Neto proteins differentially modulate the gating properties of Drosophila NMJ glutamate receptors

The formation of functional synapses requires co-assembly of ion channels with their accessory proteins which controls where, when, and how neurotransmitter receptors function. The auxiliary protein Neto modulates the function of kainate-type glutamate receptors in vertebrates as well as at the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse widely used for genetic studies on synapse development. We previously reported that Neto is essential for the synaptic recruitment and function of glutamate receptors. Here, using outside-out patch-clamp recordings and fast ligand application, we examine for the first time the biophysical properties of recombinant Drosophila NMJ receptors expressed in HEK293T cells and compare them with native receptor complexes of genetically controlled composition. The two Neto isoforms, Neto-α and Neto-β, differentially modulate the gating properties of NMJ receptors. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.


Introduction
Ionotropic glutamate receptors (iGluRs) mediate fast excitatory synaptic signaling throughout the vertebrate CNS and also at the neuromuscular junction (NMJ) of insects and crustaceans (Takeuchi and Takeuchi 1964;Jan and Jan 1976;Traynelis et al. 2010;Yu et al. 2021).iGluRs are tetrameric channels that achieve strikingly diverse biophysical properties by combining different iGluR subunits within a receptor complex and by association with a rich array of auxiliary subunits (Jackson and Nicoll 2011;Hansen et al. 2021).Auxiliary subunits bind to iGluRs at many stages of the receptor life-cycle and modulate not only channel properties but also the delivery of receptors to the cell surface, their subcellular distribution, synaptic recruitment, and association with various postsynaptic density (PSD) scaffolds.In Drosophila, multiple NMJ iGluR subunits (DiAntonio 2006), and the auxiliary protein Neto (Neuropillin and Tolloid-like) are each essential for viability (Kim et al. 2012), indicating that Neto is required for function of the NMJ.Phylogenetic studies indicate that fly NMJ iGluRs belong to the kainate receptor (KAR) clade, which has been expanded in Diptera (Li et al. 2016).Since Neto appears to be a KAR-dedicated auxiliary subunit (Zhang et al. 2009;Tomita and Castillo 2012), it would be anticipated that similar to vertebrate KARs, Drosophila Neto modulates the gating of NMJ iGluRs.However, the biophysical properties of native Drosophila iGluRs and their modulation by auxiliary proteins remain poorly understood.
In flies as in humans, synapse strength and plasticity is determined by the interplay between expression of different iGluR subtypes (DiAntonio et al. 1999).At the fly NMJ, type-A and type-B iGluRs are assembled from four different subunits: either GluRIIA (type-A) or GluRIIB (type-B), plus one copy each of GluRIIC, GluRIID and GluRIIE (Petersen et al. 1997;DiAntonio et al. 1999;Marrus et al. 2004;Featherstone et al. 2005;Qin et al. 2005).The shared subunits and Neto are both essential for viability and for iGluR synaptic recruitment (DiAntonio 2006;Kim et al. 2012).
In flies, Neto and iGluRs depend on each other for trafficking and stabilization at synaptic sites (Kim et al. 2012).Neto proteins are single-pass transmembrane proteins with a highly conserved extracellular part containing two C1r/C1s-Uegf-BMP domains (known as CUB1 and CUB2) and a low-density lipoprotein class A motif (LDLa) and variable intracellular domains.Recent cryoelectron microscopy studies indicate that the vertebrate Neto2 accesses different interfaces of GluK2 homotetrameric receptors, making tight inter-subunit connections via CUB1/GluK2-ATD and LBD domains and intra-subunit interactions within the transmembrane helices (He et al. 2021).In addition to the conserved protein interaction domains, Drosophila Neto has multiple intracellular putative docking motifs, and phosphorylation sites indicating that Neto likely controls NMJ iGluR synaptic recruitment and function via binding to iGluRs, to PSD components, as well as other interacting partners (Kim et al. 2015;Ramos et al. 2015;Sulkowski et al. 2016;Han et al. 2020).The Drosophila neto gene codes for two isoforms (Neto-α and Neto-β) with completely and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (which this version posted April 26, 2024.; https://doi.org/10.1101/2024.04.22.590603doi: bioRxiv preprint different intracellular domains of 206 and 351 residues, generated by alternative splicing (Ramos et al. 2015).Either isoform can sustain organism viability, but they have different in vivo distributions and functional roles (Kim et al. 2012;Han et al. 2015;Kim et al. 2015;Ramos et al. 2015;Han et al. 2020).
We previously reported the functional reconstitution of recombinant Drosophila NMJ iGluRs in Xenopus oocytes (Han et al. 2015).These studies showed that just as in flies, four different subunits, GluRIIA or GluRIIB, plus GluRIIC, GluRIID and GluRIIE are required for the robust surface expression of Drosophila NMJ iGluRs; complexes assembled from fewer than four subtypes do not reach the cell surface, and remain trapped in secretory compartments.Our experiments also revealed that in the absence of Neto Drosophila NMJ iGluRs are not activated by glutamate even after application of the lectin Concanavalin A (Han et al. 2015), thus establishing that with heterologous expression we can recapitulate results obtained in vivo using fly genetics.However, in contrast to vertebrate iGluRs, for which the kinetics of activation, deactivation and desensitization have been studied in depth, with extensive characterization of the properties of different subunit combinations and auxiliary proteins (Hansen et al. 2021), comparable studies on recombinant Drosophila iGluRs have not been reported.This is a big gap in the field of developmental neurobiology: the Drosophila NMJ has been a powerful system to study glutamatergic synapse assembly, development and homeostasis and extensive genetic manipulations of wild-type and mutant NMJ iGluR subunits contributed to our understanding of underlying molecular mechanisms.For example, studies of flies with GluRIIA mutations at sites that alter the kinetics of desensitization of mammalian GluA2 AMPA receptors revealed impaired trafficking behavior in vivo and abnormal distribution at postsynaptic densities (PSDs) (Petzoldt et al. 2014).However, because the recombinant expression of Drosophila NMJ GluRs had not been established, the gating behavior of these Drosophila NMJ iGluR variants was not determined, and was assumed to mimic that of their mammalian counterparts.
Here we examine the gating properties of Drosophila NMJ iGluRs using outside out patch recordings from HEK293T cells transfected with different combinations of NMJ iGluR subunits and Neto splice variants.In addition, we generated flies with genetically controlled subunit composition and recorded responses from outside out patches obtained from Drosophila larval muscle, as well as excitatory synaptic currents from their NMJ.We used rapid application of glutamate to examine the kinetics of activation, deactivation and desensitization, the effect of polyamine toxins, and the effect of the lectin Concanavalin A. We find that the two Neto isoforms differentially modulate the deactivation and desensitization of type-A and type-B receptors.Our study reveals that Drosophila Neto is not only required for channel function but also increases the repertoire of channel properties.

Drosophila NMJ iGluRs form Neto-dependent rapidly desensitizing receptors
To facilitate cell surface expression of recombinant Drosophila NMJ iGluRs, we replaced endogenous signal peptides with an optimized sequence and added a C-terminal RGSH6 epitope to all iGluR constructs.Four receptor subunits (GluRIIC, GluRIID, GluRIIE and either GluRIIA or GluRIIB) were transiently transfected in HEK293T cells with or without Neto splice variants and incubated at 30C for three days prior to outside-out patch recordings.Since these receptors have low sensitivity to glutamate (Heckmann et al. 1996;Han et al. 2015) we examined their gating properties in response to rapid application of 10 mM glutamate.In the absence of Neto, we observed no response to glutamate in 79 patches of GluRIIA/C/D/E and 46 patches of GluRIIB/C/D/E (Figure 1A -B).However, when type-A receptor complexes were co-transfected with Drosophila Neto variants (Figure 1C), a large fraction of outside-out patches (78/102) yielded macroscopic currents in response to glutamate.A similar requirement for Neto was observed for type-B receptors.Rapid application of 10 mM glutamate to outside-out patches from HEK cells transfected with various iGluR/Neto combinations revealed subunit composition-dependent gating properties and differential effects of Neto variants (Table 1).For example, for macroscopic currents recorded from type-A iGluR/Neto-α (A/α) complexes the 10%-90% rise time was 330 ± 9.64 µs (n = 8), with a deactivation time constant (off) 0.64 ± 0.07 ms, whereas type-A iGluR/Netoβ (A/β) complexes had a similar rise time, 340 ± 17.77 µs (n = 8), but 1.4 fold slower deactivation, off 0.90 ± 0.11 ms (Figure 1D).Longer applications of glutamate (100 ms) revealed rapid and profound desensitization of more than 98% for both complexes (n = 7-8), with the decay best fit by the sum of two exponential functions: A/α fast 2.01 ± 0.28 ms, slow 5.92 ± 1.07 ms, Afast 81.49 ± 4.61 %; and A/β fast 3.83 ± 0.84 ms, slow 10.37 ± 2.0 ms, Afast 80.02 ± 6.84 % (Figure 1E).and is also made available for use under a CC0 license.
These results also reveal that the cytoplasmic domain of Neto-α, but not of Neto-β, modulates the gating properties of NMJ iGluR receptors, increasing the rate of deactivation and desensitization.
Similar results were obtained for type-A receptors in complex with PM-Neto-ΔCTD (Figure 1C-E), a processing mutant variant that cannot shed its inhibitory pro-domain and fails to cluster iGluRs in vivo (Kim et al. 2015).This indicates that responses observed in outside-out patches are likely independent of receptor clustering.To allow comparison with prior studies on native extrajunctional Drosophila NMJ iGluRs, for which desensitization time constants with single exponential fits of 17.5, 18.8 and 2.0 ms were reported for wild type, type-A and type-B, respectively (DiAntonio et al. 1999), a weighted value (w) was calculated (see Methods).We find that, type-A recombinant NMJ iGluRs channels have faster desensitization compared to native wild type and native type-A receptors, with weighted decay time constants w A/α, 2.65 ± 0.35 ms and A/β, 5.18 ± 0.70 ms, while the rate of desensitization of B/α, 1.12 ± 0.15 ms and B/β, 1.83 ± 0.15 ms, respectively, is comparable to native type-B iGluRs at the NMJ.
In many recordings from patches with macroscopic currents we observed trial to trial fluctuations in amplitude for both short (1 ms) and long (100 ms) applications of glutamate.Given the large number of channels in each patch (around 10-50), these fluctuations likely occur because the open probability (Po) is relatively low.We examined this possibility using non stationary analysis of variance (Supplemental figure S1A-D, quantified in S1E-F).In many patches the open probability was too low to accurately estimate, range (0.1-0.6), consistent with a prior study that reported a value of 0.4 estimated from binomial analysis of single channel current amplitudes (Heckmann et al. 1996).Estimates of the single channel conductance obtained from non-stationary analysis of variance, 160 ± 23 pS, 164 ± 22 pS and 149 ± 39 pS for A/α, A/β and B/α, respectively, gave values comparable to those reported previously from single channel recordings from extra-junctional NMJ iGluRs (Chang et al. 1994;DiAntonio et al. 1999).In many

Modulation of Drosophila NMJ iGluR single channel responses by Concanavalin A
The lectin Concanavalin A (Con A) attenuates iGluR desensitization for a wide variety of species, including locust NMJ iGluRs (Mathers and Usherwood 1976), native vertebrate kainate receptors (Huettner 1990;Wong and Mayer 1993) and AvGluR1 from the primitive eukaryote Adineta vaga (Lomash et al. 2013).In prior work, which used Xenopus oocytes to study recombinant Drosophila NMJ iGluRs, whole cell responses to glutamate were only detectable after treatment with Con A. In the present study used single channel recording to study the effects of Con A. We found that by reducing the incubation time after transfection from three to two days, we could obtain patches where only single channel currents could be detected (Figure 2A-B).For control patches, the single channel conductance at -60 mV measured using individual openings was A/α 175 ± 12 pS (n=14), A/β 172 ± 11 pS (n=16), B/α 173 ± 12 pS (n=10) and B/β 169 ± 9 pS (n=11), similar to estimates from nonstationary analysis of variance (Figure 1-figure supplement 1).From averages of 16-57 responses from single channel patches we estimated desensitization time constants by fitting single exponentials: des A/α 2.04 ± 0.32 ms (n=6), des A/β 3.71 ± 0.40 ms (n=8), des B/α 1.05 ms (n=2) and des B/β 1.54 ± 0.20 ms (n=3); these values are comparable to those obtained for macroscopic currents recorded from multichannel patches (Figure 1 and Table 1) with faster desensitization for type-B versus type-A and for Neto-α versus Neto-β.
To study the effect of Con A on single channel activity, we treated HEK cells transfected with iGluR/Neto combinations with 0.6 mg/ml Con A for 10 min, and then excised outside-out patches to record responses to 10 mM glutamate applied for 100 ms.We found that patches from HEK cells treated with Con A were less stable, and that it was more difficult to obtain giga-ohm seals, but in exceptional cases sufficient data was obtained for kinetic analysis.In control patches not treated with Con A, single channel events were observed only at the start of the application of glutamate (Figure 2A-B) reflecting the rapid onset of desensitization observed for macroscopic responses (Figure 1).By contrast, pretreatment with Con A dramatically increased single channel activity, revealing major differences between type-A and type-B receptors and also between Neto splice variants.For A/α the response to glutamate showed much slower onset desensitization, time constant 15.5 ms (range 10-26 ms, n=3) 6-fold slower after treatment with Con A versus the value for control patches, time constant 2.65 ms, with very few openings 70 ms after the start of the application of glutamate (Figure 2C).By contrast, for A/β, B/α, and B/β there was a complete block for some patches, while the average current revealed variable extents of desensitization from patch to patch (Figure 2C-D).After treatment with Con A single channel activity for A/α and especially A/β consisted of bursts of long duration openings, with few closures within a burst (Figure 2C); by contrast, bursts for B/α and B/β were interrupted by brief closures at high frequency (Figure 2D).Following termination of the application of glutamate, the average response after treatment with Con A showed tail currents with fast decays, time constant A/α 1.9 ms ± 0.2 ms (n=4); A/β 3.8 ms ± 0.4 ms (n=5); B/α 0.9 ms ± 0.2 ms (n=4); B/β 1.2 ms ± 0.1 ms (n=3), suggesting that even after application of Con A glutamate dissociates rapidly following channel closure.2G).Calculation of the charge transfer by integration of raw data in response to application of glutamate for 100 ms revealed substantial differences between type-A and type-B receptors, and also between Neto-α versus Neto-β.For control patches the charge transfer was A/α 0.37 ± 0.16 pC (n=4), A/β 0.47 ± 0.12 pC (n=8), B/α 0.12 pC (n=2) and B/β 0.14 ± 0.01 (n=3) pC, with a large increase for patches treated with Con A (Figures 2F and 2H), charge transfer A/α 3.33 ± 0.83 pC (n=5), A/β 5.14 ± 1.38 pC (n=6), B/α 1.34 ± 0.59 pC (n=5) and B/β 2.27 ± 1.25 pC (n=5).
Estimates of open probability were obtained from analysis of all point amplitude histograms for pooled data from 2-8 patches (Figure 2I-J).Because the open state histograms were not well fit by single gaussian functions, we calculated the open probability (Po) by integration which gave control values of 0.039, 0.044, 0.015 and 0.016 for A/α, A/β, B/α and B/β and 0.37, 0.55, 0.20 and 0.29 for patches treated with Con A (Figure 2I-J).

Single channel kinetics and subconductance states
Closer inspection of single channel records for A/α and A/β complexes revealed bursts of openings, during which the current fluctuated between well-defined subconductance states with amplitudes of 50% and 75% of the open state conductance (Figure 3A-B); we did not observe openings to 25%, but it is possible these might occur with lower concentrations of glutamate.
Using a cutoff time of 200 µs, open time and burst duration histograms for A/α and A/β were well fit by the sum of two exponentials for both control patches, and for patches from HEK293T cells pretreated with Con A (Figures 3C, 3E, and Supplemental Table 1).For A/α this analysis revealed a 2-4 fold increase in the duration of the slow component of the open time, 3.75 versus 7.53 ms, and burst length, 3.12 versus 11.53 ms, for control and Con A respectively.For A/β the duration of the fast component of the burst length, 1.28 versus 3.32 ms, and open time, 1.11 versus 5.60 ms, also increased 2.6 and 5-fold, respectively for Con A treated patches (Figure 3D).Notably, for type-A/β the duration of the slow component of the open time and burst length distribution increased even more dramatically, from control values of 4.51 and 4.38 ms, to events of duration longer than 100 ms (Figure 3E and Supplemental Table 1), with for many trials channel closure only after the termination of the application of glutamate (Figure 2C).   1. Analysis of single channel kinetics for recombinant type A/α and A/β receptor complexes for control patches, and patches obtained from HEK cells treated with Con A. Closed time histograms revealed bimodal distributions for all conditions, but except for A/ + Con A the number of events was too small to allow an accurate estimate of the lifetime.Log binned open time and burst length distributions were fit with the sum of two exponentials as shown below (Figure 3).For receptor complexes with Con A we observed single openings and bursts of openings which exceeded the length of the 100 ms application of glutamate.Examination of the lifetime of openings in a burst revealed longer openings for each subconductance state after application of Con A for both A/α and A/β (Supplemental figure S2).

Native iGluRs
Closed time ms Open time ms Burst Length Nishikawa 1995 Tau crit 1 ms NR 0.069, 0.9, 4.9  substates are well represented during a burst, we conclude that, upon desensitization block, A/α channels transit between all subconductance states.In contrast, approximately 50% of A/β channel bursts showed few substate transitions, while the other 50% had ~100 transitions per burst, suggesting modal gating.Due to brief lifetime of openings observed for type-B receptors (Figure 2B) we did not attempt a similar single channel kinetic analysis, but note that inspection of the raw data suggests that the burst length of B/α is shorter than for B/β similar to the behavior observed for A/α and A/β.

Use dependent block by external philanthotoxin
Ca 2+ -permeable vertebrate kainate receptors are blocked by extracellular philanthotoxin (PhTx), a polyamine toxin derived from wasp venom (Eldefrawi et al. 1988;Bahring and Mayer 1998).Because native Drosophila NMJ iGluRs have high Ca 2+ -permeability (Chang et al. 1994) we tested the effects on PhTX on multi-channel outside-out patches at a concentration of 1 µM, while recording the response of type-A and type-B receptors to glutamate (Figures 4A and 4D).
Before application of PhTx the mean charge transfer in response to 100 ms applications of 10 mM glutamate was: A/α, 4.08 ± 1.13 pC; A/β, 4.68 ± 1.77 pC; B/α, 0.17 ± 0.04 pC; B/β, 0.40 ± 0.14 pC.Similar to the macroscopic currents shown in Supplemental figure S1, we observed trial to trial amplitude variations for all channel combinations.Inhibition by PhTx developed slowly, onset 78 s and 56 s for A/β and B/β, respectively (Supplemental figure S4), but at equilibrium, PhTx substantially reduced the charge transfer in response to a 100 ms application of glutamate to 1.6% ± 0.2 (n = 5) and 1.2% ± 0.3 (n = 5) of control, for type A/α and A/β channels respectively.
For type-B channels, block at equilibrium was weaker, with the charge transfer reduced to only 16.7% ± 2.5 for B/α (n = 4) and 25.3% ± 0.9 for B/β (n = 4) compared to control.This difference in PhTx-induced block between type-A and type-B receptors resembles that observed in prior experiments using Xenopus oocytes to study block by the structurally related channel blocker argiotoxin (Han et al. 2015).
The reduction in charge transfer by PhTX results from two effects.First, the number of channels that open in response to glutamate progressively decreases in the presence of PhTx (Figures 4A    and 4D).Second, the rate of decay of the response to glutamate, which in control conditions results from the onset of desensitization, increases; this is most likely due to a combination open channel block by PhTx combined with the onset of desensitization.Indeed in the presence of PhTx, the kinetics of decay were substantially faster, as estimated from the average response to 15-30 applications of glutamate recorded immediately before and after the start of the application of toxin, w A/α 2.84 ± 0.20 ms before and 1.87 ± 0.08 ms after PhTx (n = 5); w B/α 1.37 ± 0.14 ms versus 0.99 ± 0.09 (n = 4); w A/β 5.19 ± 0.39 ms versus 2.79 ± 0.23 (n = 5); and w B/β 1.80 ± 0.15 ms versus 1.32 ± 0.07 (n = 4).Most of this change was due to an increase in the fast component of decay of the response to glutamate (Table 2).The slow rate of onset of block by PhTx (Figures 4A and 4D

Native NMJ iGluRs
Differences between the properties of native and recombinant neurotransmitter receptors have been instrumental in the discovery of novel auxiliary proteins and synaptic modulators (Jackson and Nicoll 2011).Drosophila NMJ iGluR receptor channels are assembled from either of two alternative subunits, GluRIIA and GluRIIB, combined with two Neto isoforms.Null mutants are available for each of the four variants (DiAntonio et al. 1999;Kim et al. 2015;Han et al. 2020), permitting the in vivo isolation of individual receptor types with a single Neto isoform; this facilitates a direct comparison with recombinant iGluR/Neto channels expressed in HEK293T cells.We generated third instar larvae with single copies of either the GluRIIA or GluRIIB genes, and either neto-α or neto-β.Mutants with one or two copies of GluRIIB and neto-α (neto-β null ; GluRIIA null ) are embryonic lethal and thus could not be studied.
We recorded from outside-out patches obtained from the larval muscle membrane (muscle 6, abdominal segment 3) of animals with defined receptor complexes and compared the deactivation kinetics of responses to 1 ms applications of 10 mM glutamate with those of recombinant receptors of the same subunit composition expressed in HEK cells.We found that native, extrajunctional channels have slower deactivation kinetics than recombinant receptors (Figure 5A-B and Figure 1 and Table 1), A/α off, 1.57 ± 0.27 ms in muscle patches versus 0.64 ± 0.07 ms in HEK cells, and A/β off, 1.60 ± 0.29 ms versus 0.90 ± 0.11 ms, (n = 5 or 6).A less pronounced but similar trend was observed for B/β: recombinant receptors had faster deactivation off, 0.58 ± 0.06 ms, than the native extrajunctional complexes off, 0.97 ± 0.24 ms (Figure 5C).In contrast the single conductance : A/α, 169.3 ± 2.9 pS; A/β, 171.1 ± 3.51 pS; B/β, 172.6 ± 3.5 pS for native receptors was not different from values obtained for recombinant receptors.We next recorded mEJCs at the larval NMJ in mutants with defined receptor complexes and estimated the decay time for synaptic currents fit with single exponential functions (Figure 5D-F).The mEJC decay time constant was much slower than the deactivation time constant for both type-A and type-B extrajunctional receptors, mEJC: A/α, 4.20 ± 0.42 ms (n = 5); A/β, 6.50 ± 0.28 ms (n = 6); B/β, 4.92 ± 0.23 ms (n = 5).The slow decay cannot be explained by asynchronous release at multiple junctional sites since we recorded miniature EJCs.Instead, our data suggest that either the clearance of glutamate from the synaptic cleft is slow compared to other synapses, or that additional auxiliary membrane proteins and/or cytoplasmic modulatory proteins are present and impact the gating of synaptic receptors.
To compare the kinetics of mEJCs with the kinetics of desensitization we next recorded responses to 100 ms applications of 10 mM glutamate from native extrajunctional receptors.This revealed that the desensitization time constant for A/α, 4.60 ± 0.96 ms (n = 5) was nearly identical to the mEJC decay time constant of 4.20 ms.The desensitization time constant for native A/, 9.4 ± 2.6 ms (n = 6) was 1.4-fold slower than the mEJC decay time constant of 6.5 ms.Curiously, the desensitization time constant for native B/, 2.74 ± 0.78 ms (n = 5) was 1.8-fold faster than the mEJC decay time constant of 4.9 ms.reflecting differences in the subunit and auxiliary proteins present in different iGluR complexes (Barbour et al. 1994;Raman et al. 1994).The different kinetics of deactivation and desensitization for recombinant receptors and native extrajunctional channels observed in our experiments may reflect differences in post-translational modifications or lipid microenvironments surrounding these channels in HEK cells versus larval muscle.Alternatively, as yet unidentified additional accessory subunits or trans-synaptic proteins might modulate receptor function.
We found that the decay time constants of mEJCs, recorded from larvae with genetically controlled receptor and Neto splice variant composition, were 3-5 times slower than for deactivation measured for native extrajunctional receptor-Neto complexes of the same composition.By contrast, the rate of decay mEJC was nearly identical to the rate of desensitization for A/α, mEJC 4.2 ms, w 4.6 ms; for A/ the rate of desensitization was 1.4-fold slower than the decay of the synaptic current, mEJC 6.5 ms, w 9.4 ms; unexpectedly, for B/ the rate of desensitization was 1.8-fold faster than the synaptic current, mEJC 4.9 ms and w 2.7 ms.
Because animals with B/α receptors die during late embryogenesis, and are unable to hatch, it is likely that B/α receptors deactivate and desensitize extremely rapidly and cannot sustain normal synaptic transmission and muscle contraction.The rates of desensitization of A/α and A/ complexes were significantly slower than for the previously reported A/α+ complexes, w 4.6 ms and 9.4 ms versus 19 ms, respectively (DiAntonio et al. 1999); this may reflect an interaction between Neto-α and Neto- when bound to the same cluster of heterotetrameric receptors or may be caused by the association of A/α+ complexes with other modulators.In the larval muscle, Neto- is ~10 fold more abundant than Neto-α, but both isoforms contribute to the proper assembly of the PSDs (Ramos et al. 2015;Han et al. 2020).
Overall, our results suggest that desensitization plays a major role in determining the kinetics of synaptic transmission at the larval NMJ.This result explains previous observations that the K661E mutation within the M3-S2 linker of GluRIIA, which effectively blocks desensitization of vertebrate homomeric GluA2 (Yelshansky et al. 2004), slows synaptic current decays at GluRIIA K661E -containing larval NMJs (Petzoldt et al. 2014).By contrast, the mEPSC decay time constant in GluRIIA mutants that correspond to fast-desensitizing vertebrate GluA2 mutants resembles the fast kinetics of type-B receptors.This critical role for desensitization in the kinetics of synaptic transmission may constitute an adaptation of larval NMJ to the high concentration of glutamate (1.8 -2.0 mM) in the hemolymph, which is actively maintained (Augustin et al. 2007;He et al. 2023).Mutations that reduce the hemolymph glutamate concentration (to ~1 mM) or culturing larvae in glutamate-depleted conditions trigger a 250-500% increase in the levels of postsynaptic type-A and B receptors.Interestingly, application of Con A to glutamate-depleted larvae completely blocked the increase in iGluR clustering (Augustin et al. 2007), indicating that glutamate-mediated suppression of iGluR synaptic recruitment might depend on receptor desensitization.Of note, alterations in ambient extracellular glutamate also dramatically alters glutamatergic neurotransmission in cultured vertebrate hippocampal neurons (Lissin et al. 1999).
We are not aware of any reports for which the synaptic current decay at vertebrate synapses is determined exclusively by the rate of desensitization.However, for some vertebrate CNS glutamatergic synapses desensitization has been shown to contribute to a slow component of synaptic currents (Barbour et al. 1994;Koike-Tani et al. 2005).In addition, desensitization shapes the response to multiquantal neurotransmitter release, and to paired pulse stimulation at vertebrate glutamatergic synapses (Trussell et al. 1993).
In our experiments, mEJCs were recorded using two-electrode voltage clamp which revealed rise times of around 1.5 ms and decay time constants of 4-6 ms, which varied with subunit composition.Previous recordings from control animals, which express both type-A and type-B receptors or GluRIIA null mutants, with only type-B receptors, reported decay time constants of 7.73 ± 1.52 ms and 2.95 ± 0.65 ms, respectively (Petzoldt et al. 2014).Furthermore, a survey of the literature, for which we manually digitized published records for mEJC decay obtained using extracellular focal recording (Stewart et al. 1994;Cooper et al. 1995;Heckmann and Dudel 1998;Dawson-Scully et al. 2007;Karunanithi et al. 2018), yielded a mean value for mEJC of 4.37 ± 0.17 ms (n = 7), range 2.5 to 7.7 ms, similar to values recorded in the present experiments.In prior studies the rise time of mEJCs measured using focal recording varied from 0.35 to 1.1 ms (Heckmann and Dudel 1998;Paul et al. 2015) slightly faster than we and others (Petzoldt et al. 2014) recorded using two-electrode voltage clamp.Taken together, our results suggest that the time course of decay of the concentration of glutamate in the synaptic cleft of the larval NMJ is slow compared to conventional synapses and determined by desensitization and not deactivation.

Modulation of desensitization by Con A and single channel kinetics
Similar to vertebrate kainate receptors (Huettner 1990;Wong and Mayer 1993), application of was only possible after application of Con A (Han et al. 2015), and it remained possible that receptors expressed in the absence of Neto were activated by glutamate, but desensitized too rapidly to detect using oocyte recording.In the experiments reported here we used outside out patches with sub ms solution exchange and found that in the absence of Neto, Drosophila NMJ iGluRs are not activated by glutamate on a physiological time scale; this explains why neto null mutants are embryonic lethal (Kim et al. 2012).We cannot exclude the possibility that receptors which traffic to the membrane desensitize so rapidly in the absence of Neto that the channel does not open, as found for the AMPA receptor S750D and E755A mutants (Partin et al. 1996;Horning and Mayer 2004).
Prior studies on native Drosophila NMJ iGluRs have largely focused on measurements of single channel conductance (Broadie and Bate 1993a;Nishikawa and Kidokoro 1995;Heckmann and Dudel 1997;DiAntonio et al. 1999).There is good agreement among these studies with results obtained in the present experiments, which reveal a single channel conductance of 160 to 170 pS, with no difference between native and recombinant type-A and type-B receptors or Neto isoforms.Our experiments on recombinant Drosophila NMJ iGluRs revealed frequent transitions to subconductance states of 75% and 50% of the main state when activated by 10 mM glutamate.
This was observed for both control patches and for patches from cells pretreated with Con A, but were easier to detect in the latter condition due to an increase in the open time.The presence of subconductance states is a widely observed feature of the gating of vertebrate AMPA and kainate receptors (Rosenmund et al. 1998;Daniels et al. 2013;Coombs and Cull-Candy 2021;Baranovic et al. 2022) and it is surprising that this has not been reported before for native extrajunctional Drosophila NMJ iGluRs.Inspection of published raw data from prior single channel recording experiments reveals hints of substate activity, though it is possible that this results from brief transitions between fully open and closed states that were not resolved (Heckmann and Dudel 1995;Nishikawa and Kidokoro 1995;Heckmann and Dudel 1997;DiAntonio et al. 1999).In the future, it will be interesting to expand the analyses of substates and compare the behavior of recombinant and extrajunctional receptors at physiological glutamate concentrations.
For measurement of single channel lifetimes we used a cutoff value of 200 µs for generating the list of idealized events, and a critical time of 2 ms for burst length analysis; as a consequence we did not resolve brief events with µs lifetimes reported in prior studies on native receptors (Heckmann and Dudel 1995;Chang and Kidokoro 1996).Due to the limited number of single channel events recorded in control patches as a result of rapid desensitization, combined with their short duration, we did not calculate closed time distributions, and we limited our analysis to recombinant type-A receptors for which openings were well resolved.For control patches the open time distributions were comparable to those reported for wild type extrajunctional receptors, as summarized in Supplemental table 1 (Broadie and Bate 1993b;Heckmann and Dudel 1995;Nishikawa and Kidokoro 1995), while for patches from HEK cells treated with Con A there was a substantial increase in open time.For the burst length distribution there is only a single observation in the literature for native iGluRs, with medium and long duration events of life time 0.9 and 4.9 ms (Nishikawa and Kidokoro 1995) 1).In addition we analyzed the open time distribution of substates within bursts and found additional subtype specific differences (Figure 3 -figures supplement 1 and 2, and Table 1).

Channel block by polyamine toxins
Similar to vertebrate Ca 2+ -permeable receptors (Bowie and Mayer 1995;Kamboj et al. 1995) we previously found that recombinant Drosophila NMJ iGluRs expressed in Xenopus oocytes show biphasic rectification and were blocked by Argiotoxin (Han et al. 2015).Prior studies on Drosophila NMJ synaptic responses also revealed biphasic rectification (Broadie and Bate 1993b;Nishikawa and Kidokoro 1995), with similar voltage dependence to that produced by polyamines indicating that channel block by cytoplasmic polyamines modulates synaptic transmission in vivo.
This behavior is likely critical to flies, which live on rotten fruits and tolerate large amounts of polyamines in their food.Interestingly, disruption of the polyamine metabolism in the fly muscles causes progressive locomotor defects that can be rescued by polyamine supplementation (Coni et al. 2021).
Block of Drosophila NMJ iGluRs by philanthotoxin has particular relevance for studies of synaptic plasticity at the NMJ in response to application of PhTX.In our experiments we found that PhTx produces a slow onset cumulative block of responses to sequential applications of glutamate, as well as an acceleration in the rate of decay of individual responses to glutamate (Fig. 5 and Table S2).At the Drosophila NMJ, manipulations that reduce postsynaptic iGluR activity trigger a compensatory increase in neurotransmitter release (DiAntonio et al. 1999;Frank et al. 2006).This form of plasticity has been intensely studied using two experimental paradigms: (1) a chronic (developmental) response in GluRIIA null mutants, and (2) the acute response to PhTx

Fly strains and in vivo recordings
Four different types of mutations were used to study the gating properties of Drosophila NMJ glutamate receptor channels by receptor subunit and Neto isoform composition: GluRIIA SP16 (Petersen et al. 1997), GluRIIB[Mi03631] (BDSC# 37066), neto-α null (Han et al. 2020) and netoβ null (Ramos et al. 2015).These GluR mutants were crossed with a deficiency covering both GluRIIA and GluRIIB loci, Df(2L)cl h4 (Petersen et al. 1997), similarly neto alleles were crossed with a neto null mutant (Kim et al. 2012).To study the gating properties of extrasynaptic receptor channels using outside-out patch recording as described above, wandering third instar larvae were dissected in ice-cold, calcium-free hemolymph-like HL-3 saline, then incubated with 30 μg/ml collagenase type IV (Sigma-Aldrich) for 10 min, washed with calcium-free HL-3 saline and moved to the recording chamber.The calcium-free HL-3 saline contained (in mM): 70 NaCl, 5 KCl, 20 MgCl2, 10 HCO3, 5 trehalose, 115 sucrose, 5 HEPES (pH 7.2).To examine the properties of synaptic receptor channels, two-electrode voltage-clamp recordings were performed on from muscle 6, segment A3 at room temperature as described previously (Qin et al. 2005).The

Figure 1 .
Figure 1.Differential modulation of Drosophila NMJ iGluRs by Neto isoforms.(A-B) Responses to 10 mM glutamate applied for 1 ms (upper traces) and 100 ms (lower traces) to outside-out patches from HEK293T cells transfected with GluRIIA/C/D/E (A) and GluRIIB/C/D/E (B) without Neto or in the presence of different Neto splice variants.Black lines show the average of 35-60 responses from one patch; red lines show the decay of the responses fitted with the sum of one (upper) or two (lower) exponential functions; open tip junction currents measured at the end of the experiments are shown at the top.The holding potential was -60mV for all recordings.(C) Diagram of Neto variants utilized.Drosophila Neto isoforms are expressed as pre-proteins; their inhibitory pro-domain must be cleaved at conserved Furin processing sites (marked by arrows) before Neto can promote formation of synaptic iGluR aggregates in vivo.PM denotes a processing mutant unable to shed the inhibitory pro-domain.(D-E) Summary graphs for deactivation, *p<0.05,(D) and desensitization time constants (E) for various iGluR/Neto complexes; the Afast (%) component for desensitization is shown in blue.
patches with macroscopic currents we observed steps corresponding to the closure or desensitization of individual channels (Supplemental figure S1A-D).Supplemental figure S1.Nonstationary analysis of variance of responses to 10 mM glutamate applied for 1 ms (upper traces) and 100 ms (lower traces) to outside-out patches from HEK cells transfected with various iGluR/Neto receptor complexes as indicated.(A-D) Superimposed individual responses are shown in black; the average current in red; open tip junction currents measured at the end of the experiments are shown at the top.The holding potential was -60mV for all recordings.The insets in each panel show the peak amplitude for all trials (left) and the current-variance relationship (right) fit with the function σ 2 =iI -I 2 /N, where σ 2 is the variance, i is the mean current, N is the number of channels.(E) The mean number of channels and the open probability (F) determined by variance analyses of responses for 3-5 patches for different iGluR/Neto channel complexes as indicated, showing responses for individual patches and the mean ± SEM.

Figure 2 .
Figure 2. Concanavalin A attenuates desensitization and increases single channel activity evoked by glutamate.(A and C) Responses for outside-out patches to 10 mM glutamate applied for 100 ms to HEK293T cells transfected with type-A receptors with or without Neto splice variants, as indicated, before (A) and after (C) treatment with 0.6 mg/ml Con A for 10 min (blue line); average responses (top, red line) and three single channel responses from one patch are shown for each channel variant.The same sequence is shown for type-B receptors (B and D).The holding potential was -60 mV for all recordings.Open tip junction currents measured at the end of the experiments are shown at the top.(E-F) Single channel conductance at -60 mV and the charge transfer for responses to glutamate measured by integration of 35-55 trials shown in panels (A and C) for type-A receptors.(G-H) show the same analysis for type-B receptors.(I-J) All points amplitude histograms plotted on a log scale before (black) and after (blue) treatment with Con A, normalized to the closed state peak at 0 pA.Note the shift in the peak profiles at -10 pA (open channels) after application of Con A and the asymmetric profile for the open state.Data are represented as mean ± SEM.

Figure 3 .
Figure 3. Analysis of single-channel kinetics for type-A receptors.(A-B) Filtered and idealized traces illustrating responses to 10 mM glutamate applied for 100 ms to outside-out patches from HEK293T cells transfected with A/α and A/β receptor complexes; left: control patches, right: patches obtained after treatment of HEK cells with 0.6 mg/ml Con A for 10 min; idealized traces, shown in red and blue, respectively, reveal the full open state and two subconductance states, 1/2 open and 3/4 open, as indicated by dotted lines.(C-D) open time histograms for A/α or A/β receptor complexes for control patches (left) and patches after treatment with Con A (right) fit with the sum of two exponential functions as indicated.(E-F) burst duration histograms fit with the sum of two exponential functions for A/α and A/β for control patches (left) and after treatment with Con A (right).

(
figures S2 and S3).The distribution of burst duration versus the number of transitions among substates further captured the different channel behaviors and indicated that A/α channels undergo ~50-100 substate transitions within a burst (Supplemental figureS3C-D).Since all the ) likely reflects open channel block and the low open channel probability (Supplemental figure S1) convolved with the limited time that the channel spends in the open state due to rapid onset of desensitization in response to glutamate.

Figure 4 .
Figure 4. Slow onset of block by external PhTx.(A and D) Representative traces for type-A and type-B receptors showing responses recorded from outside-out patches to 10 mM glutamate applied for 100 ms at an interval of 1 s before (red line) and after the application of 1 µM PhTx (blue line).The amplitude variation is due to differences in the number of channels activated from trial to trial.(B and C, E and F) Averages of 20 responses before PhTx or starting 5 s after the onset of PhTx application for (B) A/α; (C) A/β; (E) B/α; (F) B/β.Red lines show fits of double exponential functions, and reveal faster decay in

Figure 5 .
Figure 5. Distinct receptor properties observed for native and recombinant receptors.(A-C) Representative individual traces (left) and the average of 20-40 responses (right) to 10 mM glutamate applied for 1 ms to outside-out patches of native, extrajunctional receptors excised from body-wall muscles of third instar larvae with distinct iGluR/Neto complexes, as indicated.The red line shows time course of deactivation fit by an exponential function; open tip junction currents measured at the end of the experiments are shown at the top.(D-F) Miniature EJCs recorded from larvae of the same genotypes fit with single exponential functions.(G-I) Comparisons of deactivation kinetics for recombinant receptors expressed in HEK cells (R), native, extrajunctional receptors from larval muscle membranes (Ext) and mEJCs (Syn).(J-L) Average of 20-40 responses to 10 mM glutamate applied for 100 ms to outside-out patches of native, extrajunctional receptors for the same genotypes; the red line shows fits of the sum of two exponential functions; open tip junction currents measured at the end of the experiments are shown at the top.(M-O) Comparisons of desensitization kinetics for recombinant receptors expressed in HEK cells (R), native, extrajunctional receptors from larval muscle membranes (Ext) and mEJCs (Syn).Data are represented as mean ± SEM. **** p<0.0001, *** p<0.001, ** p<0.01, *p<0.05,ns, p>0.05.
Con A consistently attenuated (type A/) or blocked (A/, B/ and B/) desensitization, and increased the ratio of charge transfer, open time and burst duration of single channels expressed in HEK cells.The initial characterization of Drosophila NMJ iGluRs expressed in Xenopus oocytes

(
20 μM) applied to dissected larval fillets.The two settings appear to elicit genetically distinct responses(Frank 2014).Our findings that external application of PhTx blocks both type-A (>98%) but also type-B receptors (by ~80%) indicate that both type-A and type-B receptors are impaired during acute potentiation by PhTx application.Thus the two different plasticity paradigms differ in the time frame, developmental versus acute, but also in the nature of receptors affected, more specifically type-A receptors are absent in the developmental paradigm, while both type-A and type-B mediate the acute response.Since type-A and type-B receptors are recruited and stabilized at the NMJ via genetically distinct mechanisms(Parnas et al. 2001;Liebl and Featherstone 2008;Petzoldt et al. 2014;Ramos et al. 2015;Sulkowski et al. 2016) disruption of their activity is expected to elicit different compensatory mechanisms.developmental lethality, to defects in the assembly and maintenance of PSDs, which generally induce locomotor deficits, and finally to subtle changes in postsynaptic composition and impairments in synaptic plasticity.Within this wide range of biological outcomes, the investigations on fly NMJ iGluRs and their modulation by Neto have the potential to reveal new functions for iGluRs and Neto and new modalities of regulation.Our study paves the way to parse out and elucidate the multiple functions and regulation of kainate receptors and their auxiliary proteins Neto.
recording solution was HL-3 with 0.5 mM CaCl2.Intracellular electrodes (borosilicate glass capillaries of 1 mm diameter) were filled with 3 M KCl with resistances ranging from 12 to 25 MΩ.Recordings were done from muscle cells with an initial membrane potential between -50 and -70 mV, and input resistances of ≥ 4 MΩ.To record spontaneous miniature excitatory junction currents (mEJCs) the muscle cells were clamped to -80 mV.To calculate mEJCs mean amplitudes, 50-100 events from each muscle were measured and averaged using the MiniAnalysis program (Synaptosoft).Data analysisTo calculate deactivation and desensitization time constants, 50 ~ 100 representative responses were averaged and fit using a first order exponential function for deactivationI(t) = I exp(−t/off)and a double exponential function for desensitizationI(t) = Ifast exp(−t/fast) + Islow exp(−t/slow)where Ix is the peak current amplitude and x is the corresponding decay time constant.To allow for comparison of decay times with published values fit with single exponential functions, weighted time constants w were calculated using the formula w = (Ifast/(Ifast + Islow))* fast + (Islow/(Ifast + Islow))* slow and is also made available for use under a CC0 license.wasnot certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105