Parkinson's disease-risk protein TMEM175 is a proton-activated proton channel in lysosomes.

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.

LRRC8 family proteins within lysosomes regulate cellular osmoregulation and enhance cell survival to multiple physiological stresses.

LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L706L707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s "bladder." When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.

Stac protein regulates release of neuropeptides.

Neuropeptides are important for regulating numerous neural functions and behaviors. Release of neuropeptides requires long-lasting, high levels of cytosolic Ca2+ However, the molecular regulation of neuropeptide release remains to be clarified. Recently, Stac3 was identified as a key regulator of L-type Ca2+ channels (CaChs) and excitation-contraction coupling in vertebrate skeletal muscles. There is a small family of stac genes in vertebrates with other members expressed by subsets of neurons in the central nervous system. The function of neural Stac proteins, however, is poorly understood. Drosophila melanogaster contain a single stac gene, Dstac, which is expressed by muscles and a subset of neurons, including neuropeptide-expressing motor neurons. Here, genetic manipulations, coupled with immunolabeling, Ca2+ imaging, electrophysiology, and behavioral analysis, revealed that Dstac regulates L-type CaChs (Dmca1D) in Drosophila motor neurons and this, in turn, controls the release of neuropeptides.

Degeneration of Injured Axons and Dendrites Requires Restraint of a Protective JNK Signaling Pathway by the Transmembrane Protein Raw.

The degeneration of injured axons involves a self-destruction pathway whose components and mechanism are not fully understood. Here, we report a new regulator of axonal resilience. The transmembrane protein Raw is cell autonomously required for the degeneration of injured axons, dendrites, and synapses in Drosophila melanogaster In both male and female raw hypomorphic mutant or knock-down larvae, the degeneration of injured axons, dendrites, and synapses from motoneurons and sensory neurons is strongly inhibited. This protection is insensitive to reduction in the levels of the NAD+ synthesis enzyme Nmnat (nicotinamide mononucleotide adenylyl transferase), but requires the c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase and the transcription factors Fos and Jun (AP-1). Although these factors were previously known to function in axonal injury signaling and regeneration, Raw's function can be genetically separated from other axonal injury responses: Raw does not modulate JNK-dependent axonal injury signaling and regenerative responses, but instead restrains a protective pathway that inhibits the degeneration of axons, dendrites, and synapses. Although protection in raw mutants requires JNK, Fos, and Jun, JNK also promotes axonal degeneration. These findings suggest the existence of multiple independent pathways that share modulation by JNK, Fos, and Jun that influence how axons respond to stress and injury.SIGNIFICANCE STATEMENT Axonal degeneration is a major feature of neuropathies and nerve injuries and occurs via a cell autonomous self-destruction pathway whose mechanism is poorly understood. This study reports the identification of a new regulator of axonal degeneration: the transmembrane protein Raw. Raw regulates a cell autonomous nuclear signaling pathway whose yet unknown downstream effectors protect injured axons, dendrites, and synapses from degenerating. These findings imply that the susceptibility of axons to degeneration is strongly regulated in neurons. Future understanding of the cellular pathway regulated by Raw, which engages the c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase and Fos and Jun transcription factors, may suggest new strategies to increase the resiliency of axons in debilitating neuropathies.

Concomitant differentiation of a population of mouse embryonic stem cells into neuron-like cells and schwann cell-like cells in a slow-flow microfluidic device.

BACKGROUND: To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining spiral ganglion neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived "neurons" could potentially substitute for lost or damaged SGN. mESC-derived "Schwann cells" produce MIF, as do all Schwann cells (Huang et al., a; Roth et al., 2007; Roth et al., 2008) and could attract SGN to a "cell-coated" implant. RESULTS: Neuron- and Schwann cell-like cells were produced from a common population of mESCs in an ultra-slow-flow microfluidic device. As the populations interacted, "neurons" grew over the "Schwann cell" lawn, and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing "Schwann cells" were used to coat a CI: Mouse SGN and MIF-induced "neurons" grew directionally to the CI and to a wild-type but not MIF-knockout organ of Corti explant. CONCLUSIONS: Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. Developmental Dynamics 246:7-27, 2017. © 2016 Wiley Periodicals, Inc.

The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.

Sodium and potassium currents influence Wallerian degeneration of injured Drosophila axons.

Axons degenerate after injury and in neuropathies and disease via a self-destruction program whose mechanism is poorly understood. Axons that have lost connection to their cell bodies have altered electrical and synaptic activities, but whether such changes play a role in the axonal degeneration process is not clear. We have used a Drosophila model to study the Wallerian degeneration of motoneuron axons and their neuromuscular junction synapses. We found that degeneration of the distal nerve stump after a nerve crush is greatly delayed when there is increased potassium channel activity (by overexpression of two different potassium channels, Kir2.1 and dORKΔ-C) or decreased voltage-gated sodium channel activity (using mutations in the para sodium channel). Conversely, degeneration is accelerated when potassium channel activity is decreased (by expressing a dominant-negative mutation of Shaker). Despite the effect of altering voltage-gated sodium and potassium channel activity, recordings made after nerve crush demonstrated that the distal stump does not fire action potentials. Rather, a variety of lines of evidence suggest that the sodium and potassium channels manifest their effects upon degeneration through changes in the resting membrane potential, which in turn regulates the level of intracellular free calcium within the isolated distal axon.

A role for the calmodulin kinase II-related anchoring protein (αkap) in maintaining the stability of nicotinic acetylcholine receptors.

αkap, a muscle specific anchoring protein encoded within the Camk2a gene, is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChRs). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated, and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells and thus the maintenance of postsynaptic receptor density and synaptic function.

High potency zinc modulation of human P2X2 receptors and low potency zinc modulation of rat P2X2 receptors share a common molecular mechanism.

Human P2X2 receptors (hP2X2) are strongly inhibited by zinc over the range of 2-100 µM, whereas rat P2X2 receptors (rP2X2) are strongly potentiated over the same range, and then inhibited by zinc over 100 µM. However, the biological role of zinc modulation is unknown in either species. To identify candidate regions controlling zinc inhibition in hP2X2 a homology model based on the crystal structure of zebrafish P2X4.1 was made. In this model, His-204 and His-209 of one subunit were near His-330 of the adjacent subunit. Cross-linking studies confirmed that these residues are within 8 Å of each other. Simultaneous mutation of these three histidines to alanines decreased the zinc potency of hP2X2 nearly 100-fold. In rP2X2, one of these histidines is replaced by a lysine, and in a background in which zinc potentiation was eliminated, mutation of Lys-197 to histidine converted rP2X2 from low potency to high potency inhibition. We explored whether the zinc-binding site lies within the vestibules running down the central axis of the receptor. Elimination of all negatively charged residues from the upper vestibule had no effect on zinc inhibition. In contrast, mutation of several residues in the hP2X2 middle vestibule resulted in dramatic changes in the potency of zinc inhibition. In particular, the zinc potency of P206C could be reversibly shifted from extremely high (∼10 nM) to very low (>100 µM) by binding and unbinding MTSET. These results suggest that the cluster of histidines at the subunit interface controls access of zinc to its binding site.

TRPM7 is required within zebrafish sensory neurons for the activation of touch-evoked escape behaviors.

Mutations in the gene encoding TRPM7 (trpm7), a member of the Transient Receptor Potential (TRP) superfamily of cation channels that possesses an enzymatically active kinase at its C terminus, cause the touch-unresponsive zebrafish mutant touchdown. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles harbor mutations that abolish channel activity. Through the selective restoration of TRPM7 expression in sensory neurons, we found that TRPM7's kinase activity and selectivity for divalent cations over monovalent cations were dispensable for touch-evoked activation of escape behaviors in zebrafish. Additional characterization revealed that sensory neurons were present and capable of responding to tactile stimuli in touchdown mutants, indicating that TRPM7 is not required for sensory neuron survival or mechanosensation. Finally, exposure to elevated concentrations of divalent cations was found to restore touch-evoked behaviors in touchdown mutants. Collectively, these findings are consistent with a role for zebrafish TRPM7 within sensory neurons in the modulation of neurotransmitter release at central synapses, similar to that proposed for mammalian TRPM7 at peripheral synapses.

touché Is required for touch-evoked generator potentials within vertebrate sensory neurons.

The process by which light touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing channelrhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP-positive peripheral neurites. In wild-type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild-type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that cutaneous mechanoreceptors with generator potentials are necessary for responsiveness to light touch in zebrafish.

Dstac Regulates Excitation-Contraction Coupling in Drosophila Body Wall Muscles.

Stac3 regulates excitation-contraction coupling (EC coupling) in vertebrate skeletal muscles by regulating the L-type voltage-gated calcium channel (Cav channel). Recently a stac-like gene, Dstac, was identified in Drosophila and found to be expressed by both a subset of neurons and muscles. Here, we show that Dstac and Dmca1D, the Drosophila L-type Cav channel, are necessary for normal locomotion by larvae. Immunolabeling with specific antibodies against Dstac and Dmca1D found that Dstac and Dmca1D are expressed by larval body-wall muscles. Furthermore, Ca2+ imaging of muscles of Dstac and Dmca1D deficient larvae found that Dstac and Dmca1D are required for excitation-contraction coupling. Finally, Dstac appears to be required for normal expression levels of Dmca1D in body-wall muscles. These results suggest that Dstac regulates Dmca1D during EC coupling and thus muscle contraction.

Opposite effects of zinc on human and rat P2X2 receptors.

P2X(2) receptors from rats show potentiation when a submaximal concentration of ATP is combined with zinc in the range of 10-100 microM. Alignment of the amino acid sequences of human P2X(2) (hP2X(2)) and rat P2X(2) (rP2X(2)) indicated that only one of two histidines essential for zinc potentiation in rP2X(2) is present at the homologous position in hP2X(2) (H132), with the position homologous to rat H213 instead having an arginine (R225). When expressed in Xenopus oocytes, mouse P2X(2a) and P2X(2b) receptors showed zinc potentiation indistinguishable from rat P2X(2a), but hP2X(2b) receptors were inhibited by zinc. The extent of zinc inhibition of hP2X(2b) varied with the ATP concentration, with an IC(50) of 8.4 microM zinc when ATP was applied at 10% of maximal and 87 microM zinc when ATP was applied at 99% of maximal. Site-directed mutagenesis showed that none of the nine histidines in the extracellular domain of hP2X(2b) were required for zinc inhibition, although inhibition was attenuated in the H204A and H209A mutations. Mutating R225 to a cysteine was sufficient to confer zinc potentiation onto hP2X(2b), and zinc potentiation was absent in the hP2X(2b)H132A/R225C double mutant. This suggests that zinc potentiation in the mutant hP2X(2b) uses the same mechanism as zinc potentiation in wild-type rP2X(2a). Because of the species-specific modulation by zinc, evidence for an in vivo role of P2X(2) receptors based on studies conducted on genetically modified mice needs to be viewed with caution when extrapolations are made to the function of the human nervous system.

Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors.

Two histidines are known to be essential for zinc potentiation of rat P2X2 receptors, but the chemistry of zinc coordination would suggest that other residues also participate in this zinc-binding site. There is also a second lower affinity zinc-binding site in P2X2 receptors whose constituents are unknown. To assess whether the extracellular acidic residues of the P2X2 receptor contribute to zinc potentiation or inhibition, site-directed mutagenesis was used to produce alanine substitutions at each extracellular glutamate or aspartate. Two electrode voltage clamp recordings from Xenopus oocytes indicated that 7 of the 34 mutants (D82A, E85A, E91A, E115A, D136A, D209A, and D281A) were deficient in zinc potentiation and one mutant (E84A) was deficient in zinc inhibition. Additional tests on cysteine mutants at these eight positions indicated that D136 is the only residue that is a strong candidate to be at the potentiating zinc-binding site, and that E84 is unlikely to be at the inhibitory zinc-binding site.

Responses of rat P2X2 receptors to ultrashort pulses of ATP provide insights into ATP binding and channel gating.

To gain insight into the way that P2X(2) receptors localized at synapses might function, we explored the properties of outside-out patches containing many of these channels as ATP was very rapidly applied and removed. Using a new method to calibrate the speed of exchange of solution over intact patches, we were able to reliably produce applications of ATP lasting <200 micros. For all concentrations of ATP, there was a delay of at least 80 micros between the time when ATP arrived at the receptor and the first detectable flow of inward current. In response to 200-micros pulses of ATP, the time constant of the rising phase of the current was approximately 600 micros. Thus, most channel openings occurred when no free ATP was present. The current deactivated with a time constant of approximately 60 ms. The amplitude of the peak response to a brief pulse of a saturating concentration of ATP was approximately 70% of that obtained during a long application of the same concentration of ATP. Thus, ATP leaves fully liganded channels without producing an opening at least 30% of the time. Extensive kinetic modeling revealed three different schemes that fit the data well, a sequential model and two allosteric models. To account for the delay in opening at saturating ATP, it was necessary to incorporate an intermediate closed state into all three schemes. These kinetic properties indicate that responses to ATP at synapses that use homomeric P2X(2) receptors would be expected to greatly outlast the duration of the synaptic ATP transient produced by a single presynaptic spike. Like NMDA receptors, P2X(2) receptors provide the potential for complex patterns of synaptic integration over a time scale of hundreds of milliseconds.

Restraint of presynaptic protein levels by Wnd/DLK signaling mediates synaptic defects associated with the kinesin-3 motor Unc-104.

The kinesin-3 family member Unc-104/KIF1A is required for axonal transport of many presynaptic components to synapses, and mutation of this gene results in synaptic dysfunction in mice, flies and worms. Our studies at the Drosophila neuromuscular junction indicate that many synaptic defects in unc-104-null mutants are mediated independently of Unc-104's transport function, via the Wallenda (Wnd)/DLK MAP kinase axonal damage signaling pathway. Wnd signaling becomes activated when Unc-104's function is disrupted, and leads to impairment of synaptic structure and function by restraining the expression level of active zone (AZ) and synaptic vesicle (SV) components. This action concomitantly suppresses the buildup of synaptic proteins in neuronal cell bodies, hence may play an adaptive role to stresses that impair axonal transport. Wnd signaling also becomes activated when pre-synaptic proteins are over-expressed, suggesting the existence of a feedback circuit to match synaptic protein levels to the transport capacity of the axon.

Identification of nicotinic acetylcholine receptor recycling and its role in maintaining receptor density at the neuromuscular junction in vivo.

In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChR-recycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

The zebrafish shocked gene encodes a glycine transporter and is essential for the function of early neural circuits in the CNS.

shocked (sho) is a zebrafish mutation that causes motor deficits attributable to CNS defects during the first2dof development. Mutant embryos display reduced spontaneous coiling of the trunk, diminished escape responses when touched, and an absence of swimming. A missense mutation in the slc6a9 gene that encodes a glycine transporter (GlyT1) was identified as the cause of the sho phenotype. Antisense knock-down of GlyT1 in wild-type embryos phenocopies sho, and injection of wild-type GlyT1 mRNA into mutants rescues them. A comparison of glycine-evoked inward currents in Xenopus oocytes expressing either the wild-type or mutant protein found that the missense mutation results in a nonfunctional transporter. glyt1 and the related glyt2 mRNAs are expressed in the hindbrain and spinal cord in nonoverlapping patterns. The fact that these regions are known to be required for generation of early locomotory behaviors suggests that the regulation of extracellular glycine levels in the CNS is important for proper function of neural networks. Furthermore, physiological analysis after manipulation of glycinergic activity in wild-type and sho embryos suggests that the mutant phenotype is attributable to elevated extracellular glycine within the CNS.

Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor.

P2X receptors are ATP-gated cation channels that are widely expressed in the brain. The extracellular domains of all seven P2X receptors contain 10 conserved cysteines, which could form disulfide bonds or binding sites for transition metals that modulate P2X receptors. To test whether these cysteines are critical for receptor function, we studied wild-type rat P2X(2) receptors and 10 mutant P2X(2) receptors, each containing an alanine substituted for a cysteine. Nine mutants were functional but had reduced maximum currents compared with wild-type P2X(2) expressed in either Xenopus oocytes or human embryonic kidney (HEK) 293 cells. The 10th mutant (C224A) did not respond to ATP when expressed in oocytes and gave very small currents in HEK 293 cells. Seven mutants (C113A, C124A, C130A, C147A, C158A, C164A, and C214A) showed rightward shifts (9- to 30-fold) in their ATP concentration-response relationships and very little potentiation by zinc. In contrast, C258A and C267A had EC(50) values similar to those of wild-type P2X(2) and were potentiated by zinc. Acidic pH potentiated wild-type and all mutant receptor currents. Despite the loss of zinc potentiation in seven mutants, these cysteines are unlikely to be exposed in the zinc-binding site, because [2-(trimethylammonium)ethyl] methanethiosulfonate bromide did not prevent zinc potentiation of wild-type receptor currents. On the basis of correlations in the maximum current, EC(50), zinc potentiation, and pH potentiation, we suggest that the following cysteine pairs form disulfide bonds: C113-C164, C214-C224, and C258-C267. We also suggest that C124, C130, C147, and C158 form two disulfide bonds, but we are unable to assign specific cysteine pairs to these two bonds.

Potent and long-lasting inhibition of human P2X2 receptors by copper.

P2X receptors are ion channels gated by ATP. In rodents these channels are modulated by zinc and copper. Zinc is co-released with neurotransmitter at some synapses and can modulate neuronal activity, but the role of copper in the brain is unclear. Rat P2X2 receptors show potentiation by 2-100 µM zinc or copper in the presence of a submaximal concentration of ATP but are inhibited by zinc or copper at concentrations above 100 µM. In contrast, human P2X2 (hP2X2) receptors show no potentiation and are strongly inhibited by zinc over the range of 2-100 µM. The effect of copper on hP2X2 is of interest because there are human brain disorders in which copper concentration is altered. We found that hP2X2 receptors are potently inhibited by copper (IC50 = 40 nM). ATP responsiveness recovered extremely slowly after copper washout, with full recovery requiring over 1 h. ATP binding facilitated copper binding but not unbinding from this inhibitory site. A mutant receptor in which the first six extracellular cysteines were deleted, C(1-6)S, showed normal copper inhibition, however reducing agents dramatically accelerated recovery from copper inhibition in wild type hP2X2 and the C(1-6)S mutant, indicating that the final two disulfide bonds are required to maintain the high affinity copper binding site. Three histidine residues required for normal zinc inhibition were also required for normal copper inhibition. Humans with untreated Wilson's disease have excess amounts of copper in the brain. The high copper sensitivity of hP2X2 receptors suggests that they are non-functional in these patients.