The second intracellular loop of the glycine transporter 2 contains crucial residues for glycine transport and phorbol ester-induced regulation.

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 4alpha-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.