Glycine transporter 1 associates with cholesterol-rich membrane raft microdomains

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Abstract

Membrane rafts, the highly-ordered, cholesterol-rich microdomains of the plasma membrane play important roles in cellular functions. In this study, GLYT1-CFP and GLYT2-CFP were constructed, followed by investigation of whether the tagged transporters associate with a fluorescence probe that labels membrane rafts (DilC16) by using Fluorescence Resonance Energy Transfer. A close association was observed between DiIC16 and GLYT1-CFP, but not for GLYT2-CFP. The glycine transport ability of GLYT1 is also highly dependent on the integrity of this area. Together, the results suggest that GLYT1 and membrane rafts are co-localized in the membrane, and that this influences the rate of glycine transport.

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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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