The transmembrane domain of syntaxin 1A negatively regulates voltage-sensitive Ca2+ channels

Trus, Michael; Wiser, Ofer; Goodnough, Michael C.; Atlas, Daphné

doi:10.1016/s0306-4522(01)00083-5

Cited by 41 publications

(86 citation statements)

References 52 publications

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confidence: 94%

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confidence: 98%

“…Sx1A has also been shown to modify the kinetic properties of voltage-gated Ca 2ϩ channels establishing a pivotal role of Ca 2ϩ channels in the onset and regulation of the release process (11)(12)(13). The TMD of syntaxin 1A participates in the reduction of current amplitude of N-type (Ca v 2.2) and Lc-type (Ca v 1.2) channels (12,13).…”

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confidence: 99%

“…The TMD of syntaxin 1A participates in the reduction of current amplitude of N-type (Ca v 2.2) and Lc-type (Ca v 1.2) channels (12,13). A Sx1A chimera in which the TMD domain was switched with syntaxin 2 TMD abolished the inhibitory effect of Sx1A, thereby pointing to the inhibitory function of the transmembrane domain (12,13). Furthermore, botulinum toxin C1, shown to cleave specifically Sx1A close to its transmembrane anchor, abolished Sx1A modulation of inward currents, most likely by uncoupling the Sx1A/channel interaction (12).…”

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confidence: 99%

“…The two cysteines appear to play an essential role in modulating the voltage-gated channels, Ca v 1.2 and Ca v 2.2 (12,13). In contrast, syntaxin 2, which has valine residues in the TMD in place of the two cysteines, does not modulate the channel activity (11,12). A Sx1A mutant in which the two cysteines were mutated to valine showed loss of function, with no effect on current amplitude, whereas mutating only one of the two cysteines retained the inhibitory activity of syntaxin (12).…”

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Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner

Arien¹,

Wiser²,

Arkin

et al. 2003

Journal of Biological Chemistry

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Syntaxin 1A (Sx1A) modifies the activity of voltagegated Ca 2؉ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca 2؉ channel, Ca v 1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits ␣ 1 1.2/␣ 2 ␦1/ ␤2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca 2؉ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca 2؉ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca 2؉ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.Signal transduction in the synapse is initiated by neurotransmitter release as a consequence of fusion that takes place between synaptic vesicles and the plasma membrane. It is commonly accepted that at the heart of this tightly regulated, multifarious process lies the ternary complex formed by the synaptic proteins syntaxin 1A (Sx1A), 1 SNAP-25, and synaptobrevin II (1-3). This ternary complex is thought to be responsible for juxtaposing the synaptic vesicle and the plasma membrane prior to membrane fusion, which involves the binding of soluble N-ethylmaleimide-sensitive factor (NSF) (1-3). It is for this reason that the above-mentioned ternary complex is referred to as the SNARE (receptor for soluble NSF attachment proteins) complex.Analysis of ternary complexes formed by the full-length proteins revealed that the C-terminal transmembrane domains (TMDs) of both Sx1A and synaptobrevin II were protected from trypsin digestion (4). Moreover, inclusion of these TMDs increased the stability of the ternary complexes and affected the ability of the cytoplasmic domains to join other proteins (4, 5). Co-reconstituted into liposomes, synaptobrevin II and syntaxin 1A formed a stable binary complex that required the presence of th...

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confidence: 94%

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confidence: 98%

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confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner

Arien¹,

Wiser²,

Arkin

et al. 2003

Journal of Biological Chemistry

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Reconstitution of Depolarization and Ca2+-Evoked Secretion in Xenopus Oocytes Monitored by Membrane Capacitance

Cohen

Schmitt

Atlas

2008

Methods in Molecular Biology

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The identity of the proteins that constitute the "minimal molecular machinery" required for depolarization-evoked neurotransmitter release at synapses is still not fully disclosed. Using capacitance monitoring combined with heterologous protein expression in Xenopus oocytes, we were able to reconstitute a fast (<.5 s) secretion that was triggered directly by membrane depolarization. The functional assembly of voltage-gated Ca2+ channel (Cav1.2 or Cav2.2) coexpressed with syntaxin 1A, synaptosome-associated protein of 25 kDa (SNAP-25), and synaptotagmin led to the reconstitution of depolarization-evoked secretion. Botulinum C1, botulinum A, and tetanus toxin were used to establish that this minimal set of proteins, named the excitosome complex, was necessary and sufficient for reconstituting depolarization-induced exocytosis. Similar to synaptic transmission, the capacitance changes were sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channels (Lc or N type; ionotropic glutamate GLUR3), and depended nonlinearly on extracellular divalent cation concentration. Expression of a recombinant intracellular domain of the calcium channel (Lc753-893) abolished evoked release in the reconstituted assay. Also, mutations at the synaptotagmin C2A polylysine motif, a channel interaction site, abolished depolarization-evoked capacitance transients, consistent with release studies in PC12 cells. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic and nonsynaptic exocytosis and examine the role of other proteins implicated in these processes.

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Regulation of Active Zone Ca2+ Channels

Leal

Mochida

2014

Presynaptic Terminals

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The transmembrane domain of syntaxin 1A negatively regulates voltage-sensitive Ca2+ channels

Cited by 41 publications

References 52 publications

Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner

Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner

Reconstitution of Depolarization and Ca2+-Evoked Secretion in Xenopus Oocytes Monitored by Membrane Capacitance

Regulation of Active Zone Ca2+ Channels

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