Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Abstract

Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the ␣2 or ␣3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala 254 ) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala 254 determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level.

A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy

“…To address this possibility in detail, we used transfected HEK293T cells and performed whole cell patch clamp analysis (Figure 5 ). We focused here on the GlyR α2 subunit because primary hippocampal neurons at DIV8-13 predominantly express GlyR α2 (Meier and Grantyn, 2004 ; Raltschev et al, 2016 ). However, as both splice variants of GlyR α2 (Figures 5A,B ) and the GlyR α3L subunit are expressed in the iTLE hippocampus (Eichler et al, 2008 ), we included both GlyR α2 splice variants (Figure 5B ) and GlyR α3L (Supplementary Figure 1 ) into our analyses.…”

“…Total RNA was isolated from primary hippocampal neurons from E18 rat embryos (DIV8) and reverse transcribed into cDNA as described earlier (Raltschev et al, 2016 ). Oligos were used to amplify regions of AID (5′-GTCCGCTGGGCTAAGGGTC-3′ and 5′-GCACAGTCGTAGCAGGGGC-3′), APOBEC-1 (5′-CCCTGTAGCTGTTGATCCCACTC-3′ and 5′-CAGAGTTACATGGGGGTATCGGC-3′), ABOBEC-2 (5′-CTGATCGATCTGCCGCCCTTC-3′ and 5′-CAGGTTCTTGGTCTTGCTGAGGG-3′), APOBEC-3 (5′-GCCATCGCAGACCCTATTCACCG-3′ and 5′-CTTGCTGCAGGGGCTCCAGG-3′), APOBEC-4 (5′-GGCAGGGGAGGTGAGTCTGG-3′ and 5′-GTTAGCCTCGTCACAAGGGGAG-3′), ACF (5′-TTCTGTCAGAGGGGCTGCG-3′ and 5′-AGCTTTGGGGGTGTGAAAGG-3′) and GAPDH (5′-CAGTATGACTCTACCCACGG-3′ and 5′-CTCAGTGTAGCCCAGGATG-3′).…”

Voltage-Dependent Inhibition of Glycine Receptor Channels by Niflumic Acid

“…A systematic study of I/V relations for different subunits of GlyRs was undertaken recently, but for desensitized channels, activated by high concentration of glycine (Raltschev et al, 2016 ). It was shown that currents mediated by α2 and α3 GlyRs in desensitized state are inwardly rectifying, while α1 GlyRs currents are linear.…”

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

“…Therefore, the glycine-induced Cl -current (ICl glycine ) observed here is clearly independent of GlyR. Apart from this, the biophysical properties of ICl glycine are different from GlyR-mediated Cl -currents [43], but they are identical to those of swelling-activated Cl -currents (ICl swell , ICl vol , VRAC) described in microglial cells and in a plenitude of other cells [26,27,33,44,45].…”