Na ؉ and Cl ؊ -coupled glycine transporters control the availability of glycine neurotransmitter in the synaptic cleft of inhibitory glycinergic pathways. In this report, we have investigated the involvement of the second intracellular loop of the neuronal glycine transporter 2 (GLYT2) on the protein conformational equilibrium and the regulation by 4␣-phorbol 12 myristate 13-acetate (PMA). By substituting several charged (Lys-415, Lys-418, and Lys-422) and polar (Thr-419 and Ser-420) residues for different amino acids and monitoring plasma membrane expression and kinetic behavior, we found that residue Lys-422 is crucial for glycine transport. The introduction of a negative charge in 422, and to a lower extent in neighboring N-terminal residues, dramatically increases transporter voltage dependence as assessed by response to high potassium depolarizing conditions. In addition, [2-(trimethylammonium)ethyl] methanethiosulfonate accessibility revealed a conformational connection between Lys-422 and the glycine binding/permeation site. Finally, we show that the mutation of positions Thr-419, Ser-420, and mainly Lys-422 to acidic residues abolishes the PMA-induced inhibition of transport activity and the plasma membrane transporter internalization. Our results establish a new structural basis for the action of PMA on GLYT2 and suggest a complex nature of the PMA action on this glycine transporter.

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.