Glycine transporter 1 associates with cholesterol-rich membrane raft microdomains
Section snippets
Materials and methods
Materials. DiIC16 and cell culture media and solutions were supplied by Invitrogen, Australia (Mt Waverly, VIC, Australia) unless otherwise stated. Chinese hamster ovary (CHO) cells stably transfected with human GLYT1 and GLYT2 were a gift from John Morrow at Organon Laboratories Ltd. (Cambridge, England). All other chemicals were obtained from Sigma–Aldrich (Castle Hill, NSW, Australia) unless otherwise stated.
Plasmid construction and preparation of RNA. Stop codons of GLYT1-pcDNA3.1 and
Depletion of membrane cholesterol affects the transport activity of GLYTs
3H-glycine uptake by CHO-K1 cells stably expressing GLYT1 and GLYT2 was examined in the presence and absence of the GLYT inhibitors, NFPS and ALX1393 to confirm the pharmacological profile of the transporters. One μM NFPS completely blocked 3H-glycine uptake by GLYT1 expressing cells and 1 μM ALX1393 inhibited 3H-glycine uptake by GLYT2 expressing cells by 83 ± 1%. To investigate the role of lipid rafts on glycine transporter function, we treated the cells with 1 mM methyl-β-cyclodextrin (MCD) for
Discussion
Disruption of membrane rafts using MCD reduced the maximal glycine transport currents by GLYT1 by 40 ± 1% (n = 5) in Xenopus oocytes, and the 3H-glycine uptake level of CHO-K1 cells stably expressing GLYT1 by 36 ± 4% (n = 5). This data suggests that GLYT1 function may require membrane raft location for optimal transport activity in X. laevis oocytes and CHO-K1 cells.
MCD has been widely used as a potent carrier for water insoluble cholesterol. Although MCD is water soluble, it has a hydrophobic cavity,
Acknowledgments
We thank Ms. Cheryl Handford for assistance with cell culture, Ken Wyse, Gracia Quek for frog surgeries and maintenance of the Xenopus laevis colony. A.D.M is supported by a Rolf Edgar Lake Postdoctoral Fellowship from The University of Sydney, R.J.V. is supported by a NHMRC Senior Research Fellowship and this work was supported by NHMRC project Grant 402564.
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