Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

Abstract

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.

Control of Ethanol Sensitivity of the Glycine Receptor α3 Subunit by Transmembrane 2, the Intracellular Splice Cassette and C-Terminal Domain

“…Besides the differences in protein sequence, the GlyR a3 subunit exhibits variants generated by alternative splicing that confer differential properties in the regulation by protein kinases (Lynch, 2004), extent and time course of desensitization (Breitinger et al, 2002), structural properties (Breitinger et al, 2009), ion channel gating (Breitinger et al, 2009), and receptor clustering and diffusion (Notelaers et al, 2012). In this framework, we analyzed the involvement of the intracellular domain and the GlyR a3L cassette in ethanol and Gbg modulation.…”

Intracellular glycine receptor function facilitates glioma formation in vivo

“…The protein region where RNA splicing changes amino acid sequences is interesting as it is located immediately downstream of the NLS region. It has been established that this protein region influences GlyR protein structure as, for example, inclusion of exon 8A (coding for TAEFALEKFYRFSDT) changes receptor desensitization kinetics, clustering and subcellular trafficking in neurons (Eichler et al, 2009;Nikolic et al, 1998;Notelaers et al, 2012;Winkelmann et al, 2014). The tumor-cell-specific preponderant expression of GlyR a1ins and a3K RNA splice variants identifies a functional role for GlyR a1ins and sets glioma cells apart from neurons with regard to RNA splicing of GlyR a3-coding gene transcripts (Eichler et al, 2009).…”

Changes in neural network homeostasis trigger neuropsychiatric symptoms

“…Targeted presynaptic GlyR HA-α3L 185L protein expression in vivo was confirmed using the HA epitope tag at the receptor N terminus (24,27,28) in immunohistochemical and ultrastructural analyses. Presynaptic GlyR expression is known to facilitate synaptic transmission due to a depolarized chloride reversal potential for axonal versus perisomatic ligand-gated chloride channels (49)(50)(51)(52)(53), and consistently, presynaptic GlyR HA-α3L 185L influenced the paired-pulse ratio of postsynaptic currents and decreased the amplitude of evoked excitatory field potentials when expressed in glutamatergic or parvalbumin-positive neurons, respectively.…”

“…Unlike the short GlyR α3K variant, GlyR α3L contains exon 8A (30,31), which codes for the TEAFALEKFYRFSDT peptide located in the large cytoplasmic loop between transmembrane domains 3 and 4 (TM3-4). Exon 8A was shown to confer particular subcellular trafficking and clustering properties on GlyR α3 (27,28). To further investigate the relevance of this exon, we searched for interaction partners of GlyR α3L.…”

“…In fact, expression of RNA-edited GlyR α3 is increased in patients with temporal lobe epilepsy and leads to P185L amino acid substitution and neurotransmitter receptor gain of function. Furthermore, it has been established that the RNA-spliced long GlyR α3L variant is preponderantly expressed in the hippocampus of patients with epilepsy and shows particular synaptic clustering and physiological receptor properties (27,28). To address the question of whether α3L 185L -GlyR triggers clinical symptoms of epilepsy, we generated a corresponding knockin mouse model and investigated the neuron type-specific functional impact of this particular molecule on bidirectional synaptic plasticity, network excitability and gamma oscillatory activity,…”