[show abstract][hide abstract] ABSTRACT: Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.
[show abstract][hide abstract] ABSTRACT: Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming α1 and the regulatory α2δ, γ and β subunits. Molecular and functional studies have revealed which α1 subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by α1A subunit, mediate evoked transmitter release. The generation of α1A-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs α1 and β subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The α1A subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits α1B, α1D and α1E were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the α1A. On the other hand, the β1b, β2a and β4 were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the α1A subunit, P/Q channel under certain pathological conditions.
[show abstract][hide abstract] ABSTRACT: At synapses of the mammalian central nervous system, release of neurotransmitter occurs at rates transiently as high as 100 Hz, putting extreme demands on nerve terminals with only tens of functional vesicles at their disposal. Thus, the presynaptic vesicle cycle is particularly critical to maintain neurotransmission. To understand vesicle cycling at the most fundamental level, we studied single vesicles undergoing exo/endocytosis and tracked the fate of newly retrieved vesicles. This was accomplished by minimally stimulating boutons in the presence of the membrane-fluorescent styryl dye FM1-43, then selecting for terminals that contained only one dye-filled vesicle. We then observed the kinetics of dye release during single action potential stimulation. We found that most vesicles lost only a portion of their total dye during a single fusion event, but were able to fuse again soon thereafter. We interpret this as direct evidence of "kiss-and-run" followed by rapid reuse. Other interpretations such as "partial loading" and "endosomal splitting" were largely excluded on the basis of multiple lines of evidence. Our data placed an upper bound of <1.4 s on the lifetime of the kiss-and-run fusion event, based on the assumption that aqueous departitioning is rate limiting. The repeated use of individual vesicles held over a range of stimulus frequencies up to 30 Hz and was associated with neurotransmitter release. A small percentage of fusion events did release a whole vesicle's worth of dye in one action potential, consistent with a classical picture of exocytosis as fusion followed by complete collapse or at least very slow retrieval.
[show abstract][hide abstract] ABSTRACT: Vesicle fusion and recycling are particularly critical for ongoing neurotransmitter release in the small nerve terminals of the brain, which typically contain about 30 functional vesicles. However, the modes of exocytosis and endocytosis that operate at synapses of the central nervous system are incompletely understood. Here we show real-time visualization of a single vesicle fusing at a small synapse of the central nervous system, made possible by highly intensified charge-coupled device imaging of hippocampal synaptic terminals, in which a single vesicle was labelled with the fluorescent membrane marker FM1-43 (ref. 6). In a small number of cases, full loss of fluorescent membrane dye was elicited by a single action potential, consistent with classical complete collapse. In most cases, however, action potentials triggered only partial loss of fluorescence, suggesting vesicular retention of membrane marker, consistent with 'kiss-and-run' vesicle cycling. An alternative hypothesis of independent fusion of partially stained vesicles arising from endosomal splitting could be excluded by observations on the size and timing of successive fusion events. Thus, our experimental evidence supports a predominance of kiss-and-run fusion events and rapid vesicular re-use.
[show abstract][hide abstract] ABSTRACT: The neuronal nucleus plays a vital role in information processing, but whether it supports computational functions such as paired-pulse facilitation, comparable to synapses, is unclear. Ca(2+)-dependent movement of calmodulin (CaM) to the nucleus is highly responsive to Ca(2+) entry through L-type channels and promotes activation of the transcription factor CREB (cAMP-responsive element binding protein) through phosphorylation by CaM-sensitive kinases. We characterized key features of this CaM translocation and its possible role in facilitation of nuclear signaling. Nuclear CaM was elevated within 15 s of stimulus onset, preceding the first signs of CREB phosphorylation in hippocampal pyramidal neurons. Depolarization-induced elevation of nuclear CaM also was observed in cerebellar granule cells, neocortical neurons, and dentate gyrus granule cells. Nuclear translocation of CaM was not blocked by disruption of actin filaments or microtubules, or by emptying endoplasmic reticulum Ca(2+) stores with thapsigargin. Translocation of fluorescently tagged CaM was prevented by fusing it with the Ca(2+)/CaM binding peptide M13, suggesting that nuclear CaM accumulation depends on association with endogenous Ca(2+)/CaM binding proteins. To determine whether increased nuclear [CaM] might influence subsequent nuclear signal processing, we compared responses to two consecutive depolarizing stimuli. After a weak "priming" stimulus that caused CaM translocation, CREB phosphorylation caused by a subsequent stimulus was significantly faster, more sensitive to Ca(2+) elevation, and less specifically dependent on Ca(2+) influx through L-type channels. CaM translocation not only supports rapid signaling to the nucleus, but also could provide a "memory" for facilitatory effects of repeated neural activity, seen in altered phosphorylated CREB dynamics and Ca(2+) channel dependence.
Proceedings of the National Academy of Sciences 01/2002; 98(26):15342-7. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca(2+) channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. We assayed ion channel function by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reached or exceeded the pathogenic range for SCA6. This gain at the protein level was not a consequence of changes in RNA stability, as indicated by Northern blot analysis. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yielded functional ion channels that supported inward Ca(2+) channel currents, with a sharp increase in P/Q Ca(2+) channel current density relative to WT. Our results showed that Ca(2+) channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface.
Journal of Neuroscience 01/2002; 21(23):9185-93. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3'-diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC+) and PC-negative (PC-). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC+ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC+ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.
Proceedings of the National Academy of Sciences 11/2001; 98(22):12748-53. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Memory storage in mammalian neurons probably depends on both biochemical events and morphological alterations in dendrites. Here we report an activity-dependent stabilization of the MAP kinase (MAPK) pathway, prominent in hippocampal dendrites. The longevity of the signal in these dendrites was increased to hours when multiple spaced stimuli were used. Likewise, spaced stimuli and MAPK activation were critical for protrusion of new dendritic filopodia that also remained stable for hours. Our experiments define a new role for stimulus-specific responses of MAPK signaling in activity-dependent neuronal plasticity. The local biochemical signaling in dendrites complements MAPK signaling in gene expression. Together, these processes may support long-lasting behavioral changes.
[show abstract][hide abstract] ABSTRACT: The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca(2+) transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca(2+) signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only approximately 1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.
Proceedings of the National Academy of Sciences 03/2001; 98(5):2808-13. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca(2+) channels in biochemical studies and affects gating of these Ca(2+) channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming alpha(1B) subunit of N-type Ca(2+) channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. Our analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appeared critical for the channel modulation but not for biochemical association with the "synprint site" in the II/III loop of alpha(1B). These results suggest that syntaxin and the alpha(1B) subunit engage in two kinds of interactions: an anchoring interaction via the II/III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca(2+) channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis.
Proceedings of the National Academy of Sciences 01/2001; 97(25):13943-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Functional presynaptic vesicles have been subdivided into readily releasable (RRP) and reserve (RP) pools. We studied recycling properties of RRP vesicles through differential retention of FM1-43 and FM2-10 and by varying the time window for FM dye uptake. Both approaches indicated that vesicles residing in the RRP underwent rapid endocytosis (tau approximately 1s), whereas newly recruited RP vesicles were recycled slowly (tau approximately 30 s). With repeated challenges (hypertonic or electrical stimuli), the ability to release neurotransmitter recovered 10-fold more rapidly than restoration of FM2-10 destaining. Finding neurotransmission in the absence of destaining implied that rapidly endocytosed RRP vesicles were capable of reuse, a process distinct from repopulation from the RP. Reuse would greatly expand the functional capabilities of a limited number of vesicles in CNS terminals, particularly during intermittent bursts of activity.
[show abstract][hide abstract] ABSTRACT: L-type Ca(2+) channels are unusual in displaying two opposing forms of autoregulatory feedback, Ca(2+)-dependent inactivation and facilitation. Previous studies suggest that both involve direct interactions between calmodulin (CaM) and a consensus CaM-binding sequence (IQ motif) in the C terminus of the channel's alpha(1C) subunit. Here we report the functional effects of an extensive series of modifications of the IQ motif aimed at dissecting the structural determinants of the different forms of modulation. Although the combined substitution by alanine at five key positions (Ile(1624), Gln(1625), Phe(1628), Arg(1629), and Lys(1630)) abolished all Ca(2+) dependence, corresponding single alanine replacements behaved similarly to the wild-type channel (77wt) in four of five cases. The mutant I1624A stood out in displaying little or no Ca(2+)-dependent inactivation, but clear Ca(2+)- and frequency-dependent facilitation. An even more pronounced tilt in favor of facilitation was seen with the double mutant I1624A/Q1625A: overt facilitation was observed even during a single depolarizing pulse, as confirmed by two-pulse experiments. Replacement of Ile(1624) by 13 other amino acids produced graded and distinct patterns of change in the two forms of modulation. The extent of Ca(2+)-dependent facilitation was monotonically correlated with the affinity of CaM for the mutant IQ motif, determined in peptide binding experiments in vitro. Ca(2+)-dependent inactivation also depended on strong CaM binding to the IQ motif, but showed an additional requirement for a bulky, hydrophobic side chain at position 1624. Abolition of Ca(2+)-dependent modulation by IQ motif modifications mimicked and occluded the effects of overexpressing a dominant-negative CaM mutant.
Journal of Biological Chemistry 08/2000; 275(28):21121-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: When the presynaptic membrane protein syntaxin is coexpressed in Xenopus oocytes with N- or P/Q-type Ca(2+) channels, it promotes their inactivation (Bezprozvanny et al., 1995; Wiser et al., 1996, 1999; Degtiar et al., 2000) (I. B. Bezprozvanny, P. Zhong, R. H. Scheller, and R. W. Tsien, unpublished observations). These findings led to the hypothesis that syntaxin influences Ca(2+) channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca(2+) channels to the release machinery. We examined this effect in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely blocked neurotransmitter release. This treatment altered the pattern of Ca(2+) entry monitored with fura-2. Whereas the initial Ca(2+) rise induced by depolarization with K(+)-rich solution was unchanged, late Ca(2+) entry was strongly augmented by syntaxin cleavage. Similar results were obtained when Ca(2+) influx arose from repetitive firing induced by the K(+)-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca(2+) entry. The BoNtC1-induced alteration in Ca(2+) signaling was specific to voltage-gated Ca(2+) channels, not Ca(2+) extrusion or buffering, and it involved N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin was not immediately manifest when synaptosomes had been K(+)-predepolarized in the absence of external Ca(2+), but developed with a delay after admission of Ca(2+), suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca(2+) channel inactivation.
Journal of Neuroscience 07/2000; 20(12):4368-78. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca(2+) channels. This biochemical association is proposed to colocalize Ca(2+) channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca(2+) channels according to the state of the associated release machinery (Bezprozvanny et al., 1995; Wiser et al., 1996; see also Mastrogiacomo et al., 1994). Here we report that syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promoted slow inactivation gating, but had little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depended strongly on voltage protocol. Slow inactivation was found for N-type channels even in the absence of syntaxin and could be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence (Q(10), approximately 0.8). Trains of action potential-like stimuli were more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decreased the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuated the modulatory effects on Ca(2+) channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca(2+) influx in nerve terminals.
Journal of Neuroscience 07/2000; 20(12):4355-67. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: 'Silent synapses' show responses from high-affinity NMDA receptors (NMDARs) but not low-affinity AMPA receptors (AMPARs), but gain AMPAR responses upon long-term potentiation (LTP). Using the rapidly reversible NMDAR antagonist l-AP5 to assess cleft glutamate concentration ([glu]cleft), we found that it peaked at <170 microM at silent neonatal synapses, but greatly increased after potentiation. Cyclothiazide (CTZ), a potentiator of AMPAR, revealed slowly rising AMPA EPSCs at silent synapses; LTP shortened their rise times. Thus, LTP at silent synapses increased rate-of-rise and peak amplitude of [glu]cleft. Release probability reported by NMDARs remained unchanged during LTP, implying that [glu]cleft increases arose from immediately presynaptic terminals. Our data suggest that changes in the dynamics of fusion-pore opening contribute to LTP.
[show abstract][hide abstract] ABSTRACT: Activity-dependent gene expression in neurons shows a remarkable ability to differentiate between different types of stimulation: orthodromic inputs that engage synaptic transmission are much more effective than antidromic stimuli that do not. We have studied the basis of such selectivity in cultured hippocampal neurons in which nuclear cAMP response element-binding protein (CREB) phosphorylation is induced by synaptic activity but not by action potential (AP) stimulation in the absence of EPSPs, although spikes by themselves generate large elevations in intracellular Ca(2+). Previous work has shown that Ca(2+) entry through L-type Ca(2+) channels plays a dominant role in triggering calmodulin mobilization and activation of calmodulin-dependent kinases that phosphorylate CREB, raising the possibility that L-type channels contribute to the selective response to EPSPs rather than APs. Accordingly, we performed voltage-clamp experiments to compare the currents carried by L-type channels during depolarizing waveforms that approximated APs or dendritic EPSPs. The integrated current generated by L-type channels was significantly less after mock APs than with EPSP-like depolarizations. The difference was traced to two distinct factors. Compared with other channels, L-type channels activated at relatively negative potentials, favoring their opening with EPSP stimulation; they also exhibited relatively slow activation kinetics, weighing against their contribution during an AP. The relative ineffectiveness of APs as a stimulus for CREB phosphorylation could be overcome by exposure to the agonist Bay K8644, which potentiated the AP-induced influx through L-type channels by approximately 10-fold. Under normal conditions, the unique biophysical properties of L-type channels allow them to act as a kinetic filter to support spike-EPSP discrimination.
Journal of Neuroscience 02/2000; 20(1):266-73. · 6.91 Impact Factor