Alternative splicing generates two isoforms of the α2 subunit of the inhibitory glycine receptor

Kuhse, Jochen; Kuryatov, Alexander; Maulet, Yves; Malosio, Maria Luisa; Schmieden, Volker; Betz, Heinrich

doi:10.1016/0014-5793(91)80557-j

Cited by 130 publications

(90 citation statements)

References 21 publications

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“…Likewise, two ␣3 variants, ␣3K and ␣3L, are generated by alternative splicing of GlyR ␣3 exon 8A (TEAFALEKFYRFSDT) located in the TM3-TM4 loop (12). For GlyR ␣2, alternative splicing of exon 3 generates two different receptor variants that differ by two amino acids (␣2A (IA) and ␣2B (VT)) in the extracellular ligand binding domain (13).…”

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confidence: 99%

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Raltschev

Hetsch

Winkelmann

et al. 2016

Journal of Biological Chemistry

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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.

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confidence: 99%

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Raltschev

Hetsch

Winkelmann

et al. 2016

Journal of Biological Chemistry

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“…The ␣ 2 homomeric GlyR subunit is an embryonic receptor form (18,19) and plays an important role during synaptogenesis and cell differentiation. Furthermore, the ␣ 2 homomeric GlyR has slow kinetic properties and opens mainly with a single large conductance state (100 -120 pS) (20); this property makes this receptor a good model for analyzing the effects of picrotoxinin and picrotin on GlyR kinetics.…”

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confidence: 99%

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

Wang

Buckinx

Le-Corronc

et al. 2007

Journal of Biological Chemistry

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Contrary to its effect on the ␥-aminobutyric acid type A and C receptors, picrotoxin antagonism of the ␣ 1 homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and nonselective between the picrotoxin components picrotoxinin and picrotin. Picrotoxin antagonism of the embryonic ␣ 2 homomeric GlyR is known to be use-dependent and reflects a channel-blocking mechanism, but the selectivity of picrotoxin antagonism of the embryonic ␣ 2 homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin-and picrotoxinin-induced inhibition of currents, which were evoked by the activation of ␣ 2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents, picrotin had a 30 times higher IC 50 than picrotoxinin. Picrotin-evoked inhibition displayed voltage dependence, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentrationdependent manner, indicating that these picrotoxin components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were coapplied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but both could be trapped at their binding site when the channel closed during glycine dissociation. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking ␣ 2 homomeric GlyR.Glycine and GABA 4 are the most important inhibitory neurotransmitters in the adult central nervous system. The glycine receptors (GlyRs) belong to the cysteine-loop family of ligandgated ion channels. The GlyR is a pentameric transmembrane protein complex, which forms an anion-selective channel. So far five different subunits have been cloned in mammals, one ␤ subunit and four ␣ subunits (␣ 1 -␣ 4 ) (1). Each subunit is composed of a large external N-terminal domain and four transmembrane domains termed M1-M4, with the M2 transmembrane domain forming the pore of the channel (1). These subunits can be associated in two different ways to form functional receptor channels. The homomeric receptors consist of five ␣ subunits, whereas the heteromeric receptors are a combination of two ␣ and three ␤ subunits with the agonist-binding site at the interface of the ␣ and the ␤ subunits (2). Although the plant alkaloid picrotoxin (PTX) was first used to discriminate between functional GABA A Rs and GlyRs, it is now well established that PTX can inhibit both cation-selective (nicotinic acetylcholine receptor and 5-hydroxytryptophan receptor type 3) and anion-selective (GABA A R, GABA C R, GlyR, and GluCl) receptor channels (3-7). The action of PTX has been extensively studied on GABA A Rs, on GABA C R...

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“…GlyRa2 Splicing in Nova-1 Null Mice Since GlyRa2 gene expression and physiology in rodent spinal cord and brainstem undergo regulation after the first week of postnatal life (Kuhse et al, 1991;Bechade et al, 1994;Singer et aL, 1998), we bred Nova-1 null mice into a CD1 genetic background, yielding Nova-1 null mice that lived for an average of 2-3 weeks after birth. In these mice, the motor phenotype became particularly pronounced after ~7-10 days, with weakness and tremulousness, and with some mice showing clear signs of atrophy in the hindlimbs.…”

Section: Resultsmentioning

confidence: 99%

“…Full-length Nova-1 binds with low-nanomolar affinity to a stem loop RNA harboring a sequence-specific motif (UCAUY) 3 . This sequence led to the identification of a candidate Nova-1 RNA target in (GlyRa2) pre-mRNA present inanrintron 85 nt up* stream of the inhibitory glycine receptor a2 exon 3A (GlyRa2 E3A; ; This exon is alternatively spliced in a mutually exclusive fashion with a downstream exon (E3B; Kuhse et al, 1991). Immunoprecipitation of Nova from brain extracts specifically coprecipitated GlyRa2 pre-mRNA , providing evidence that a Nova'-GlyRa2 protein-RNA complex forms in neurons.…”

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Functional Analysis of Nova Proteins: Gene Regulation and Breast Tumor Immunity.

Jensen¹

1998

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Alternative splicing generates two isoforms of the α2 subunit of the inhibitory glycine receptor

Cited by 130 publications

References 21 publications

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

Functional Analysis of Nova Proteins: Gene Regulation and Breast Tumor Immunity.

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