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

Juan-Sanz, Jaime de; Núñez, Enrique; Zafra, Francisco; Berrocal, María Cruz; Corbacho, Isaac; Ibáñez, Ignacio; Arribas-González, Esther; Marcos, Daniel; López-Corcuera, Beatriz; Mata, Ana M.; Aragón, Carmen

doi:10.1074/jbc.m114.586966

Cited by 27 publications

(23 citation statements)

References 98 publications

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“…These values were corrected against local background staining and compared to neighboring untransfected somas to correct for variations in global staining intensity. Immunocytochemistry to ensure expression of STIM1-myc or D76A/E87A STIM1-myc in STIM1-KD neurons using an antibody against the myc tag (mouse, Abcam, #ab18185; 1:500) was performed as described previously (de Juan-Sanz et al, 2014), visualized using an Alexa-546 anti-mouse secondary antibody (Life Technologies).…”

Section: Star Methodsmentioning

confidence: 99%

Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals

Juan-Sanz

Holt

Schreiter

et al. 2017

Neuron

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233

273

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Although the endoplasmic reticulum (ER) extends throughout axons and axonal ER dysfunction is implicated in numerous neurological diseases, its role at nerve terminals is poorly understood. We developed novel genetically-encoded ER-targeted lowaffinity Ca2+ indicators optimized for examining axonal ER Ca2+. Our experiments revealed that presynaptic function is tightly controlled by ER Ca2+ content. We found that neuronal activity drives net Ca2+ uptake into presynaptic ER although this activity does not contribute significantly to shaping cytosolic Ca2+ except during prolonged repetitive firing. In contrast we found that axonal ER acts as an actuator of plasma membrane (PM) function: [Ca2+]ER controls STIM1 activation in presynaptic terminals, which results in the local modulation of presynaptic function, impacting activity-driven Ca2+ entry and release probability. These experiments reveal a critical role of presynaptic ER in the control of neurotransmitter release and will help frame future investigations into the molecular basis of ER-driven neuronal disease states.

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Section: Star Methodsmentioning

confidence: 99%

Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals

Juan-Sanz

Holt

Schreiter

et al. 2017

Neuron

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233

273

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“…Then, GlyT2 is sorted to the plasma membrane in lipid rafts, microdomains where it interacts with proteins like neuronal plasma membrane Ca 2+ -ATPase (PMCA), and Na + /Ca 2+ -exchanger 1 (NCX1). Endogenous PMCA and NCX activities are necessary for GlyT2 activity and this modulation depends on lipid raft integrity [18]. In neurons, GlyT2 is concentrated at presynaptic elements in a process dependent on the PDZ-interacting motif located in its C-terminus [19].…”

Section: Introduction: the Building Of A Glycinergic Neuronmentioning

confidence: 99%

Glycinergic transmission: glycine transporter GlyT2 in neuronal pathologies

2016

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Glycinergic neurons are major contributors to the regulation of neuronal excitability, mainly in caudal areas of the nervous system. These neurons control fluxes of sensory information between the periphery and the CNS and diverse motor activities like locomotion, respiration or vocalization. The phenotype of a glycinergic neuron is determined by the expression of at least two proteins: GlyT2, a plasma membrane transporter of glycine, and VIAAT, a vesicular transporter shared by glycine and GABA. In this article, we review recent advances in understanding the role of GlyT2 in the pathophysiology of inhibitory glycinergic neurotransmission. GlyT2 mutations are associated to decreased glycinergic function that results in a rare movement disease termed hyperekplexia (HPX) or startle disease. In addition, glycinergic neurons control pain transmission in the dorsal spinal cord and their function is reduced in chronic pain states. A moderate inhibition of GlyT2 may potentiate glycinergic inhibition and constitutes an attractive target for pharmacological intervention against these devastating conditions.

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“…In neurons, LNX1 may act as a presynaptic scaffold, promoting the formation of multimolecular complexes that could allow the correct subcellular localization of GlyT2 and other interacting partners 31,63 . GlyT2 has been previously shown to interact with several presynaptic proteins, including syntaxin1 16 , Plasma Membrane Calcium ATPases PMCA2 and PMCA3 18 and sodium/potassium ATPase subunits α3 (α3NKA) and β2 (β2NKA) 17 . It is possible that LNX1 may act as a scaffold to facilitate the interaction of GlyT2 with the mentioned proteins, and this idea of a GlyT2-LNX1-PMCA/NKA multimolecular complex is supported by a recent proteomic study that found PMCA2 and β2NKA as candidate interacting partners of LNX1 32 .…”

Section: Discussionmentioning

confidence: 99%

“…Understanding the molecular regulation of GlyT2 transport would provide insight into the molecular and cellular basis of glycinergic neurotransmission and potentially lead to identifying new therapeutic targets for presynaptic Hyperekplexia. Previous studies on GlyT2 regulatory mechanisms revealed that GlyT2 activity is regulated by PKC activation 10,11 , calnexin function 12,13 , P2Y and P2X purinergic receptors 14,15 and interaction with syntaxin1 16 , Na+/K+-ATPase 17 and PMCAs 18 . In addition, we previously described that GlyT2 trafficking and surface expression are regulated by ubiquitination 11,19 , a process in which the small protein ubiquitin is covalently attached to a cytoplasmic lysine residue of a target protein.…”

Section: Introductionmentioning

confidence: 99%

Ubiquitin ligase LNX1 is a major regulator of glycine recapture by the presynaptic transporter GlyT2

Núñez

Arribas-González

López-Corcuera

et al. 2017

Preprint

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The neuronal glycine transporter GlyT2 is an essential regulator of glycinergic neurotransmission that recaptures glycine in presynaptic terminals to facilitate quantal transmitter packaging in synaptic vesicles. Alterations in GlyT2 expression or activity result in lower cytosolic glycine levels, emptying glycinergic synaptic vesicles and impairing neurotransmission. Lack of glycinergic neurotransmission caused by GlyT2 loss-of-function mutations results in Hyperekplexia, a rare neurological disease characterized by generalized stiffness and motor alterations that may result in sudden infant death. Although the importance of GlyT2 in pathology is known, how this transporter is regulated at the molecular level is poorly understood, limiting current therapeutic strategies. Guided by an unbiased screening, we discovered that the E3 ubiquitin ligase Ligand of Numb protein X1 (LNX1) modulates the ubiquitination status of GlyT2. LNX1 ubiquitinates a cytoplasmic C-terminal lysine cluster in GlyT2 (K751, K773, K787 and K791) through its N-terminal RING-finger domain, and this process regulates the expression levels and transport activity of GlyT2 in neurons. These experiments reveal for the first time the identity of an E3 ubiquitin-ligase acting on GlyT2 and identify a novel regulatory mechanism by which neurons regulate GlyT2 expression and activity.

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

Cited by 27 publications

References 98 publications

Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals

Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals

Glycinergic transmission: glycine transporter GlyT2 in neuronal pathologies

Ubiquitin ligase LNX1 is a major regulator of glycine recapture by the presynaptic transporter GlyT2

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