Paul Brehm

Regulation of Neuronal Traits by a Novel Transcriptional Complex

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Calcium‐mediated inactivation of calcium current in Paramecium

1. The Ca current seen in response to depolarization was investigated in Paramecium caudatum under voltage clamp. Inactivation of the current was measured with the double pulse method; a fixed test pulse of an amplitude sufficient to evoke maximal inward current was preceded by a conditioning pulse of variable amplitude (0‐120 mV).

Properties of non‐junctional acetylcholine receptor channels on innervated muscle of Xenopus laevis.

Patch‐clamp recordings of current through acetylcholine‐activated channels were made from non‐junctional membrane of innervated myotomal muscle from Xenopus laevis. Two classes of acetylcholine (ACh) receptor channels were identified on the basis of current amplitudes. Both amplitude classes exhibited current‐voltage relations which deviated from linearity as the extrapolated reversal potential was approached (‐5 to ‐12 mV). Over the range of greatest linearity the conductances of the two classes were 64 and 44 pS. Both event classes were blocked by alpha‐bungarotoxin. At the normal resting membrane potential (approximately ‐95 mV) the larger conductance channel (gamma) exhibited an apparent mean channel open time of less than 1 ms, compared to approximately 2 ms for the smaller gamma class. The apparent open time was voltage‐dependent, changing e‐fold with a 63 mV hyperpolarization for the high gamma channel and 93 mV hyperpolarization for the low gamma channel. At low ACh concentrations (0.1‐0.3 microM) both amplitude classes exhibited bursts of successive openings separated by brief closures of less than 0.5 ms. Bursts were separated by longer closed intervals of 1 to greater than 100 ms. Closed interval histograms revealed corresponding populations of brief and long closures, indicating that at least two kinetic processes are required to describe the distribution of closed intervals. In the absence of exogenous ACh, channels were observed in an occasional patch which showed a conductance and extrapolated reversal potential similar to ACh‐activated channels. In such patches the event frequency could occasionally be altered by adjusting the negative pressure applied to the patch. The two main conductance classes of ACh activated channels were observed to coexist in most patches. However, the most frequent event observed in non‐junctional membrane of innervated muscle corresponded to the high gamma class. In this respect, the non‐junctional ACh receptors bore a greater similarity to junctional ACh receptors than to non‐junctional receptors reported for denervated muscle.

Voltage-Dependent Sodium Channel Function Is Regulated Through Membrane Mechanics

Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na(+) channel alpha subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na(+) channels involve mechanical destabilization. Our observation that the beta1 subunit causes similar changes in function of the Na(+) channel alpha subunit suggests that beta1 may act through interaction with cytoskeleton.

Regulation of acetylcholine receptor channel function during development of skeletal muscle

The nicotinic acetylcholine (ACh) receptor channel mediates synaptic transmission at the neuromuscular junction. During the development of skeletal muscle, ACh receptors undergo changes in distribution, antigenic determinants, degradation rate, and function. Now that these developmental hallmarks have been identified, attention has turned toward understanding both the structural bases for such changes and the role of nerve in triggering these changes. Recently, a much clearer understanding of one of these developmental processes, namely, the alterations in channel function, has emerged through both sensitive patch-clamp measurements and the application of recombinant DNA technology. In light of these new advances, we now reevaluate the processes governing the developmental changes in the functional properties of the ACh receptor.

Tethering Naturally Occurring Peptide Toxins for Cell-Autonomous Modulation of Ion Channels and Receptors In Vivo

The physiologies of cells depend on electrochemical signals carried by ion channels and receptors. Venomous animals produce an enormous variety of peptide toxins with high affinity for specific ion channels and receptors. The mammalian prototoxin lynx1 shares with alpha-bungarotoxin the ability to bind and modulate nicotinic receptors (nAChRs); however, lynx1 is tethered to the membrane via a GPI anchor. We show here that several classes of neurotoxins, including bungarotoxins and cobratoxins, retain their selective antagonistic properties when tethered to the membrane. Targeted elimination of nAChR function in zebrafish can be achieved with tethered alpha-bungarotoxin, silencing synaptic transmission without perturbing synapse formation. These studies harness the pharmacological properties of peptide toxins for use in genetic experiments. When combined with specific methods of cell and temporal expression, the extension of this approach to hundreds of naturally occurring peptide toxins opens a new landscape for cell-autonomous regulation of cellular physiology in vivo.

Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

Increased neuromuscular activity causes axonal defects and muscular degeneration.

Before establishing terminal synapses with their final muscle targets, migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twister mutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor α-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.

Acetylcholine Receptors Direct Rapsyn Clusters to the Neuromuscular Synapse in Zebrafish

Clustering of nicotinic muscle acetylcholine receptors (AChRs) requires association with intracellular rapsyn, a protein with an intrinsic ability to self-cluster. Previous studies on sofa potato (sop), an AChR null line of zebrafish, have suggested that AChRs may play an active role in subsynaptic localization of rapsyn clusters. To test this proposal directly, we identified and cloned the gene responsible for the sop phenotype and then attempted to rescue subsynaptic localization of the receptor-rapsyn complex in mutant fish. sop contains a leucine to proline mutation at position 28, near the N terminus of the zebrafish AChR δ subunit. Transient expression of mutant δ subunit in sop fish was unable to restore surface expression of muscle AChRs. In contrast, expression of wild-type δ subunit restored the ability of muscle to assemble surface receptors along with the ability of fish to swim. Most importantly, the ability of rapsyn clusters to localize effectively to subsynaptic sites also was rescued in large part. Our results point to direct involvement of the AChR molecule in restricting receptor-rapsyn clusters to the synapse.

Calcium‐dependent repolarization in Paramecium

1. Intracellular injection, recording and current‐passing methods were used to investigate the role of intracellular Ca in the modulation of electrical behaviour in the ciliate Paramecium caudatum.