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Molecular and functional characterization of an Na + ‐independent choline transporter in rat astrocytes
Author(s) -
Inazu Masato,
Takeda Hiroshi,
Matsumiya Teruhiko
Publication year - 2005
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2005.03299.x
Subject(s) - choline , organic cation transport proteins , chemistry , extracellular , acetylcholine , transporter , biochemistry , ouabain , sodium , biophysics , endocrinology , biology , organic chemistry , gene
In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na + . Na + ‐independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis–Menten constant ( K m ) of 35.7 ± 4.1 µ m and a maximal velocity ( V max ) of 49.1 ± 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na + ‐independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium‐3. The prototypical organic cation tetrahexylammonium (TEA), and other n ‐tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na + ‐independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1‐methyl‐4‐tetrahydropyridinium (MPP + ), clonidine, quinine, quinidine, guanidine, N ‐methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na + ‐independent choline transport system. Corticosterone and 17β‐estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na + ‐independent choline uptake. RT‐PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high‐affinity Na + ‐dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT‐PCR. Furthermore, mRNA for choline transporter‐like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na + ‐independent choline uptake. We conclude that rat astrocytes express an intermediate‐affinity Na + ‐independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.