Functional Implications of the Exon 9 Splice Insert in GluK1 Kainate Receptors

Reviewer #1 (Public Review):

Kainate receptors play various important roles in synaptic transmission. The receptors can be divided into low affinity kainate receptors (GluK1-3) and high affinity kainate receptos (GluK4-5). The receptors can assemble as homomers (GluK1-3) or low-high affinity heteromers (GluK4-5). The functional diversity is further increased by RNA splicing. Previous studies have investigated C-terminal splice variants of GluK1, but GluK1 N-terminal (exon 9) insertions have not been previously characterized. In this study Dhingra et al investigate the functional implications of a GluK1 splice variant that inserts a 15 amino acid segment into the extracellular N-terminal region of the protein using whole-cell and excised outside-out electrophysiology.
The authors produce solid data to show that the insertion profoundly impacts the function of GluK1-1a - the channels that have the insertion are slower to desensitize. The data also shows that the insertion changes the modulatory effects of Neto proteins, resulting in altered rates of desensitization and recovery from desensitization. To determine the mechanism by which the insertion exerts these functional effects, the authors perform pull-down assays of Neto proteins, and extensive mutagenesis on the insert.

The electrophysiological part of the study is very rigorous and meticulous.

The biggest weakness of the manuscript is the structural work. Due to issues with preferred orientation (a common problem in cryo-EM), the 3D reconstructions are at a low resolution (in the 5-8 Å range) and cannot offer much mechanistic insight into the effects of the insertion. Based on the available data, the authors posit that the insertion does not change the arrangement of the subunits in the desensitized state. However, there is no comparison with a structure that does not contain the insertion, so while the statement may well be true, no data is shown to support it.

Overall, the cryo-EM contributes little and distracts from the good parts of the manuscript.

Another part that does not contribute much is the RNAseq data that has been pulled from a database and analyzed for the paper. It is being used to show that the exon 9 insertion variant is predominantly expressed in the cerebellar cortex at early stages of brain development. The methods do not describe in detail how the data has been analyzed (e.g., is the data scaled per sample/gene or globally?) so it is hard to know what we can compare in the heat plots. In Figure 1- supplement 1 there aren't striking differences in expression (at least not obviously visible in the current illustration).

Despite these weaknesses, the study is an important contribution to the field because it characterizes a GluK1 variant that has not been studied before and highlights the functional diversity that exists within the kainate receptor family.

Reviewer #2 (Public Review):

Among ionotropic glutamate receptors, kainate receptors (KAR) are still the object of intense investigation to understand their role in normal and pathological excitatory synaptic transmission. Like other receptors, KAR appear under different splicing variants and their respective physiological function is still debated. In this manuscript Dhingra et al explored the impact of the presence and of the absence of Exon9 of the GluK1 receptors on the pharmacological, biophysical and structural properties of the receptors. They further investigated how it is impacted by the association of KAR with their cognate auxiliary subunit Neto 1 and 2. This study represents a large body of work and data. The authors addressed the issue in a very systematic and rigorous manner.

First, by exploring RNAseq database, authors showed that GluK1 transcripts containing the exon 9 are present in many brain structures and especially in the cerebellum suggesting that a large part of GluK1 contains effectively this exon9.
Using HEK cells as an expression system, they characterized many gating and biophysical properties of GluK1 receptors containing or not the exon9. Evaluated parameters were desensitization, relative potency of glutamate versus kainite, polyamine block.

It is known that the association of GluK1 with auxiliary proteins Neto1/2 modulate the properties of the receptors. Authors investigated systematically whether Neto1 and 2 similarly alter GluK1 properties in function of the presence of exon9. This study provides many quantitative data that could be reused for modeling the role of kainate receptors. Given the change shown by the authors, the presence of exon in GluK1 is noticeable and likely should have an impact of synaptic transmission.
Interestingly, authors used a mutational approach to identify critical residue encoded by exon9 that are responsible for the functional differences between the two splice variants. In many cases, the replacement of a single amino acid lead to the absence of current confirming the crucial role of the segment of the receptor. However, it made the comparison and the identification of critical residues more challenging.
Authors attempted to establish the structure GluK1 receptors comprising the exon9 using different preparation methods. They succeeded in obtaining structures with equivalent or lower resolution compared with previous report on GluK1 and GluK2 receptors. However, the organization of the peptide coded by exon is poorly defined and limited possible analyses. Despite this they could observe that the presence of the exon9 does not alter significantly the structure of GluK1.

Reviewer #3 (Public Review):

GluK1 forms glutamate-gated ion channels with an important function in synaptic transmission and neuron excitability. Particularly, a GluK1 splice-variant (Gluk1-1) with significant expression in different regions of the brain has not been characterized before. The paper of Dhingra et al. aims to evaluate the role of the Exon 9 splice insert in GluK1 on channel function. This study relies mainly on electrophysiological approaches to determine the effect of the splice insert on GluK1 gating properties, including desensitization, agonist efficacy, recovery, and rectification. Overall, this work provides two major milestones: 1) the first functional characterization of the Gluk1-1a variant and 2) the first structure of this channel. The functional data supporting the role of the insert on channel properties are solid, although the current data does not provide significant insights about the mechanisms behind this. Also, the little information associated with the resolved structure precludes providing further insights about the structural basis that account for the impact of the insert on channel function. Overall, I consider this an interesting paper that represents an important advance in the study of glutamate receptors.