The half-maximal effective concentration for disruption of synaptic homeostasis is 32 μM ( Figure S3), which is within the range expected for amiloride derivatives to act on Drosophila pickpocket channels ( Chen et al., 2010 and Boiko et al., 2012). We have also confirmed a block of synaptic homeostasis with a
second amiloride derivative EIPA (5-(n-ethyl-n-isopropyl)amiloride) at 20 μM ( Figure S4). From these data, we can make several conclusions. First, these Enzalutamide data demonstrate that the conductance of the pickpocket channel is required for synaptic homeostasis, since functional blockade is sufficient to erase homeostatic plasticity. Second, since homeostasis is restored upon washout, it demonstrates that the induction process remains intact, but expression is blocked by inhibition of the DEG/ENaC channel. Third, we now have a pharmacological reagent that blocks homeostatic plasticity. Since synaptic transmission reverts to the levels seen in an uncompensated synapse when Benzamil is applied ( Figure 7C), it demonstrates that synaptic homeostasis is, at least initially, a process that is layered on top of normal synaptic transmission. Finally, in
our assay, the axon is cut and only ∼200 μM of axon exists between our stimulation electrode and the NMJ. Therefore, the DEG/ENaC channel must DAPT function within these 200 μM of axon or within the NMJ itself. We next asked whether the PPK channel conductance is continuously required for the expression of synaptic homeostasis. First, Benzamil was applied after a 10 min incubation in PhTx and synaptic homeostasis was blocked (Figure 7D). Thus, ENaC channel function is necessary for the expression of synaptic homeostasis, not during the induction of the process. Next, Benzamil was applied Bay 11-7085 to the GluRIIA mutant and, again, synaptic homeostasis was completely blocked ( Figure 7E). Remarkably, quantal content in the GluRIIA mutant in the presence of Benzamil was identical to that observed for a wild-type NMJ in the presence of Benzamil ( Figure 7F). Thus, even when synaptic homeostasis has been persistently
engaged for the life of the synapse, it can be erased by blocking the function of the Benzamil-sensitive DEG/ENaC channel. Several controls were performed. It is well established that Benzamil acts to inhibit DEG/ENaC channels (Kleyman and Cragoe, 1988, Garty and Palmer, 1997 and Cuthbert and Fanelli, 1978). However, we sought additional evidence that Benzamil acts on a Drosophila PPK11/PPK16-containing channel. To do so, we tested whether animals that are doubly heterozygous for the ppk11 and ppk16 mutations (in cis) might have increased sensitivity to Benzamil. The double heterozygote has normal synaptic homeostasis ( Figure 7G). When a low concentration of Benzamil (25 μM) is applied to a wild-type NMJ, synaptic homeostasis is also normal ( Figure 7G).
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