Trafficking properties and activity regulation of the neuronal glycine transporter GLYT2 by protein kinase C

Abstract

The neuronal glycine transporter GLYT2 controls the availability of the neurotransmitter in glycinergic synapses, and the modulation of its function may influence synaptic transmission. The active transporter is located in membrane rafts and reaches the cell surface through intracellular trafficking. In the present study we prove that GLYT2 constitutively recycles between the cell interior and the plasma membrane by means of a monensin-sensitive trafficking pathway. Also, a regulated trafficking can be triggered by PMA. We demonstrate that PMA inhibits GLYT2 transport by causing net accumulation of the protein in internal compartments through an increase of the internalization rate. In addition, a small increase of plasma membrane delivery and a redistribution of the transporter to non-raft domains is triggered by PMA. A previously identified phorbol-ester-resistant mutant (K422E) displaying an acidic substitution in a regulatory site, exhibits constitutive traffic but, in contrast with the wild-type, fails to show glycine uptake inhibition, membrane raft redistribution and trafficking modulation by PMA. We prove that the action of PMA on GLYT2 involves PKC (protein kinase C)-dependent and -independent pathways, although an important fraction of the effects are PKC-mediated. We show the additional participation of signalling pathways triggered by the small GTPase Rac1 on PMA action. GLYT2 inhibition by PMA and monensin also take place in brainstem primary neurons and synaptosomes, pointing to a GLYT2 trafficking regulation in the central nervous system.

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

“…A marked constitutive internalization has also been shown for other members of the SLC6 transporter family such as the DAT, GLYT2, and norepinephrine transporter (11,12,42). However, the fate of constitutively internalized SLC6 transporters, and ultimately the consequences of the internalization, remains elusive.…”

“…Other membrane proteins such as the EGF receptor and the ␦-opioid receptor are destined to late endosomes and lysosomes for degradation upon endocytosis (14,15). For the SLC6 transporters, it has been suggested based on use of recycling inhibitors that both DAT and the glycine transporter 2 (GLYT2) are sorted to a recycling pathway, permitting reinsertion in the membrane (11,12,16). It was therefore proposed that internalization of these transporters serve to maintain an intracellular pool of transporters that can be recruited to the surface during periods of high signaling activity (12).…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…Indeed, DAT has a high constitutive internalization rate, both when expressed in cell lines (Loder and Melikian, 2003;Sorkina et al, 2005) and for the endogenous transporter in cultured dopaminergic neurons (Eriksen et al, 2009). Likewise, endogenous GAT1 and GLYT2 were shown to constitutively internalize (Fornés et al, 2008). For DAT, GLYT2, and GAT1, the constitutively internalized transporter was suggested to sort mainly to a recycling pathway, permitting reinsertion of the transporter into the plasma membrane.…”

“…The suggested phosphorylation sites in NET (Thr258 and Ser259) are situated in IL2, which also have been connected with PMA-induced internalization of GLYT2. Mutation of the corresponding residues, Thr419 and Ser420, in GLYT2 partially impaired PMAmediated internalization (Fornés et al, 2004), and mutation of the nearby Lys442 abolished PMA-mediated internalization of GLYT2 without affecting constitutive internalization (Fornés et al, 2004(Fornés et al, , 2008.…”

P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

“…Analysis of knock-out animals proved that the modulation of glycine transporter expression and/or transport activity influences glycine-mediated neurotransmission and opened a way to find therapeutic applications (5,6). The levels of active glycine transporters in the plasma membrane are regulated by several mechanisms, including protein trafficking (7)(8)(9), Ca 2ϩ modulation (10,11), protein-protein interaction (11), and membrane raft association (9), among others (7,12). In the central nervous system (CNS), these regulatory pathways must be triggered by physiological stimuli such as the neural function or the activity of appropriate receptors.…”