pgp showed enhanced efflux
of the model substrate digoxin. In contrast, analysis of the function of five nonsyn-
onymous variants, including Ala893Ser, in HeLa cells (human cervical cancer cell
line) using seven different substrates showed no differences in transport compared
to the reference Pgp.
80
Two subsequent studies looking at Ala893Ser also concluded
that there was no in vitro functional difference using established Pgp substrates,
such as calcein-AM and verapamil.
10
,
81
It is possible that the varying results for
the Ser893 variant reflect differences between the heterologous expression systems
used in these analyses or result from substrate-dependent effects of this polymor-
phism. Other Pgp variants have been characterized in vitro, including Asn21Asp,
Phe103Leu, Ser400Asn, and Ala998Thr.
80
,
82
,
83
In most cases there were no differ-
ences in function between the reference and variant Pgps; however, Ser400Asn shows
increased resistance to vinblastine and vincristine and decreased transepithelial flux of
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