Modulation of glycine receptor single-channel conductance by intracellular phosphorylation

Abstract

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKAdependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKAtargeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLic and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs. Glycine receptors (GlyRs) belong to the pentameric ligand-gated ion channel (pLGIC) family. GlyRs are anion-permeable channels, allowing the fast influx of chloride and the control of neuronal excitability. An individual GlyR subunit is composed by an extracellular domain (ECD), four transmembrane domains (TM1-4) and an intracellular domain between the TM3 and TM4 domains (ICD) 1-4. To date, a single β subunit and four α subunits (α1-4) has been described. The α subunits share a high degree of sequence identity (≈75%). Nevertheless, they exhibit important differences in their biophysical and pharmacological properties as well as in their distribution along the CNS 1,3,4. In the mammalian CNS, GlyR activity critically controls neurophysiological functions such as motor coordination, respiratory control, muscle tone, as well as pain processing 2,3,5-13. The importance of glycinergic inhibition was first recognized in studies using the GlyR antagonist strychnine 14,15. Later, genetic studies found that mutations in the GlyR α1 and β genes are linked to hyperekplexia in humans 16. More recent evidence has shown that specific GlyR subunits may play key roles in several diseases. For example, while the α1 subunit has been linked to tumorigenesis and alcohol intoxication 17,18 , mutations in the α2 subunit have been linked to autism 19. Alterations in the RNA processing of α3 subunits generates hyperactive receptors, which have been rela...

Presence of ethanol‐sensitive and ethanol‐insensitive glycine receptors in the ventral tegmental area and prefrontal cortex in mice

“…Thus, the present results support a predominant presence of the 1 subunit in the VTA, but not in the PFC. Although, phosphorylation and receptor density can affect some physiological characteristic of GlyRs (Taleb and Betz, 1994;Moraga-Cid et al, 2020) and might explain some of the distinct properties found in VTA and PFC neurons, we believe that these differences are related to differences on subunit expression as discussed previously. This is further supported by the sensitivity of GlyRs to ethanol in the 1 KI and 2 KO mice summarized in table 1.…”

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin

“…The large intracellular loop of the inhibitory GlyR contains numerous motifs for intracellular interaction [2] , [4] , [17] , [18] , [42] , [43] , [44] , [45] and modulation of receptor function [7] , [17] , [34] , [46] , [47] . Here, we investigated the function of a proline-rich motif, 365 PPPAPSKSP 373 , which forms a PPII helix that is located in the intracellular TM3-4 loop of the human GlyRα1 subunit.…”

Allosteric modulation of alternatively spliced Ca2+-activated Cl−channels TMEM16A by PI(4,5)P2and CaMKII

“…We further showed that the values are increased by channel dephosphorylation induced by CaMKII inhibition or by intracellular ATP removal. In a similar way, the single-channel conductances of ligand-gated glycine receptors, glutamate receptors, and 5-HT3 receptors are modulated by phosphorylation (54)(55)(56).…”