An electrophysiology-based forward genetic screen has identified two genes, pickpocket11 (ppk11) and pickpocket16 (ppk16), as being necessary for the homeostatic modulation of presynaptic neurotransmitter release at the Drosophila neuromuscular junction (NMJ). Pickpocket genes encode Degenerin/Epithelial Sodium channel subunits (DEG/ENaC). This study demonstrates that ppk11 and ppk16 are necessary in presynaptic motoneurons for both the acute induction and long-term maintenance of synaptic homeostasis. ppk11 and ppk16 are cotranscribed as a single mRNA that is upregulated during homeostatic plasticity. Acute pharmacological inhibition of a PPK11- and PPK16-containing channel abolishes the expression of short- and long-term homeostatic plasticity without altering baseline presynaptic neurotransmitter release, indicating remarkable specificity for homeostatic plasticity rather than NMJ development. Finally, presynaptic calcium imaging experiments support a model in which a PPK11- and PPK16-containing DEG/ENaC channel modulates presynaptic membrane voltage and, thereby, controls calcium channel activity to homeostatically regulate neurotransmitter release (Younger, 2013).

Homeostatic signaling systems are believed to interface with the mechanisms of learning-related plasticity to achieve stable, yet flexible, neural function and animal behavior. Experimental evidence from organisms as diverse as Drosophila, mouse, and humans demonstrates that homeostatic signaling systems stabilize neural function through the modulation of synaptic transmission, ion channel abundance, and neurotransmitter receptor trafficking. In each experiment, the cells respond to an experimental perturbation by modulating ion channel abundance or synaptic transmission to counteract the perturbation and re-establish baseline function. Altered homeostatic signaling is hypothesized to contribute to the cause or progression of neurological disease. For example, impaired or maladaptive homeostatic signaling may participate in the progression of autism-spectrum disorders, posttraumatic epilepsy, and epilepsy (Younger, 2013).

The homeostatic modulation of presynaptic neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. The Drosophila neuromuscular junction (NMJ) is a prominent model system for the study of this form of homeostatic plasticity (Petersen, 1997; Davis, 1998; Davis, 2006; Weyhersmuller, 2011). At the Drosophila NMJ, decreased postsynaptic neurotransmitter receptor sensitivity is precisely counteracted by a homeostatic potentiation of neurotransmitter release, thereby maintaining appropriate muscle excitation. The homeostatic enhancement of presynaptic release is due to increased vesicle release without a change in active zone number (Petersen, 1997; Frank, 2006; Muller, 2012b; Younger, 2013 and references therein).

This process is referred to as 'synaptic homeostasis,' recognizing that it reflects a subset of homeostatic regulatory mechanisms that have been shown to stabilize neural function through modulation of ion channel gene expression and neurotransmitter receptor abundance (quantal scaling; Turrigiano, 2008; Marder, 2006; Younger, 2013 and references therein).

An electrophysiology-based, forward genetic screen has been pioneered to identify the mechanisms of synaptic homeostasis (Dickman, 2009; Muller, 2011). To date, a role has been ascribed for several genes in the mechanism of synaptic homeostasis including the Eph receptor (Frank, 2006), the schizophrenia-associated gene dysbindin (Dickman, 2009), the presynaptic CaV2.1 calcium channel (Frank, 2006, 2009), presynaptic Rab3 GTPase-activating protein (Rab3-GAP; Muller, 2011), and Rab3-interacting molecule (RIM; Muller, 2012b). An emerging model suggests that, in response to inhibition of postsynaptic glutamate receptor function, a retrograde signal acts upon the presynaptic nerve terminal to enhance the number of synaptic vesicles released per action potential to precisely offset the severity of glutamate receptor inhibition. Two components of the presynaptic release mechanism are necessary for the execution of synaptic homeostasis, increased calcium influx through presynaptic CaV2.1 calcium channels (Muller, 2012a) and a RIM-dependent increase in the readily releasable pool of synaptic vesicles (Muuller, 2012b). Many questions remain unanswered. In particular, how is a change in presynaptic calcium influx induced and sustained during synaptic homeostasis (Younger, 2013)?

This study reports the identification of two genes, pickpocket16 and pickpocket11, that, when mutated, block homeostatic plasticity. Drosophila pickpocket genes encode Degenerin/Epithelial Sodium channel (DEG/ENaC) subunits. Channels in this superfamily are voltage insensitive and are assembled as either homomeric or heteromeric trimers. Each channel subunit has two transmembrane domains with short cytoplasmic N and C termini and a large extracellular loop implicated in responding to diverse extracellular stimuli (Younger, 2013).

Little is known regarding the function of pH-insensitive DEG/ ENaC channels in the nervous system. DEG/ENaC channels have been implicated as part of the mechanotransduction machinery and in taste perception in both invertebrate and vertebrate systems. In Drosophila, PPK11 has been shown to function as an ENaC channel subunit that is required for the perception of salt taste (Liu, 2003b) and fluid clearance in the tracheal system, a function that may be considered analogous to ENaC channel activity in the mammalian lung (Liu, 2003a; Younger, 2013 and references therein).

This study demonstrates that ppk11 and ppk16 are coregulated during homeostatic synaptic plasticity and that homeostatic plasticity is blocked when gene is genetically deleted, when gene expression is disrupted in motoneurons, or when pickpocket channel function is pharmacologically inhibited. Advantage was taken of the fact that presynaptic homeostasis can be blocked pharmacologically to demonstrate that the persistent induction of homeostatic plasticity does not interfere with synapse growth and development. Homeostatic plasticity can be acutely and rapidly erased, leaving behind otherwise normal synaptic transmission. Finally, pharmacological inhibition of this pickpocket channel was demonstrated to abolish the homeostatic modulation of presynaptic calcium influx that was previously shown to be necessary for the homeostatic increase in neurotransmitter release (Muller, 2012a; Younger, 2013).

A model for DEG/ENaC channel function can be based on the well-established regulation of ENaC channel trafficking in the kidney during the homeostatic control of salt balance. Enhanced sodium reabsorption in the principle cells of the cortical collecting duct of the kidney is achieved by increased ENaC channel transcription and trafficking to the apical cell surface, which enhances sodium influx. Sodium is then pumped out of the basolateral side of the cell, accomplishing sodium reabsorption (Schild, 2010). By analogy, a model is proposed for synaptic homeostasis in which the trafficking of DEG/ENaC channels to the neuronal membrane, at or near the NMJ, modulates presynaptic membrane potential to potentiate presynaptic calcium channel activity and thereby achieve precise homeostatic modulation of neurotransmitter release (Younger, 2013).

This study provides evidence that a presynaptic DEG/ENaC channel composed of PPK11 and PPK16 is required for the rapid induction, expression, and continued maintenance of homeostatic synaptic plasticity at the Drosophila NMJ. Remarkably, ppk11 and ppk16 genes are not only required for homeostatic plasticity but are among the first homeostatic plasticity genes shown to be differentially regulated during homeostatic plasticity. Specifically, it was shown that expression of both ppk11 and ppk16 is increased 4-fold in the GluRIIA mutant background. It was also demonstrated that ppk11 and ppk16 are transcribed together in a single transcript and behave genetically as an operon-like, single genetic unit. This molecular organization suggests a model in which ppk11 and ppk16 are cotranscribed to generate DEG/ ENaC channels with an equal stoichiometric ratio of PPK11 and PPK16 subunits. This is consistent with previous models for gene regulation in Drosophila. However, the possibility cannot be ruled out that two independent DEG/ ENaC channels are upregulated, one containing PPK11 and one containing PPK16. The upregulation of ppk11 and ppk16 together with the necessity of DEG/ENaC channel function during the time when synaptic homeostasis is assayed, indicates that these genes are probably part of the homeostat and not merely necessary for the expression of synaptic homeostasis (Younger, 2013).

DEG/ENaC channels are voltage-insensitive cation channels that are primarily permeable to sodium (Ben-Shahar, 2011; Bianchi, 2002) and can carry a sodium leak current. A model for DEG/ENaC channel function during synaptic homeostasis can be based on the well-established regulation of ENaC channel trafficking in the kidney during the homeostatic control of salt balance. Enhanced sodium reabsorption in the principle cells of the cortical collecting duct of the kidney is triggered by aldosterone binding to the mineralocorticoid receptor. This increases ENaC channel transcription and trafficking to the apical cell surface, which enhances sodium influx. Sodium is then pumped out of the basolateral side of the cell, accomplishing sodium reabsorption (Schild, 2010; Younger, 2013 and references therein).

It is speculated that a retrograde, homeostatic signal from muscle triggers increased trafficking of a PPK11/16-containing DEG/ENaC channel to the neuronal plasma membrane, at or near the NMJ. Since the rapid induction of synaptic homeostasis is protein synthesis independent (Goold, 2007), the existence is hypothesized of a resting pool of PPK11/16 channels that are inserted in the membrane in response to postsynaptic glutamate receptor inhibition. If postsynaptic glutamate receptor inhibition is sustained, as in the GluRIIA mutant, then increased transcription of ppk11/16 supports a persistent requirement for this channel at the developing NMJ. Once on the plasma membrane, the PPK11/16 channel would induce a sodium leak and cause a moderate depolarization of the nerve terminal. This subthreshold depolarization would lead, indirectly, to an increase in action potential-induced presynaptic calcium influx through the CaV2.1 calcium channel and subsequent neurotransmitter release (Younger, 2013).

There are two major possibilities for how ENaC-dependent depolarization of the nerve terminal could potentiate calcium influx and evoked neurotransmitter release. One possibility, based on work in the ferret prefrontal cortex and Aplysia central synapses, is that presynaptic membrane depolarization causes action potential broadening through potassium channel inactivation, thereby enhancing both calcium influx and release. A second possibility is that subthreshold depolarization of the nerve terminal causes an increase in resting calcium that leads to calcium-dependent calcium channel facilitation. Consistent with this model, it has been shown at several mammalian synapses that subthreshold depolarization of the presynaptic nerve terminal increases resting calcium and neurotransmitter release through low-voltage modulation of presynaptic P/Q-type calcium channels. However, at these mammalian synapses, the change in resting calcium does not lead to an increase in action potential-evoked calcium influx, highlighting a difference between the mechanisms of homeostatic potentiation and the type of presynaptic modulation observed at these other synapses. The mechanism by which elevated basal calcium potentiates release at these mammalian synapses remains under debate. It should be noted that small, subthreshold depolarization of the presynaptic resting potential, as small as 5 mV, are sufficient to cause a 2-fold increase in release at both neuromuscular and mammalian central synapses. This is within a reasonable range for modulation of presynaptic membrane potential by pickpocket channel insertion. Unfortunately, it is not technically feasible to record directly from the presynaptic terminal at the Drosophila NMJ. Finally, it is noted that it remains formally possible that a PPK11/16-containing DEG/ENaC channel passes calcium, based upon the ability of mammalian ASIC channels to flux calcium (Younger, 2013).

This model might provide insight regarding how accurate tuning of presynaptic neurotransmitter release can be achieved. There is a supralinear relationship between calcium influx and release. Therefore, if changing calcium channel number is the mechanism by which synaptic homeostasis is achieved, then there must be very tight and tunable control of calcium channel number within each presynaptic active zone. By contrast, if homeostatic plasticity is achieved by ENaC-dependent modulation of membrane voltage, then variable insertion of ENaC channels could uniformly modulate calcium channel activity, simultaneously across all of the active zones of the presynaptic nerve terminal. Furthermore, if the ENaC channel sodium leak is small, and if presynaptic calcium channels are moderately influenced by small changes in resting membrane potential, then relatively coarse modulation of ENaC channel trafficking could be used to achieve precise, homeostatic control of calcium influx and neurotransmitter release. Again, these are testable hypotheses that will be addressed in the future (Younger, 2013).