The Drosophila Postsynaptic DEG/ENaC Channel ppk29 Contributes to Excitatory Neurotransmission

Abstract

The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.

Keywords: DEG/ENaC; Drosophila melanogaster; NMJ; fruit fly; synapse.

Figure 1.

ppk29 mutants display normal homeostatic synaptic plasticity yet decreased response to spontaneous release at the larval NMJ. A, Representative traces of mEJPs and EJPs before and after treatment with the glutamate receptor antagonist PhTX. B, Compared with wild-type controls, ppk29 mutants display decreased mEJP amplitude. Both wild-type and ppk29 mutants display decreased mEJP amplitude following PhTX treatment. C, There is no effect of genotype or PhTX treatment on amplitude of evoked EJPs. D, ppk29 mutants display increased quantal content compared with wild-type controls, and both genotypes display increased quantal content by PhTX treatment compared with baseline. Data are presented as the average ± SEM. n = 8–16 recordings/group for all experiments. Letters above bars represent statistical significance, p < 0.05 (one-way ANOVA followed by Tukey's post hoc test).

Figure 2.

ppk29 plays a postsynaptic role in the response to spontaneous release. A, The decreased mEJP amplitude observed in ppk29 mutants is rescued by the expression of ppk29 cDNA in muscles (using the BG57-Gal4 driver) but not in neurons (using the elav-Gal4 driver). B, ppk29 mutation has no impact on mEJP frequency, yet overexpression of ppk29 cDNA in neurons (using the elav-Gal4 driver) decreases mEJP frequency. C, There is no effect of ppk29 mutation, rescue of ppk29 expression in muscles, or rescue in neurons on EJP amplitude. D, ppk29 mutation and rescue in muscle or neurons has no significant effect on quantal content. Data are presented as the average ± SEM. n = 8–19 recordings/genotype for all experiments. *p < 0.05 (one-way ANOVA followed by Tukey's post hoc tests).

Figure 3.

ppk29 mutants display impaired larval roll behavior, which is rescued by muscle overexpression. A, There are no significant differences in average crawling velocity between wild-type controls and ppk29 mutants. n = 8–9 larvae/genotype. B, ppk29 mutants display increased rollover time in a larval rollover assay. n = 12 larvae/genotype. C, ppk29 mutant phenotype in larval rolling is rescued by the overexpression of ppk29 cDNA in muscles (BG57-GAL4 promoter). n = 48 larvae/genotype. D, ppk29 mutant phenotype is not rescued by the overexpression of ppk29 cDNA in neurons (elav-Gal4 promoter). n = 48 larvae/genotype. E, sei mutants display no significant difference in larval roll behavior compared with wild-type controls. n = 24 larvae/genotype. F, Overexpression of sei cDNA in muscles decreases rollover time. n = 24 larvae/genotype. G, Overexpression of sei cDNA in neurons has no impact on larval roll behavior. n = 24 larvae/genotype. Data are presented as the average ± SEM. Letters above bars represent statistical significance, p < 0.05 (one-way ANOVA followed by Tukey's post hoc tests); *p < 0.05 (Student's t test).

Figure 4.

ppk29 mutants have normal presynaptic morphology and ultrastructure at the larval NMJ. A, B, Representative images of muscle 4 NMJs stained for HRP (blue), glutamate receptor subunit GluRIIC (red), and presynaptic active zone-associated protein BRP (green). C, D, ppk29 mutants display no significant difference in the number of presynaptic boutons or branches per NMJ, as assessed by HRP staining. E, ppk29 mutants display no significant difference in the number of active zones per NMJ, as assessed by BRP puncta. F, ppk29 mutants display no significant difference in the number of active zones unopposed by glutamate receptor clusters, as assessed by coimmunostaining for BRP and GluRIIC. Data analyzed using Student's t test and presented as the average ± SEM. n = 13–16 NMJs analyzed/genotype. Scale bars, 5 μm.

Figure 5.

ppk29 mutants have normal ultrastructure and synaptic vesicle size. A–D, Representative EM images of the larval NMJ show similar synaptic ultrastructure in wild-type (A, B) and ppk29 mutant (C, D) NMJs. E, Histogram of all synaptic vesicles counted shows similar distributions between wild-type and ppk29 mutants. F, Comparison of average vesicle diameter across genotypes shows similar average vesicle diameters. n = 7 boutons per genotype. Data analyzed using Student's t test and presented as average ± SEM. Scale bar, 500 nm.

Figure 6.

In ppk29 mutants, GluRIIA and GluRIIB mRNA expression levels are altered but display normal localization to the NMJ. A, Images of muscle 4 NMJs show normal clustering of GluRIIA (red) and GluRIIB (green) subunits. B, Quantification of fluorescence intensity from immunostained NMJ z-stacks does not reveal statistically significant differences in levels of glutamate receptor subunits. n = 4–6 images/genotype. C, qRT-PCR for glutamate receptor subunits shows that ppk29 mutants have increased levels of GluRIIA and decreased levels of GluRIIB, with no change in GluRIIC levels. n = 4 samples/genotype. Data are presented as the average ± SEM. *p < 0.05 (Student's t test). Scale bar, 5 μm.

Figure 7.

ppk29 mutants display decreased amplitude of spontaneous synaptic current, with no change in kinetics. A, Representative traces of two-electrode voltage-clamp recordings of spontaneous mEJCs from wild-type and ppk29 mutant muscles. B, Compared with wild-type controls, ppk29 mutants display decreased mEJC amplitude. C, ppk29 mutants display decreased current flow (charge) compared with wild-type controls. D, There is no effect of genotype on the decay time constant tau. n = 8–11 recordings/group for all experiments. Data are presented as the average ± SEM. *p < 0.05 (Student's t test).