Tài liệu Báo cáo khoa học: The role of N-glycosylation in the stability, trafficking and GABA-uptake

The role of N-glycosylation in the stability, trafficking and

GABA-uptake of GABA-transporter 1

Terminal N-glycans facilitate efficient GABA-uptake activity

of the GABA transporter

Guoqiang Cai1,2, Petrus S. Salonikidis3

, Jian Fei1

, Wolfgang Schwarz3

, Ralf Schu¨ lein4

,

Werner Reutter2 and Hua Fan2

1 Institute of Biochemistry and Cell Biology, SIBS, CAS, Shanghai, China

2 Institut fu¨r Molekularbiologie und Biochemie, CBF, Charite´ Universita¨tsmedizin Berlin, Berlin-Dahlem, Germany

3 Max-Planck Institut fu¨r Biophysik, Frankfurt, Germany

4 Forschungsinstitut fu¨r Molekulare Pharmakologie, Berlin-Buch, Germany

The cellular membrane transporter for the inhibitory

neurotransmitter c-aminobutyric acid (GABA) belongs

to a family of secondary active systems that are driven

by electrochemica1 gradients of Na+ and Cl– [1]. The

main physiological function of the transporter is

believed to be the control of the concentration and

dwell time of GABA in the synaptic cleft. Because the

transport of one molecule of GABA is coupled to the

Keywords

GABA transporter; N-glycosylation; N-glycan

trimming; membrane trafficking; patch￾clamp

Correspondence

H. Fan, Institut fu¨r Molekularbiologie und

Biochemie, Campus Bejamin Franklin,

Charite´ Universita¨tsmedicin Berlin,

Arnimallee 22, D-14195 Berlin-Dahlem,

Germany

Fax: +49 30 84451541

Tel: +49 30 84451544

E-mail: [email protected]

(Received 17 July 2004, revised 24 January

2005, accepted 2 February 2005)

doi:10.1111/j.1742-4658.2005.04595.x

Neurotransmitter transporters play a major role in achieving low concen￾trations of their respective transmitter in the synaptic cleft. The GABA

transporter GAT1 belongs to the family of Na+- and Cl–

-coupled trans￾port proteins which possess 12 putative transmembrane domains and three

N-glycosylation sites in the extracellular loop between transmembrane

domain 3 and 4. To study the significance of N-glycosylation, green fluor￾escence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation

defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO

cells. Compared with the wild type, all N-glycosylation mutants showed

strongly reduced protein stability and trafficking to the plasma membrane,

which however were not affected by 1-deoxymannojirimycin (dMM). This

indicates that N-glycosylation, but not terminal trimming of the N-glycans

is involved in the attainment of a correctly folded and stable conformation

of GAT1. All N-glycosylation mutants were expressed on the plasma mem￾brane, but they displayed markedly reduced GABA-uptake activity. Also,

inhibition of oligosaccharide processing by dMM led to reduction of this

activity. Further experiments showed that both N-glycosylation mutations

and dMM reduced the Vmax value, while not increasing the Km value for

GABA uptake. Electrical measurements revealed that the reduced transport

activity can be partially attributed to a reduced apparent affinity for extra￾cellular Na+ and slowed kinetics of the transport cycle. This indicates that

N-glycans, in particular their terminal trimming, are important for the

GABA-uptake activity of GAT1. They play a regulatory role in the GABA

translocation by affecting the affinity and the reaction steps associated with

the sodium ion binding.

Abbreviations

CHO, Chinese hamster ovary; dMM, 1-deoxymannojirimycin; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorting; GABA,

c-aminobutyric acid; GAT1, GABA transporter type I; GFP, green fluorescence protein.

FEBS Journal 272 (2005) 1625–1638 ª 2005 FEBS 1625