Synaptic Calcium-Channel Function in<i>Drosophila</i>: Analysis and Transformation Rescue of Temperature-Sensitive Paralytic and Lethal Mutations of<i>Cacophony</i>

Kawasaki, Fumiko; Collins, Stephen C.; Ordway, Richard W.

doi:10.1523/jneurosci.22-14-05856.2002

Cited by 84 publications

(105 citation statements)

References 61 publications

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“…The dab EC1 mutation was recovered in a classic ethane methyl sulfonate mutagenesis screen for modifiers of the TS presynaptic voltage-gated calcium channel α1 subunit mutant cac TS2 (20)(21)(22) that had been recovered previously as a modifier of comatose (23). dab EC1 is an enhancer of cac TS2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Kawasaki

Iyer

Posey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Members of the DISABLED (DAB) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric CLATHRIN-associated sorting proteins that recruit cargo proteins into endocytic vesicles. Here, we report a Drosophila disabled mutant revealing a novel role for DAB proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activitydependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila DAB and CLATHRIN were highly colocalized within two distinct classes of puncta, including relatively dim puncta that were located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including DYNAMIN, supported a general role for CLATHRIN-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool.I ntensive study of chemical synaptic transmission has led to detailed molecular models of synaptic vesicle endocytosis (1-4). Ongoing efforts seek to define the underlying molecular components and interactions further as well as their spatial organization with respect to neurotransmitter release sites. The present study reveals novel molecular mechanisms and interactions in synaptic vesicle endocytosis involving a member of the DISABLED family of Phosphotyrosine Binding (PTB) domain proteins. In Drosophila, the disabled (dab) gene, the founding member of the dab gene family, was first identified in a screen for genetic modifiers of abelson (5). Although putative dab mutations were subsequently mapped to a different gene (6), recent studies have established functional interactions of Drosophila DISABLED (dDAB) and ABELSON (7). Proteins closely related to dDAB, including mouse DAB-2 (mDAB-2) and the single Caenorhabditis elegans family member ceDAB-1, function as CLATHRIN-associated adaptors in endocytic trafficking of cell surface receptors (8-10). These DABs have been shown to interact with CLATHRIN, the α-ADAPTIN (AP-2) adaptor complex, and other components of the endocytic machinery through conserved binding motifs (8-10) (Fig. 1A). Notably, mDAB-2, ceDAB-1, and dDAB share a PTB/DAB Homology (DH) domain near the N terminus (Fig. 1B), which interacts with specific vesicle cargo proteins and phosphatidylinositol-4,5-diphosphate (11, 12). Neither DAB proteins nor PTB domain interactions have been previously implicated in synaptic vesicle trafficking.The present study reveals a role for DAB proteins in synaptic vesicle endocytosis and extends previous work on interactions of endocytic and exocytic mechanisms in neurotransmitter release. Here, we use a glutamatergic neuromuscular synapse of the Drosophila adult as a model for genetic analysis of molecular mechanisms determining conserved properties of glutamatergic synapse function (13,14). Previous analysis i...

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Section: Resultsmentioning

confidence: 99%

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Kawasaki

Iyer

Posey

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…As described previously (Kawasaki et al, 2002) HC129 . Wild-type flies were Canton S. Stocks and crosses for this study were maintained at 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…More recently, isolation and analysis of a cac mutant exhibiting rapid temperaturesensitive paralysis has demonstrated that cac encodes a primary presynaptic calcium channel in Drosophila (Dellinger et al, 2000;Kawasaki et al, 2000Kawasaki et al, , 2002. To further examine the role of this calcium channel gene product in neurotransmitter release, we used transgenic methods to express enhanced green fluorescent protein (EGFP)-tagged cac-encoded ␣1 subunits in vivo.…”

Section: Introductionmentioning

confidence: 99%

Active Zone Localization of Presynaptic Calcium Channels Encoded by thecacophonyLocus ofDrosophila

et al. 2004

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Presynaptic calcium channels play a central role in chemical synaptic transmission by providing the calcium trigger for evoked neurotransmitter release. These voltage-gated calcium channels are composed of a primary structural subunit, ␣1, as well as auxiliary ␤ and ␣2␦ subunits. Our previous genetic, molecular, and functional analysis has shown that the cacophony (cac) gene encodes a primary presynaptic calcium channel ␣1 subunit in Drosophila. Here we report that transgenic expression of a cac-encoded ␣1 subunit fused with enhanced green fluorescent protein efficiently rescues cac lethal mutations and allows in vivo analysis of calcium channel localization at active zones. The results reported here further characterize the primary role of cac-encoded calcium channels in neurotransmitter release. In addition, these studies provide a unique genetic tool for live imaging of functional presynaptic calcium channels in vivo and define a molecular marker for immunolocalization of other presynaptic proteins relative to active zones. These findings are expected to facilitate additional analysis of synaptic development and function in this important model system.

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“…In both cases, the R876C charge neutralization reduces the sensitivity of the channel to voltage, shifting the conductance-voltage relationship and requiring more depolarization for channel activation. Indeed, the third arginine in the S4 voltage domain has been shown to contribute more than any other S4 charged residue in (19), and the P1385S change found in Dmca1A TS2 (62). B, the Arg-876 amino acid (circled) is conserved in all voltage-gated ion channels through evolution and forms the third charged residue in the S4 voltage sensor transmembrane helix.…”

Section: Fig 1 Temperature-sensitive Mutants Inmentioning

confidence: 99%

Presynaptic N-type Calcium Channels Regulate Synaptic Growth

Rieckhof

Yoshihara

Guan

et al. 2003

Journal of Biological Chemistry

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Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest Ntype channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calciumsensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.

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Synaptic Calcium-Channel Function inDrosophila: Analysis and Transformation Rescue of Temperature-Sensitive Paralytic and Lethal Mutations ofCacophony

Cited by 84 publications

References 61 publications

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

The DISABLED protein functions in CLATHRIN-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Active Zone Localization of Presynaptic Calcium Channels Encoded by thecacophonyLocus ofDrosophila

Presynaptic N-type Calcium Channels Regulate Synaptic Growth

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