Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling.

Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca(2+) influx without significantly altering the Ca(2+) sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca(2+)]i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca(2+) ([Ca(2+)]e). Lowering external Ca(2+) to match the isoflurane-induced reduction in Ca(2+) entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca(2+) entry without significant direct effects on Ca(2+)-exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca(2+) influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system.

The voltage dependence of gating currents of the neuronal CA(v)3.3 channel is determined by the gating brake in the I-II loop.

Low-voltage-activated Ca(v)3 Ca(2+) channels have an activation threshold around -60 mV, which is lower than the activation threshold of other voltage-dependent calcium channels (VDCCs). The kinetics of their activation at membrane voltages just above the activation threshold is much slower than the activation kinetics of other VDCCs. It was demonstrated recently that the intracellular loop connecting repeats I and II of all three Ca(v)3 channels contains a so-called gating brake. Disruption of this brake yields channels that activate at even more hyperpolarized potentials with significantly accelerated kinetics. We have compared gating of a wild-type Ca(v)3.3 channel and a mutated ID12 channel, in which the putative gating brake at the proximal part of the I-II loop was removed. Voltage dependence of the gating current activation was shifted by 34.6 mV towards more hyperpolarized potentials in ID12 channel. ON-charge movement was significantly faster in the ID12 channel, while the kinetics of the off-charge was not altered by the mutation. We conclude that the putative gating brake in I-II loop hinders not only the opening of the conducting pore but also the activating movement of voltage-sensing S4 segments, stabilizing the channel in its closed state.

Rap2-JNK removes synaptic AMPA receptors during depotentiation.

The related small GTPases Ras and Rap1 are important for signaling synaptic AMPA receptor (-R) trafficking during long-term potentiation (LTP) and long-term depression (LTD), respectively. Rap2, which shares 60% identity to Rap1, is present at excitatory synapses, but its functional role is unknown. Here, we report that Rap2 activity, stimulated by NR2A-containing NMDA-R activation, depresses AMPA-R-mediated synaptic transmission via activation of JNK rather than Erk1/2 or p38 MAPK. Moreover, Rap2 controls synaptic removal of AMPA-Rs with long cytoplasmic termini during depotentiation. Thus, Rap2-JNK pathway, which opposes the action of the NR2A-containing NMDA-R-stimulated Ras-ERK1/2 signaling and complements the NR2B-containing NMDA-R-stimulated Rap1-p38 MAPK signaling, channels the specific signaling for depotentiating central synapses.

Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate.

T-type Ca2+ channels (T-channels) are involved in the control of neuronal excitability and their gating can be modulated by a variety of redox agents. Ascorbate is an endogenous redox agent that can function as both an anti- and pro-oxidant. Here, we show that ascorbate selectively inhibits native Ca(v)3.2 T-channels in peripheral and central neurons, as well as recombinant Ca(v)3.2 channels heterologously expressed in human embryonic kidney 293 cells, by initiating the metal-catalyzed oxidation of a specific, metal-binding histidine residue in domain 1 of the channel. Our biophysical experiments indicate that ascorbate reduces the availability of Ca(v)3.2 channels over a wide range of membrane potentials, and inhibits Ca(v)3.2-dependent low-threshold-Ca2+ spikes as well as burst-firing in reticular thalamic neurons at physiologically relevant concentrations. This study represents the first mechanistic demonstration of ion channel modulation by ascorbate, and suggests that ascorbate may function as an endogenous modulator of neuronal excitability.

Alternative splicing within the I-II loop controls surface expression of T-type Ca(v)3.1 calcium channels.

Molecular diversity of T-type/Ca(v)3 Ca2+ channels is created by expression of three genes and alternative splicing of those genes. Prompted by the important role of the I-II linker in gating and surface expression of Ca(v)3 channels, we describe here the properties of a novel variant that partially deletes this loop. The variant is abundantly expressed in rat brain, even exceeding transcripts with the complete exon 8. Electrophysiological analysis of the Delta8b variant revealed enhanced current density compared to Ca(v)3.1a, but similar gating. Luminometry experiments revealed an increase in the expression of Delta8b channels at the plasma membrane. We conclude that alternative splicing of Ca(v)3 channels regulates surface expression and may underlie disease states in which T-channel current density is increased.

I-II loop structural determinants in the gating and surface expression of low voltage-activated calcium channels.

The intracellular loops that interlink the four transmembrane domains of Ca(2+)- and Na(+)-channels (Ca(v), Na(v)) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I-II loop) in high voltage-activated (HVA) Ca(2+) channels possesses the binding site for Ca(v)beta subunits and plays significant roles in channel function, including trafficking the alpha(1) subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I-II loop of Ca(v)1/Ca(v)2 HVA channels and Ca(v)3 LVA/T-type channels, evidence for a regulatory role of the I-II loop in T-channel function has recently emerged for Ca(v)3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Ca(v)3 channels, we have performed a structure-function analysis of the I-II loop in Ca(v)3.1 and Ca(v)3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Ca(v)3.1 and Ca(v)3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Ca(v)3 channels. In contrast to findings in Ca(v)3.2, deletion of the central region of the I-II loop in Ca(v)3.1 and Ca(v)3.3 yielded a modest increase (+30%) and a reduction (-30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Ca(v)3 function by highlighting the unique role played by the intracellular I-II loop in Ca(v)3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Ca(v)3.3.

Orientation of the calcium channel beta relative to the alpha(1)2.2 subunit is critical for its regulation of channel activity.

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.

Characterization of the gating brake in the I-II loop of Ca(v)3.2 T-type Ca(2+) channels.

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.

State-dependent Ras signaling and AMPA receptor trafficking.

Synaptic trafficking of AMPA-Rs, controlled by small GTPase Ras signaling, plays a key role in synaptic plasticity. However, how Ras signals synaptic AMPA-R trafficking is unknown. Here we show that low levels of Ras activity stimulate extracellular signal-regulated kinase kinase (MEK)-p42/44 MAPK (extracellular signal-regulated kinase [ERK]) signaling, whereas high levels of Ras activity stimulate additional Pi3 kinase (Pi3K)-protein kinase B (PKB) signaling, each accounting for approximately 50% of the potentiation during long-term potentiation (LTP). Spontaneous neural activity stimulates the Ras-MEK-ERK pathway that drives GluR2L into synapses. In the presence of neuromodulator agonists, neural activity also stimulates the Ras-Pi3K-PKB pathway that drives GluR1 into synapses. Neuromodulator release increases with increases of vigilance. Correspondingly, Ras-MEK-ERK activity in sleeping animals is sufficient to deliver GluR2L into synapses, while additional increased Ras-Pi3K-PKB activity in awake animals delivers GluR1 into synapses. Thus, state-dependent Ras signaling, which specifies downstream MEK-ERK and Pi3K-PKB pathways, differentially control GluR2L- and GluR1-dependent synaptic plasticity.

Fluorescence imaging of mobility shifts: an expression cloning method for identification of cell signaling targets.

There is a need for a simple global approach to identify signaling targets that are posttranslationally modified in response to physiologic or pathologic stimuli within living cells. Reported here is a simple method, fluorescence imaging of mobility shifts (FIMS), which relies on in-gel detection of cell-expressed green fluorescent protein fusion proteins undergoing electrophoretic mobility shifts. This detection method is applied to a small pool cDNA library screening protocol. The readout is essentially a differential display of posttranslational modifications. Unlike biochemical approaches to identifying signaling targets, the screen is performed in living cells using standard methods for transient transfection. This enables detection of intracellular targets modified in response to either molecularly defined stimuli, such as growth factors or drugs, or complex pathologic stimuli, such as oxidative stress or hypoglycemia. FIMS is rapid, sensitive, inexpensive, and nonradioactive and easily adapted to automated high throughput methods, including capillary electrophoresis. The technique is sufficiently sensitive to easily detect fluorescent proteins expressed in a single well in 384-well format. FIMS is applicable to traditional cDNA library screening, but the method will be especially attractive for screening preselected collections of autofluorescent fusion proteins. A bonus of the technique is that examination of transfected cells by fluorescence microscopy provides immediate information about intracellular localization and stimulus-induced translocation of putative targets. We illustrate the utility of the technique with pilot screens for apoptotic and mitogenic targets modified by staurosporine and serum stimulation, respectively.