Na+/K+-ATPase Is a New Interacting Partner for the Neuronal Glycine Transporter GlyT2 That Downregulates Its Expression In Vitro and In Vivo

Abstract

The neuronal glycine transporter GlyT2 plays a fundamental role in the glycinergic neurotransmission by recycling the neurotransmitter to the presynaptic terminal. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is absolutely necessary to preserve quantal glycine content in synaptic vesicles. Alterations in GlyT2 activity modify glycinergic neurotransmission and may underlie several neuromuscular disorders, such as hyperekplexia, myoclonus, dystonia, and epilepsy. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans and produce congenital muscular dystonia type 2 (CMD2) in Belgian Blue cattle. GlyT2 function is strictly coupled to the sodium electrochemical gradient actively generated by the Naϩ /Cl Ϫ /glycine generating large rises of Na ϩ inside the presynaptic terminal that must be efficiently reduced by the NKA to preserve Na ϩ homeostasis. In this work, we have used high-throughput mass spectrometry to identify proteins interacting with GlyT2 in the CNS. NKA was detected as a putative candidate and through reciprocal coimmunoprecipitations and immunocytochemistry analyses the association between GlyT2 and NKA was confirmed. NKA mainly interacts with the raft-associated active pool of GlyT2, and low and high levels of the specific NKA ligand ouabain modulate the endocytosis and total expression of GlyT2 in neurons. The ouabainmediated downregulation of GlyT2 also occurs in vivo in two different systems: zebrafish embryos and adult rats, indicating that this NKA-mediated regulatory mechanism is evolutionarily conserved and may play a relevant role in the physiological control of inhibitory glycinergic neurotransmission.

Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)

“…5, A and B). In agreement with previously reported results, GlyT2 is enriched in raft fractions (9,15), showing here a relative distribution in rafts of 77.26 Ϯ 4.46% S.E., whereas PMCA2 and PMCA3 distributions are 51.09 Ϯ 6.25% and 49.46 Ϯ 7.57% S.E., respectively (Fig. 5B).…”

“…5, C-H) (61). We observed a clear co-localization between GlyT2 and Thy-1, as reported previously (15). Interestingly, these GlyT2-Thy-1 clusters extensively co-localized with PMCA2 or PMCA3, leading to a high degree of triple colocalization as shown by the white color in the overlay image (denoted as Merge in Fig.…”

“…Thus, the native transporter was immunoprecipitated from brainstem and spinal cord synaptosomes using a specific GlyT2 antibody (40), and co-purified proteins were identified by high throughput mass spectrometry. GlyT2 immunoprecipitates were found to selectively enhance the detection of several proteins, including some of the previously described GlyT2 interacting partners such as syntaxin-1 (16) or Na ϩ /K ϩ -ATPase (15). We also found other molecules that are known to be implicated in GlyT2 trafficking, such as clathrin heavy chain (10).…”

“…Primary neurons or purified synaptosomes were analyzed by immunofluorescence as reported previously (15). The primary antibodies used in this work were incubated overnight at 4°C using the following dilutions: GlyT2 (1:500); PSD95 (1:500); PMCA2 (1:250); PMCA3 (1:150); NCX1 (1:200); and Thy-1 (1:500).…”

“…GlyT2 has a crucial role in the pathophysiology of inhibitory glycinergic neurotransmission, and studying modulatory factors that regulate its activity might help the success of future therapies. Indeed, studies of endogenous GlyT2 regulatory mechanisms have revealed that GlyT2 activity is regulated by PKC activation (9,10), lipid raft environment (10,11), P2Y purinergic receptors (12), ubiquitination (13), calnexin function (14), or Na ϩ /K ϩ -ATPase interaction (15). In addition, we have also described that GlyT2 exocytosis is regulated by syntaxin-1A and calcium (Ca 2ϩ ) increases (16).…”