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E F Stanley

Publications and source records attributed to E F Stanley.

At least 19 recordsLinked to original sources
PubMed2006

N type Ca2+ channels and RIM scaffold protein covary at the presynaptic transmitter release face but are components of independent protein complexes.

Fast neurotransmitter release at presynaptic terminals occurs at specialized transmitter release sites where docked secretory vesicles are triggered to fuse with the membrane by the influx of Ca2+ ions that enter through local N type (CaV2.2) calcium channels. Thus, neurosecretion involves two key processes: the docking of vesicles at the transmitter release site, a process that involves the scaffold protein RIM (Rab3A interacting molecule) and its binding partner Munc-13, and the subsequent gating of vesicle fusion by activation of the Ca2+ channels. It is not known, however, whether the vesicle fusion complex with its attached Ca2+ channels and the vesicle docking complex are parts of a single multifunctional entity. The Ca2+ channel itself and RIM were used as markers for these two elements to address this question. We carried out immunostaining at the giant calyx-type synapse of the chick ciliary ganglion to localize the proteins at a native, undisturbed presynaptic nerve terminal. Quantitative immunostaining (intensity correlation analysis/intensity correlation quotient method) was used to test the relationship between these two proteins at the nerve terminal transmitter release face. The staining intensities for CaV2.2 and RIM covary strongly, consistent with the expectation that they are both components of the transmitter release sites. We then used immunoprecipitation to test if these proteins are also parts of a common molecular complex. However, precipitation of CaV2.2 failed to capture either RIM or Munc-13, a RIM binding partner. These findings indicate that although the vesicle fusion and the vesicle docking mechanisms coexist at the transmitter release face they are not parts of a common stable complex.

Animals↗
PubMed2006

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins.

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Na/Ca exchanger and PMCA localization in neurons and astrocytes: functional implications.

Immunocytochemistry reveals that the Na/Ca exchanger (NCX) in neuronal somata and astrocytes is confined to plasma membrane (PM) microdomains that overlie sub-PM (junctional) endoplasmic reticulum (jER). By contrast, the PM Ca(2+) pump (PMCA) is more uniformly distributed in the PM. At presynaptic nerve terminals, the NCX distribution is consistent with that observed in the neuronal somata, but the PMCA is clustered at the active zones. Thus, the PMCA, with high affinity for Ca(2+) (K(d) congruent with 100 nM), may keep active zone Ca(2+) very low and thereby "reprime" the vesicular release mechanism following activity. NCX, with lower affinity for Ca(2+) (K(d) congruent with 1,000 nM), on the other hand, may extrude Ca(2+) that has diffused away from the active zones and been temporarily sequestered in the endoplasmic reticulum. The PL microdomains that contain the NCX also contain Na(+) pump high ouabain affinity alpha2 (astrocytes) or alpha 3 (neurons) subunit isoforms (IC(50) congruent with 5-50 nM ouabain). In contrast, the alpha1 isoform (low ouabain affinity in rodents; IC(50) >10,000 nM), like the PMCA, is more uniformly distributed in these cells. The sub-PM endoplasmic reticulum in neurons (and probably glia and other cell types as well) and the adjacent PM form junctions that resemble cardiac muscle dyads. We suggest that the PM microdomains containing NCX and alpha 2/alpha 3 Na(+) pumps, the underlying jER, and the intervening tiny volume of cytosol (<10(-18) l) form functional units (PLasmERosomes); diffusion of Na(+) and Ca(2+) between these cytosolic compartments and "bulk" cytosol may be markedly restricted. The activity of the Na(+) pumps with alpha 2/alpha 3 subunits may thus regulate NCX activity and jER Ca(2+) content. This view is supported by studies in mice with genetically reduced (by congruent with 50%) alpha 2 Na(+) pumps: evoked Ca(2+) transients were augmented in these cells despite normal cytosolic Na(+) and resting Ca(2+) concentrations ([Na(+)](CYT) and [Ca(2+)](CYT)). We conclude that alpha 2/alpha 3 Na(+) pumps control PLasmERosome (local) [Na(+)](CYT). This, in turn, via NCX, modulates local [Ca(2+)](CYT), jER Ca(2+) storage, Ca(2+) signaling, and cell responses.

Animals↗
PubMed2000

G-Protein types involved in calcium channel inhibition at a presynaptic nerve terminal.

The inhibition of presynaptic calcium channels via G-protein-dependent second messenger pathways is a key mechanism of transmitter release modulation. We used the calyx-type nerve terminal of the chick ciliary ganglion to examine which G-proteins are involved in the voltage-sensitive inhibition of presynaptic N-type calcium channels. Adenosine caused a prominent inhibition of the calcium current that was totally blocked by pretreatment with pertussis toxin (PTX), consistent with an exclusive involvement of G(o)/G(i) in the G-protein pathway. Immunocytochemistry was used to localize these G-protein types to the nerve terminal and its transmitter release face. We used two approaches to test for modulation by other G-protein types. First, we treated the terminals with ligands for a variety of G-protein-linked neurotransmitter receptor types that have been associated with different G-protein families. Although small inhibitory effects were observed, these could all be eliminated by PTX, indicating that in this terminal the G(i) family is the sole transmitter-induced G-protein inhibitory pathway. Second, we examined the kinetics of calcium channel inhibition by uncaging the nonselective and irreversible G-protein activator GTPgammaS, bypassing the receptors. A large fraction of the rapid GTPgammaS-induced inhibition persisted, consistent with a G(o)/G(i)-independent pathway. Immunocytochemistry identified G(q), G(11), G(12), and G(13) as potential PTX-insensitive second messengers at this terminal. Thus, our results suggest that whereas neurotransmitter-mediated calcium channel inhibition is mainly, and possibly exclusively, via G(o)/G(i), other rapid PTX-insensitive G-protein pathways exist that may involve novel, and perhaps transmitter-independent, activating mechanisms.

Adenosine↗
PubMed2000

Location of calcium transporters at presynaptic terminals.

The plasma membrane ATP-driven Ca2+ pump (PMCA) and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion at presynaptic nerve terminals, but little is know about the location of these transporters relative to the major sites of Ca2+ influx, the transmitter release sites. We used immunocytochemistry to identify these transport proteins in a calyx-type presynaptic nerve terminal from the ciliary ganglion of the chick. The PMCA clusters were localized to the transmitter release sites, as identified by staining for the secretory vesicle-specific protein synaptotagmin I. This colocalization was not due to the presence of the pump on the secretory vesicle itself because membrane fractionation of chick brain synaptosomes demonstrated comigration of the pump with surface membrane and not vesicle markers. In contrast, the NCX did not colocalize with synaptotagmin but tended to be located at nonsynaptic regions of the terminal. The PMCA location, near the transmitter release sites, suggests that it plays a role in priming the release site by maintaining a low free Ca2+ level, facilitating the dissociation of the ion from its binding sites. The PMCA may also replenish external Ca2+ in the synaptic cleft following periods of synaptic activity. In contrast, the NCX location suggests a role in the rapid emptying of cytoplasmic Ca2+ uptake organelles which serve as the main line of defence against high free Ca2+.

Animals↗
PubMed2000

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions.

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.

Animals↗
PubMed1999

Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal.

1. A high-conductance calcium-activated potassium channel (BK KCa) was characterized at a cholinergic presynaptic nerve terminal using the calyx synapse isolated from the chick ciliary ganglion. 2. The channel had a conductance of 210 pS in a 150 mM:150 mM K+ gradient, was highly selective for K+ over Na+, and was sensitive to block by external charybdotoxin or tetraethylammonium (TEA) and by internal Ba2+. At +60 mV it was activated by cytoplasmic calcium [Ca2+]i with a Kd of approximately 0.5 microM and a Hill coefficient of approximately 2.0. At 10 microM [Ca2+]i the channel was 50 % activated (V) at -8.0 mV with a voltage dependence (Boltzmann slope-factor) of 32.7 mV. The V values hyperpolarized with an increase in [Ca2+]i while the slope factors decreased. There were no overt differences in conductance or [Ca2+]i sensitivity between BK channels from the transmitter release face and the non-release face. 3. Open and closed times were fitted by two and three exponentials, respectively. The slow time constants were strongly affected by both [Ca2+]i and membrane potential changes. 4. In cell-attached patch recordings BK channel opening was enhanced by a prepulse permissive for calcium influx through the patch, suggesting that the channel can be activated by calcium ion influx through neighbouring calcium channels. 5. The properties of the presynaptic BK channel are well suited for rapid activation during the presynaptic depolarization and Ca2+ influx that are associated with transmitter release. This channel may play an important role in terminating release by rapid repolarization of the action potential.

Algorithms↗
PubMed1999

High-voltage-activated calcium channel messenger RNA expression in the 140-3 neuroblastoma-glioma cell line.

Expression of calcium channel alpha1 subunits in oocytes or cell lines has proven to be a powerful method in the analysis of structure-function relations, but these experimental systems are of limited value in the examination of neuron-specific functions such as transmitter release. Cell lines derived from neurons are often capable of these functions, but their intrinsic calcium channel alpha1 subunits are complicating factors in experimental design. We have examined the biophysical and molecular properties of calcium channels in a little studied neuroblastoma-glioma hybrid cell line, 140-3, a close relative of the NG108-15 cell line, to test whether this cell line might serve a role as an expression system for neural mechanisms. This cell was selected as it contains an intact transmitter release mechanism yet secretes little in response to depolarization. Patch-clamp recording revealed only a prominent low-threshold, rapidly inactivating calcium current with a single-channel conductance of approximately 7 pS that was identified as T type. A search for calcium channel alpha1 subunit messenger RNA in the 140-3 cells with three different tests only revealed alpha1C, whereas alpha1A-alpha1C were present in the parent NG108-15 line. We made a particular effort to search for alpha1E, since this subunit has been associated with a low-voltage-activated current. Our findings suggest that, since the principal nerve terminal-associated calcium channels (alpha1A, alpha1B, alpha1E) are absent in the 140-3 cell, this cell line may prove a particularly useful model for the analysis of the role of high-voltage-activated calcium channels in complex functions of neuronal cells.

Animals↗
PubMed1997

Cleavage of syntaxin prevents G-protein regulation of presynaptic calcium channels.

Neurotransmitter release into the synapse is stimulated by calcium influx through ion channels that are closely associated with the transmitter release sites. This link may involve the membrane protein syntaxin, which is known to be associated with the release sites and to bind to the calcium channels. There is evidence that presynaptic calcium channels are downregulated by second messenger pathways involving G proteins. Here we use the patch-clamp technique to test whether calcium current is regulated by G proteins in a vertebrate presynaptic nerve terminal, and whether this regulation is affected by the linkage to syntaxin. The calcium current in the nerve terminal showed typical G-protein-mediated changes in amplitude and activation kinetics which were reversed by a preceding depolarization. These effects of the G protein were virtually eliminated if syntaxin was first cleaved with botulinum toxin C1. Our findings indicate that this sensitivity of the current to modulation by G proteins requires the association of the presynaptic calcium channel with elements of the transmitter release site, which may ensure that channels tethered at release sites are preferentially regulated by the G-protein second messenger pathway.

Animals↗
PubMed1997

The calcium channel and the organization of the presynaptic transmitter release face.

Calcium influx through ion channels located on the release face of the presynaptic nerve terminal gates the release of neurotransmitters by the fusion of the secretory vesicle and the discharge of its contents. Recently, several lines of research have indicated that the relationship between the Ca2+ channel and the release site might be more complex than dictated simply by its role as an ion conduit. The evidence suggests that the channel and the transmitter-release mechanism exist as a multimolecular entity and that this interaction has functional consequences, not only on the mechanisms and properties of transmitter release, but also on the behavior of the presynaptic Ca2+ channel itself.

Animals↗
PubMed1996

Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons.

1. Single L-type calcium channels in chick ciliary ganglion neurons were studied at high current resolution in cell-attached patch recordings using quartz-glass micropipettes. 2. A single open-channel current amplitude was observed when Ba2+ was the charge carrier with a conductance of 26 pS at 110 mM barium. However, with 110 mM calcium two current fluctuation amplitudes were observed. These were termed low and high fluctuation amplitudes, CaL and CaH, and had conductances of 8.8 and 12 pS, respectively. These two levels probably reflect two different channel species. CaL was identified as an L-type calcium channel on the basis of resistance to inactivation, conductance, and dihydropyridine sensitivity. 3. Single-channel current fluctuations could be detected with calcium concentrations as low as 1.0 mM. Although the unitary conductance (gamma) was much greater with barium than calcium at every concentration tested, the concentration dependence of conductance was similar for gamma Ba, gamma CaH and gamma CaL. Fitting the concentration dependencies of these conductances with a Langmuir isotherm gave KD estimates of 4.7, 5.6 and 5.0 mM for barium, CaL and CaH, respectively 4. The single-channel conductance of the L-type channel (gamma L) can be described by the relation: conductance (in pS) = 9.2/(1 + 5.6/[Ca]) where [Ca] is the external calcium concentration in the 1.0-110 mM range. Thus, at a physiological external calcium concentration of 2 mM the conductance is 2.4 pS. 5. Ca2+ transport through the L-type calcium channel is particularly sensitive to changes in external calcium concentration in the physiological range but approaches saturation at about 10 mM. this characteristic may optimize the responsiveness of the cell to small changes in ambient calcium concentrations while limiting excess entry in the presence of abnormally high calcium levels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

An ATP-activated, ligand-gated ion channel on a cholinergic presynaptic nerve terminal.

ATP has recently been identified as a fast neurotransmitter in both the central and peripheral nervous systems. Several studies have suggested that ATP can also affect the release of classical neurotransmitters, including acetylcholine with which it is co-released. We have searched for ATP receptors on a cholinergic presynaptic nerve terminal using the calyx-type synapse of the chicken ciliary ganglion. ATP was pulsed onto the terminals under voltage clamp and induced a short latency cation current that exhibited inward rectification and marked desensitization. This current was not seen with adenosine but was mimicked by several sterically restricted ATP analogs and was blocked by suramin. ATP-activated single ion channels exhibited prominent flickering and had a conductance of approximately 17 pS. Our results demonstrate a ligand-gated P2X-like purinergic receptor on a cholinergic presynaptic nerve terminal.

Adenosine Triphosphate↗
PubMed1996

Single-domain/bound calcium hypothesis of transmitter release and facilitation.

1. We describe a model of transmitter release that is based on the finding that release can be gated during the opening of individual Ca2+ channels, suggesting that the release site can be activated by the Ca2+ domain under a single channel. In this model each release site contains four independent Ca2+ binding sites or gates with unbinding kinetics graded from slow to fast and affinities ranging from high to low. All four gates must be bound for release to occur. Thus synaptic dynamics are governed by the kinetics of Ca2+ binding and unbinding from release sites, not Ca2+ diffusion. 2. Fast facilitation occurs when an action potential invades a terminal with one or more ions remaining bound to the release sites. Residual free Ca2+ is not necessary for facilitation with this mechanism, but if present it would enhance facilitation by binding to high-affinity gates between pulses. 3. This model can account for key features of release. These include fourth-power cooperativity with regard to external Ca2+; a release time course that is virtually independent of an increase in quantal content; an inverse relation between external Ca2+ and the degree of facilitation; and a steplike increase in facilitation with increasing stimulus frequency, with each step corresponding to a unitary decline in the Ca2+ cooperativity. 4. Facilitation of single-channel-based secretion is shown to be robust even if channel opening is stochastic. Spontaneous release of transmitter, assumed to be due in part to spontaneous Ca2+ channel openings, is shown to be elevated during and after a train of impulses. 5. An extension of the model to include multiple Ca2+ channels per release site demonstrates that one role of overlapping Ca2+ domains may be to accentuate depolarization-evoked release relative to spontaneous release.

Action Potentials↗
PubMed1995

Calcium binding sites of the transmitter release mechanism: clues from short-term facilitation.

The binding of multiple, probably four, calcium ions to an intraterminal protein is believed to be an integral step in the gating of neurotransmitter release. We have reexamined the clues to this ion-protein interaction inferred from experimental results on transmitter release and its facilitation. It is argued that while one of the four calcium binding sites required to activate transmitter release may have a relatively low affinity, results obtained from studies on short-term facilitation suggest that the other sites have affinities that range from intermediate to relatively high. A low calcium affinity should not, therefore, be regarded as obligatory requirement in the identification of the calcium binding protein.

Animals↗
PubMed1994

Localization of individual calcium channels at the release face of a presynaptic nerve terminal.

Studies using biophysical techniques suggest a highly structured organization of calcium channels at the presynaptic transmitter release face (Llinás et al., 1981; Stanley, 1993), but it has not as yet proved possible to localize identified channels at the required nanometer level of resolution. We have used atomic force microscopy on the calyx-type nerve terminal of the chick ciliary ganglion to localize single calcium channels tagged via biotinylated omega-conotoxin GVIA to avidin-coated 30 nm gold particles. Calcium channels were in low (modal value approximately < or = 1 per micron 2) and high (modal value approximately 55 per micron 2) density areas and exhibited a prominent interchannel spacing of 40 nm, indicating an intermolecular linkage. Particles were observed in clusters and short linear or parallel linear arrays, groupings that may reflect calcium channel organization at the transmitter release site.

Animals↗
PubMed1993

Single calcium channels and acetylcholine release at a presynaptic nerve terminal.

The relationship between calcium influx and the gating of transmitter release was examined at the release face of a cholinergic presynaptic nerve terminal using a technique that allows the simultaneous recording of both calcium channels at the single-channel level and quantal acetylcholine secretion. Acetylcholine release occurred during large inward calcium currents through many simultaneously open channels but was also gated by very small calcium transients, admitting less than 200 ions, when only one channel was open at a time. These findings provide functional support for a highly structured model of the transmitter release face in which the synaptic vesicle release mechanism is closely tethered to one or more presynaptic calcium channels and the opening of only one of these may be sufficient to trigger quantal secretion.

Acetylcholine↗
PubMed1992

Magainin 2, a natural antibiotic from frog skin, forms ion channels in lipid bilayer membranes.

We have examined the ion channel forming properties of magainin 2 by incorporating the peptide into artificial lipid bilayers held under voltage clamp. Magainin 2 increased lipid bilayer conductance in a concentration dependent manner with a Hill coefficient of 1.7. The magainin 2 conductance was selective for monovalent cations over anions with a ratio of 5:1 and had both voltage-sensitive and -insensitive components. Two structurally related but antibiotically less potent analogues, magainin 1 and Z-12, also increased lipid bilayer conductance with a similar ion selectivity but these peptides were less potent than magainin 2. We propose that the weak cation selectivity of the magainin channels can be accounted for by the inclusion of negatively charged lipids in the channel complex and suggest two possible structures for such a channel. The ionophoric properties of these peptides are likely to be proximal to their antibiotic activities.

Animals↗