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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 dORKA-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. [ABSTRACT FROM AUTHOR]

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 &agr;novel function of kap in stabilizing nicotinic acetylcholine receptors (AChRs). Knockdown of kap expression with shRNA significantly enhanced the degradation of AChR -subunits (AChR), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that kap promoted AChR stability by a ubiquitin-dependent mechanism. In the absence ofkap, AChR was heavily ubiquitinated, and the number of AChR was increased by proteasome inhibitors. However, in the presence of kap, AChR was less ubiquitinated and proteasome inhibitors had almost no effect on AChR accumulation. The major sites of AChR ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChR stability in the absence of kap. These results provide an unexpected mechanism by which kap controls receptor trafficking onto the surface of muscle cells and thus the maintenance of postsynaptic receptor density and synaptic function [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Several zebrafish P2X receptors (zP2X1, zP2X2, and zP2X5.1) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X1 and zP2X5.1 produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X2 was still nonfunctional. zP2X2 lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X5.1. Antisense knockdown of P2X5.1 eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction. [ABSTRACT FROM AUTHOR]

P2X2 receptors from rats show potentiation when a submaximal concentration of ATP is combined with zinc in the range of 10 -100μM. Alignment of the amino acid sequences of human P2X2 (hP2X2) and rat P2X2 (rP2X2) indicated that only one of two histidines essential for zinc potentiation in rP2X2 is present at the homologous position in hP2X2 (H132), with the position homologous to rat H213 instead having an arginine (R225). When expressed in Xenopus oocytes, mouse P2X2a and P2X2b receptors showed zinc potentiation indistinguishable from rat P2X2a , but hP2X2b receptors were inhibited by zinc. The extent of zinc inhibition of hP2X2b varied with the ATP concentration, with an IC50 of 8.4 μM zinc when ATP was applied at 10% of maximal and 87 μM zinc when ATP was applied at 99% of maximal. Site-directed mutagenesis showed that none of the nine histidines in the extracellular domain of hP2X2b 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 hP2X2b, and zinc potentiation was absent in the hP2X2bH132A/R225C double mutant. This suggests that zinc potentiation in the mutant hP2X2b uses the same mechanism as zinc potentiation in wild-type rP2X2a. Because of the species-specific modulation by zinc, evidence for an in vivo role of P2X2 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To gain insight into the way that P2X2 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 μs. For all concentrations of ATP, there was a delay of at least 80 Its between the time when ATP arrived at the receptor and the first detectable flow of inward current. In response to 200-us pulses of ATP, the time constant of the rising phase of the current was ∼600 μs. Thus, most channel openings occurred when no free ATP was present. The current deactivated with a time constant of ∼60 ms. The amplitude of the peak response to a brief pulse of a saturating concentration of ATP was ∼70% of that obtained dining a long application of the same concentration of ATP. Thus, ATP leaves fully liganded channels without producing an opening at least 30% ot 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 P2X2 receptors would be expected to greatly outlast the duration of the synaptic ATP transient produced by a single presynaptic spike. Like NMDA receptors, P2X2 receptors provide the potential for complex patterns of synaptic integration over a time scale of hundreds of milliseconds. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

shocked (sho) is a zebrafish mutation that causes motor deficits attributable to CNS defects during the first 2 d of 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. [ABSTRACT FROM AUTHOR]

Reports on the cloning of a family of genes encoding subunits of the non-N-methyl-D-aspartate (non-NMDA) type of glutamate receptor, and on the identification of a site that regulates the permeation properties of these receptors. Study of structural features controlling ion permeation; Results of mutation of a certain amino acid.