Abstract
The plasma membrane monoamine transporter (PMAT) and organic cation transporter 3 (OCT3) are the two most prominent low-affinity, high-capacity (i.e., uptake2) transporters for endogenous biogenic amines. Using the Flp-in system, we expressed human PMAT (hPMAT) and human OCT3 (hOCT3) at similar levels in human embryonic kidney 293 cells. Parallel and detailed kinetics analysis revealed distinct and seemingly complementary patterns for the two transporters in transporting monoamine neurotransmitters. hPMAT is highly selective toward serotonin (5-HT) and dopamine, with the rank order of transport efficiency (Vmax/Km) being: dopamine, 5-HT ≫ histamine, norepinephrine, epinephrine. The substrate preference of hPMAT toward these amines is substantially driven by large (up to 15-fold) distinctions in its apparent binding affinities (Km). In contrast, hOCT3 is less selective than hPMAT toward the monoamines, and the Vmax/Km rank order for hOCT3 is: histamine > norepinephrine, epinephrine > dopamine >5-HT. It is noteworthy that hOCT3 demonstrated comparable (≤2-fold difference) Km toward all amines, and distinctions in Vmax played an important role in determining its differential transport efficiency toward the monoamines. Real-time reverse transcription-polymerase chain reaction revealed that hPMAT is expressed at much higher levels than hOCT3 in most human brain areas, whereas hOCT3 is selectively and highly expressed in adrenal gland and skeletal muscle. Our results suggest that hOCT3 represents a major uptake2 transporter for histamine, epinephrine, and norepinephrine. hPMAT, on the other hand, is a major uptake2 transporter for 5-HT and dopamine and may play a more important role in transporting these two neurotransmitters in the central nervous system.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medicine Sciences [Grants GM066233, GM066233-07S1].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.170142.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- 5-HT
- serotonin
- SERT
- 5-HT transporter
- PMAT
- plasma membrane monoamine transporter
- hPMAT
- human PMAT
- CNS
- central nervous system
- SLC
- solute carrier family
- D22
- decynium-22 (1,1′-diethyl-2,2′-cyanine)
- DAT
- dopamine transporter
- GUSB
- β-glucuronidase
- MPP+
- 1-methyl-4-phenylpyridinium
- NET
- norepinephrine transporter
- OCT
- organic cation transporter
- hOCT
- human OCT
- TEA
- tetraethylammonium
- HEK
- human embryonic kidney
- RT-PCR
- reverse transcription-polymerase chain reaction
- FBS
- fetal bovine serum
- DPBS
- Dulbecco's phosphate-buffered saline
- GBR12909
- 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine
- FRT
- flippase recognition target.
- Received May 7, 2010.
- Accepted September 20, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|