Ring opening of a sterically crowded 1,2-oxaphosphetane complex.

The first example of a ring opening reaction of a 1,2-oxaphosphetane complex is reported, i.e., water in the presence of [Li(12-crown-4)]Cl furnished a C-OH functional phosphinito complex. Employment of the latter in ring forming reactions with Me2ECL2 (E = Si, Ge) using different nitrogen bases is also described.

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.

Paralytic zebrafish lacking acetylcholine receptors fail to localize rapsyn clusters to the synapse.

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato, reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed, showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

Calcium channels in Xenopus spinal neurons differ in somas and presynaptic terminals.

Calcium channels play dual roles in cell signaling by promoting membrane depolarization and allowing entry of calcium ions. Patch-clamp recordings of calcium and calcium-dependent currents from the soma of Xenopus spinal neurons indicate key functional differences from those of presynaptic terminals. Both terminals and somas exhibit prominent high-voltage-activated (HVA) calcium current, but only the soma expresses additional low-voltage-activated (LVA) T-type current. Further differences are reflected in the HVA current; N- and R-type channels are predominant in the soma while the terminal calcium current is composed principally of N type with smaller contribution by L- and R-type channels. Potential physiological significance for these different distributions of channel types may lie in the differential channel kinetics. Activation of somatic HVA calcium current occurs more slowly than HVA currents in terminals. Additionally, somatic LVA calcium current activates and deactivates much more slowly than any HVA calcium current. Fast-activating and -deactivating calcium current may be critical to processing the rapid exocytotic response in terminals, whereas slow LVA and HVA calcium currents may play a central role in shaping the somatic firing pattern. In support of different kinetic behavior between these two compartments, we find that somatic calcium current activates a prominent slow chloride current not observed in terminal recordings. This current activates in response to calcium entering through either LVA or HVA channels and likely functions as a modulator of excitability or synaptic input. The restriction of this channel type to the soma lends further support to the idea that differential expression of fast and slow channel types in these neurons is dictated by differences in signaling requirements for somatic and terminal compartments.

Progesterone treatment abolishes exogenously expressed ionic currents in Xenopus oocytes.

Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na(+) and K(+) channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na(+) and K(+) channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na(+) currents temporally coincided with development of germinal vesicle breakdown, while elimination of K(+) currents was delayed by approximately 2 h. Coexpression of human beta(1)-subunit with rat skeletal muscle alpha-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na(+) channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na(+) and K(+) channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na(+) channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.

Calcium channel isoforms underlying synaptic transmission at embryonic Xenopus neuromuscular junctions.

Studies on the amphibian neuromuscular junction have indicated that N-type calcium channels are the sole mediators of stimulus-evoked neurotransmitter release. We show, via both presynaptic and postsynaptic voltage-clamp measurements, that dihydropyridine (DHP)-sensitive calcium channels also contribute to stimulus-evoked release at developing Xenopus neuromuscular junctions. Whereas inhibition of postsynaptic responses by omega-conotoxin (omega-Ctx) GVIA has been taken previously as evidence that only N-type channels mediate transmitter release, we find that both N-type and DHP-sensitive calcium currents are sensitive to this toxin. The unusual sensitivity of DHP-sensitive calcium channels to omega-Ctx GVIA in presynaptic terminals raises the possibility that this channel type may have escaped detection in previous physiological studies on adult frog neuromuscular junctions. Alternatively, the additional channel isoforms may be present only during early development, when they may serve to strengthen collectively presynaptic release during critical periods of synaptogenesis.

Distinction among neuronal subtypes of voltage-activated sodium channels by mu-conotoxin PIIIA.

The functional properties of most sodium channels are too similar to permit identification of specific sodium channel types underlying macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the mu-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability to block different types of heterologously expressed sodium channels. PIIIA mu-conotoxin blocked rat brain type II/IIA (rBII/IIA) and skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the differential block by PIIIA also applied to native sodium current. The sensitivity to block by PIIIA toxin was then used to discriminate between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in which both mRNAs are induced. During the first 24 hr of NGF-treatment, PN1 sodium channels accounted for over 90% of the sodium current. However, over the ensuing 48 hr period, a sharp rise in the proportion of rBII/IIA sodium current occurred, confirming the idea, based on previous mRNA measurements, that two distinct sodium channel types appear sequentially during neuronal differentiation of PC12 cells.

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.

Long-term desensitization of nicotinic acetylcholine receptors is regulated via protein kinase A-mediated phosphorylation.

During prolonged application of transmitter, ligand-gated ion channels enter a nonconducting desensitized state. Studies on Torpedo electroplax nicotinic acetylcholine (ACh) receptors have shown that entry into the desensitized state is accelerated by protein kinase A-dependent (PKA) receptor phosphorylation. To examine the effects of phosphorylation on desensitization of muscle-type ACh receptors, we expressed the frog embryonic receptor type in Xenopus oocytes. Treatment of embryonic muscle ACh receptors with 8-Br cAMP had no measurable effect on the rate of entry into a desensitized state, but it greatly accelerated the recovery from desensitization. Three complementary approaches to reduce the levels of receptor phosphorylation provided additional evidence for a role of PKA-dependent phosphorylation in rescuing receptors from long-term desensitization. Inactivation of the endogenous PKA activity by coexpression of an inhibitor protein, treatment of receptors with phosphatase, and removal of phosphorylation sites by site-specific subunit mutation all resulted in slowed recovery. Our findings point to the existence of two distinct desensitized states: one requiring several seconds for full recovery and a second state from which recovery requires minutes. Receptors lacking PKA phosphorylation sites exhibit a pronounced increase in the slowly recovering component of desensitization, suggesting that receptor phosphorylation speeds overall recovery by reducing the entry into a deep desensitized state. This newly described effect of phosphorylation on ACh receptor function may serve as an important modulator of postsynaptic receptor sensitivity.

Two types of ACh receptors contribute to fast channel gating on mouse skeletal muscle.

Single-channel recordings from mouse C2 myotubes indicate that maturation of skeletal muscle is accompanied by the appearance of two types of fast acetylcholine (ACh) receptor channels that are each functionally distinct from the embryonic receptor type present at early stages of differentiation. The embryonic receptor type has a low conductance (45 pS) and long channel open time, rendering slowly decaying synaptic currents. One fast channel type that appears during muscle maturation is distinguished from the embryonic receptor type on the basis of both higher conductance (65 pS) and shorter open time. However, single-channel recordings from differentiated mouse skeletal muscle cell line (C2) point to the existence of a second fast receptor type, which has a conductance similar to the embryonic receptor type (45 pS), yet significantly reduced mean channel open time. Analyses of individual channel function at high ACh concentrations directly demonstrate the coexistence of two kinetically distinct types of 45 pS ACh receptors. Openings by fast type and slow embryonic type of 45 pS receptors occurred in bursts, allowing distinction on the basis of both mean open time and open probability for individual receptors. The embryonic type of 45 pS receptor has an open time approximately twofold longer than the fast-receptor counterpart. Additional differences were reflected in the open probability distributions for fast and slow 45 pS receptor types. Both types of 45 pS receptor were kinetically distinguishable from the 65 pS receptor. We found no support for the idea that the slow and fast 45 pS receptor types result from the interconversion of dual gating modes involving the same receptor protein. Our results are consistent with the idea that the acquisition of fast synaptic current decay, required at mature neuromuscular synapses, is the result of the up-regulation of two distinct fast types of nicotinic ACh receptors during skeletal muscle development.

A single site on the epsilon subunit is responsible for the change in ACh receptor channel conductance during skeletal muscle development.

Four critically positioned amino acids on each of the alpha, beta, delta, and gamma subunits of the Torpedo nicotinic acetylcholine receptor are determinants of channel conductance. Our results show that the gamma and epsilon subunits of Xenopus muscle receptors are identical at all four positions, despite the fact that alpha 2 beta delta epsilon receptors have a 50% greater conductance than alpha 2 beta delta gamma receptors. Instead, the functional difference is conferred by a single charged residue that lies extracellular to all four positions, corresponding to a location in the Torpedo receptor previously shown to have no influence on conductance. Substitution of a positively charged lysine residue in gamma by the neutral methionine in epsilon at this extra-cellular position is responsible for the increased conductance during maturation of the amphibian neuromuscular junction.

A single pulse of nerve growth factor triggers long-term neuronal excitability through sodium channel gene induction.

The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a brief exposure is to induce selectively expression of the peripheral nerve-type sodium channel gene PN1, through a distinct signaling pathway requiring immediate-early genes. A 1 min exposure of PC12 cells to interferon-gamma also causes PN1 gene induction, suggesting that the "triggered" NGF and interferon-gamma signaling pathways share common molecular intermediates.

Two subcellular mechanisms underlie calcium-dependent facilitation of bioluminescence.

Epithelial calcium action potentials in Obelia geniculata trigger brief light flashes from specialized cells by direct activation of cytoplasmic calcium-activated photoprotein obelin. During a series of action potentials, sequential flashes undergo characteristic facilitation and decrement with no change in associated spike waveform. Analysis of the subcellular light distribution shows that facilitation results from two processes: recruitment of calcium entry sites and increased light from previously responding localized sites. We propose a model that accounts for the localized flash facilitation and decrement observed in vivo and is based upon the kinetics of calcium binding and emission of obelin. In this model, obelin emits light only when three calcium ions are bound. Changes in flash intensity during successive action potentials result from calcium bound persistently to unexpended obelin, effectively lowering the number of calcium ions required for subsequent activation. Accordingly, facilitation or decrement results from the time-dependent availability of singly and doubly bound obelin.

Adult forms of nicotinic acetylcholine receptors are expressed in the absence of nerve during differentiation of a mouse skeletal muscle cell line.

Changes in the functional properties of acetylcholine receptor (AchR) channels were followed in the C2 muscle cell line over the period of 1 to 17 days following myotube formation. Up to 1 week after myotube formation, the predominant class of channel exhibited low (45 pS) conductance and long mean channel open time (14 msec), characteristic of the major type of AchR in embryonic skeletal muscle. Three additional Ach-activated currents with conductances lower than 45 pS and long channel open times were also observed. Seven to 10 days following myotube formation, channels of 45 pS and 65 pS and short (2-6 msec) mean open duration were observed, characteristic of receptor channels in adult muscle. Increases in epsilon subunit mRNA levels preceded the functional expression of channels with brief open durations. Our results show that C2 muscle cells maintained in culture express a full range of functionally distinct AchR types in the absence of nerve.

Expression of subunit-omitted mouse nicotinic acetylcholine receptors in Xenopus laevis oocytes.

1. Nicotinic acetylcholine (ACh) receptors in developing vertebrate skeletal muscle exhibit functional heterogeneity in both conductance and kinetics. To assess the contributions of receptors differing in subunit composition to the heterogeneity, various combinations of mouse alpha beta delta gamma epsilon subunit RNAs were tested for the ability to express functional receptors in Xenopus oocytes. 2. Two combinations of dual-subunit RNAs (alpha delta and alpha gamma) resulted in detectable ACh-activated currents and six different combinations of three or more subunit RNAs produced significant numbers of functional channels. The order of combinations yielding the greatest amount of current was alpha beta gamma > alpha beta delta = alpha delta epsilon > alpha delta gamma > alpha delta > alpha gamma. 3. The extent to which a channel type with three different subunits was expressed was highly dependent upon the ratios of RNAs coding for the different subunits. For alpha beta delta receptors the efficiency of expression was alpha: beta: delta (1/5:1/5:1) >> (1:1:1) >> (1/5:1:1/5) > (1:1/5:1/5). 4. The level of expression of three-subunit combinations was also critically dependent upon the order of RNAs injected. When alpha delta or alpha gamma RNA combinations were co-injected 2 days prior to the injection of beta RNA, the expression was 2-5 times greater than when alpha beta injection was followed by injection of delta or gamma RNA. 5. Single-channel measurements revealed that alpha beta delta channels were not expressed in the presence of alpha beta delta epsilon RNAs, even under conditions when the amount of delta RNA injected was 5-fold higher than the amount of epsilon RNA. 6. These data indicate that the functional expression of subunit-omitted receptors depends critically upon the relative amounts of the five different subunit RNAs. Receptors composed of three different subunits express in the presence of the subunit RNAs characteristic of embryonic muscle (alpha beta delta gamma), but are not observed with the combination of RNAs characteristic of adult muscle (alpha beta delta epsilon).

Neuronal growth factor regulation of two different sodium channel types through distinct signal transduction pathways.

Neuronal growth factors regulate the expression of voltage-activated sodium current in differentiating sympathetic neurons and PC12 cells. We show that, in PC12 cells, the NGF- and FGF-induced sodium current results from increased expression of two distinct sodium channel types. Sodium current results from the rapid induction of a novel sodium channel transcript, also found in peripheral neurons, and from the long term induction of brain type II/IIA mRNA. Expression of the type II/IIA sodium channel requires activation of the cyclic AMP-dependent protein kinase (A-kinase), whereas induction of the peripheral neuron type sodium channel occurs through an A-kinase-independent signal transduction pathway. These findings suggest that the two sodium channel types act in concert to ensure the generation of action potentials during neuronal differentiation.

Modal shifts in acetylcholine receptor channel gating confer subunit-dependent desensitization.

During the transition from embryonic to adult skeletal muscle, a decreased mean channel open time and accelerated desensitization of nicotinic acetylcholine (ACh) receptors result from the substitution of an epsilon subunit for gamma. A single ACh receptor channel of the embryonic type, expressed in Xenopus oocytes, interconverts between gating modes of short and long open time, whereas the adult receptor channel resides almost exclusively in the gating mode with short open time. Differences in the fraction of time spent in either gating mode account for the subunit dependence of both receptor open time and desensitization. Therefore, developmental changes in the kinetics of muscle ACh receptors may be imparted through subunit-dependent stabilization of intrinsic gating modes.

The epsilon subunit confers fast channel gating on multiple classes of acetylcholine receptors.

During vertebrate skeletal muscle development, multiple forms of long-open-time (slow-type) ACh receptor channels are replaced by at least two different types of short-open-time (fast-type) ACh receptors. Expression of ACh receptors in Xenopus oocytes indicates that the substitution of an epsilon subunit for a gamma subunit may account for both types of fast-gated channel types in adult muscle. Unlike the various forms of the embryonic receptor, in which functional diversity is achieved through alterations in subunit composition, the two major fast-gated forms expressed in oocytes have identical subunit composition. These findings provide a structural basis for both types of short-open-time ACh receptor types found in adult muscle.

Contributions of the gamma and epsilon subunit family to nicotinic acetylcholine receptor function.

Hybrid acetylcholine (ACh) receptors expressed in Xenopus oocytes were used to study the role of the gamma/epsilon subunit family in determining channel function. The receptors were composed of either a mouse gamma, Torpedo gamma, Xenopus gamma or mouse epsilon subunit in combination with mouse alpha, beta and delta subunits. The effects of different gamma and epsilon subunits on channel conductance and open time were paralleled by their structural relatedness. Of the four subunits studied, mouse gamma and mouse epsilon are the most divergent in structure and the receptors containing these subunits were also the most divergent in function. Torpedo and Xenopus gamma subunits have structural features intermediate to both mouse epsilon and gamma and correspondingly imparted intermediate functional properties. Of particular interest, ACh receptors formed in combination with a Torpedo gamma subunit had a significantly lower conductance than those containing a mouse epsilon subunit, despite the presence of identical amino acids in all charge positions previously shown to affect conductance.