ORGANIC CATION TRANSPORTERS - Drug Transporters: Molecular Characterization and Role in Drug Disposition - page 22

additional studies have shown that the majority of this effect is due to activation of
phospatidylinositol 3-kinase (PI3K), as inhibition of PI3K almost completely prevents
the inhibitory effect of carbachol on OCT2-mediated transport. These studies suggest
that PKA, PI3K, and the Ca 2 + -calmodulin complex are endogenous regulators of
OCT2; however, in contrast to the effect of protein kinases on OCT1, the inhibition of
transport activity is not explained by effects on substrate-binding affinity. It is there-
fore thought that these proteins regulate other aspects of OCT2, such as maturation
and trafficking of OCT2 to the plasma membrane, or stimulation of endocytosis. 54
Glycosylation of OCT2 may be an additional mechanism for regulation of organic
cation transport. The rabbit OCT2 isoform was shown to contain three N-glycosylation
sites (Asn71, Asn96, and Asn112), each of which was found to be necessary for op-
timal transport activity by mutational analysis. 59 The Asn112Gln mutant resulted in
a significant decrease in membrane expression (to approximately 20% of wild-type)
in stably transfected Chinese hamster ovary (CHO) cells. Asn71Gln and Asn96Gln
did not alter OCT2 surface expression, but the unglycosylated triple mutant
(Asn71Gln/Asn96Gln/Asn112Gln) was retained intracellularly. Affinity for TEA was
increased approximately twofold by mutation of each of the three glycosylation sites.
Maximal transport rate was decreased fivefold by the Asn112Gln mutation (explained
by the reduced surface expression of Asn112Gln) and threefold by Asn96Gln (pre-
sumably, due to a lower transporter turnover number). Thus, N-glycosylation may
regulate maturation and trafficking of OCT2 as well as substrate recognition and
transporter turnover.
There is also evidence for hormonal regulation of OCT2. Functional studies of rat
renal cortical slices showed higher basolateral transport of TEA in slices from male
rats versus female rats, and rOCT2 mRNA and protein expression was also shown to
be higher in male rats than in female rats. 60 Further, treatment of rats with testosterone
increased rOCT2 and protein expression in the kidney of both male and female rats. 61
In Madin-Darby canine kidney (MDCK) cells, treatment with dexamethasone, hydro-
cortisone, or testosterone increased both OCT2 mRNA and protein levels as well as
uptake of TEA across the basolateral membrane. 62 Conversely, treatment with estra-
diol led to decreased OCT2 expression and activity in male rats and in MDCK cells.
The physiological significance of hormonal regulation of OCT2 is unclear but may
be related to gender-specific differences in clearance of endogenous metabolites. 32
2.5.3. OCT3
Although OCT3 contains several conserved target sequences for PKA, PKC, and
PKG, studies of heterologously expressed OCT3 have shown that activation of these
pathways has no apparent effect on OCT3 activity. 63 However, inhibition of the p44/42
mitogen-activated protein kinase (MAPK) has been shown to decrease MPP + trans-
port by OCT3, suggesting that MAPK is a positive regulator of OCT3. Similarly,
inhibition of calmodulin as well as of the downstream effector CaMKII results in
decreased OCT3 activity. Selective inhibition of the calmodulin-dependent phospho-
diesterase PDE1 also results in decreased OCT3 activity, suggesting PDE1 stimulation
as an additional mechanism for Ca 2 + - and calmodulin-dependent regulation of OCT3.