The Second Intracellular Loop of the Glycine Transporter 2 Contains Crucial Residues for Glycine Transport and Phorbol Ester-induced Regulation

Abstract

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.

P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

“…Regulation of the transport activity by protein kinase G has been reported to be controlled by homologous serine residues present in the N-terminal portion of the fifth transmembrane domain (64,65), a cytoplasmic protein region located within or near by the permeation pathway of these 12 transmembrane domain proteins (63). The homologous serine residue in GLYT2 is Ser-420, which lies close to a protein kinase C regulatory region (7). In a previous work, we generated mutant S420A in GLYT2 and showed that it has similar properties than wild type, although the replacement by Glu produced a PKCresistant transporter (7).…”

“…The homologous serine residue in GLYT2 is Ser-420, which lies close to a protein kinase C regulatory region (7). In a previous work, we generated mutant S420A in GLYT2 and showed that it has similar properties than wild type, although the replacement by Glu produced a PKCresistant transporter (7). To test the ability of the homologous serine residues in the GLYT modulation by P2Y 1 R, we constructed the homologous mutant in GLYT1 (S244A) and characterized the transport activity of the mutants expressed in COS7 cells (Fig.…”

“…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.…”

“…The P2Y receptors (P2YR) are G protein-coupled receptors that may regulate two important second messengers cytoplasmic Ca 2ϩ and cyclic adenosine monophosphate (cAMP), depending on the particular subtype (14). The seven mammalian P2X receptor subunits (P2X [1][2][3][4][5][6][7] ) and eight mammalian P2YRs (P2Y 1,2,4,6,11,12,13,14 ) so far cloned (15,16) are widely expressed in the mammalian CNS, and their activation generally results in excitatory neuronal responses (17). Purinergic signaling modulates extracellular neurotransmitter levels (acetylcholine, the monoamines, glutamate, GABA, adenosine, or glycine) (16,18,19).…”

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

“…On the other hand, amino-terminal phosphorylation has been shown to be important for reversed dopamine transport by DAT (23). In contrast, PKC-dependent phosphorylation of the second intracellular loop of glycine transporter 1 has been implicated in the phorbol estermediated down-regulation of this transporter (24), thus illustrating differences in the regulation of endocytosis of different members of this transporter family (25).…”