Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans

Huang, Yu‐Tung; Pirri, Jennifer K.; Rayes, Diego; Gao, Shangbang; Mulcahy, Ben; Grant, Jeff; Saheki, Yasunori; Francis, Michael M.; Zhen, Mei; Alkema, Mark J

doi:10.7554/elife.45905

Cited by 32 publications

(54 citation statements)

References 79 publications

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“…We first generated the cim112 animal, in which an S218L point mutation was introduced into the coding sequence of UNC-2 of the cim104 animal ( Figure 1A ). This S218L mutation and the corresponding mutation in human CaV2 have been shown to enhance calcium channel function therefore providing a sensitized background for the screen (van den Maagdenberg et al, 2007; Huang et al, 2019). Because the BK channel ortholog SLO-1 can modulate the function of UNC-2 channels making it difficult to interpret the nature of genetic interactions with genes obtained from the screen (Oh et al, 2015; Oh et al, 2017), we introduced a slo-1 null mutation, eg142 , into the cim112 animal.…”

Section: Resultsmentioning

confidence: 99%

UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α inCaenorhabditis elegans

Krout

Richmond

et al. 2021

Preprint

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Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

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Section: Resultsmentioning

confidence: 99%

UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α inCaenorhabditis elegans

Krout

Richmond

et al. 2021

Preprint

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“…We found that jph-1(0); unc-2(0) double mutants grew substantially more slowly than jph-1(0) single mutants and were sterile as adults ( Table 2 ). The unc-2(zf35gf) gain-of-function mutation causes the channel to open at a lower membrane potential, causing hyperactive locomotion but otherwise normal growth and development (Huang et al, 2019). jph-1(0); unc-2(gf) double mutants displayed significantly slower growth than jph-1 single mutants ( Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

Caenorhabditis ElegansJunctophilin has Tissue-Specific Functions and Regulates Neurotransmission with Extended-Synaptotagmin

Piggott

Nurrish

et al. 2020

Preprint

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The junctophilin family of proteins tether together plasma membrane (PM) and endoplasmic reticulum (ER) membranes, and couple PM- and ER-localized calcium channels. Understanding in vivo functions of junctophilins is of great interest for dissecting the physiological roles of ER-PM contact sites. Here, we show that the sole C. elegans junctophilin JPH-1 localizes to discrete membrane contact sites in neurons and muscles and has important tissue-specific functions. jph-1 null mutants display slow growth and development due to weaker contraction of pharyngeal muscles, leading to reduced feeding. In the body wall muscle, JPH-1 co-localizes with the PM-localized EGL-19 voltage-gated calcium channel and ER-localized UNC-68/RyR calcium channel, and is required for animal movement. We also find an unexpected cell non-autonomous effect of jph-1 in axon regrowth after injury. In neurons, JPH-1 co-localizes with the membrane contact site protein Extended-SYnaptoTagmin 2 (ESYT-2) and modulates neurotransmission. Interestingly, jph-1 and esyt-2 null mutants display mutual suppression in their response to aldicarb, suggesting that JPH-1 and ESYT-1 have antagonistic roles in neuromuscular synaptic transmission. Our genetic double mutant analysis also reveals that jph-1 functions in overlapping pathways with two PM-localized voltage-gated calcium channels, egl-19 and unc-2, and unc-68/RyR for animal health and development. Finally, we show that unc-68/RyR is required for JPH-1 localization to ER-PM puncta. Our data demonstrate important roles for junctophilin in cellular physiology, and also provide insights into how junctophilin functions together with other calcium channels in vivo.

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“…Thus, mutations in proteins that orchestrate the anchoring and tight apposition of docked SVs and Ca v 2 VGCCs are likely to negatively affect this process. Also, such proteins are probable targets for regulation of synaptic transmission, and could be utilized to set the excitation-inhibition balance also in complex neural circuits (Huang et al, 2019; Liu et al, 2018). Here, we characterized in detail the function of the C. elegans RIMB-1 protein, as well as its (functional) interaction with the Ca v 2/UNC-2 VGCC, and determinants of VGCC localization at NMJ synapses, in cholinergic and GABAergic neurons.…”

Section: Discussionmentioning

confidence: 99%

“…Such differential functions were also found in different central synapses in the mouse (Brockmann et al, 2019). In C. elegans , a gain-of-function mutation in UNC-2/Ca v 2 was recently shown to induced increased cholinergic transmission but a reduction in GABA transmission through effects on GABA A receptors, or rather, GABA synapse numbers (Huang et al, 2019). Here, we probed post-synaptic nAChRs through levamisole paralysis assays, which did not show a defect in rimb-1 mutants.…”

Section: Discussionmentioning

confidence: 99%

RIM and RIM-binding protein localize synaptic CaV2 channels to differentially regulate transmission in neuronal circuits

Jánosi

et al. 2021

Preprint

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Synapses are intricately organized subcellular compartments in which molecular machines cooperate to ensure spatiotemporally precise transmission of chemical signals. Key components of this machinery are voltage-gated Ca2+-channels (VGCCs), that translate electrical signals into a trigger for fusion of synaptic vesicles (SVs) with the plasma membrane. The VGCCs and the Ca2+ microdomains they generate must be located in the right distance to the primed SV, to elicit transmitter release without delay. Rab3 interacting molecule (RIM) and RIM-binding protein (RIM-BP) were shown in different systems to contribute to the spatial organization of the active zone protein scaffold, and to localize VGCCs next to docked SVs by binding to each other and to the C-terminal region of the Cav2 VGCC α-subunit. We asked how this machinery is organized at the neuromuscular junction (NMJ) of Caenorhabditis elegans, and whether it can differentially regulate transmission in circuits composed of different neuron types. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of the UNC-2 VGCC and delaying the onset of SV fusion, while RIM deletion had much more severe defects. rimb-1 mutants caused increased cholinergic but reduced GABAergic transmission, while overall transmission at the NMJ was reduced, as shown by voltage imaging. The UNC-2 channel could further be untethered by removing its C-terminal PDZ binding motif, and this untethering could be exacerbated by combining the ΔPDZ mutant with the rimb-1 mutation. Similar phenotypes resulted from acute degradation of the UNC-2 β-subunit, indicating that destabilization of the VGCC complex causes the same phenotypes as its untethering.

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Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans

Cited by 32 publications

References 79 publications

UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α inCaenorhabditis elegans

UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α inCaenorhabditis elegans

Caenorhabditis ElegansJunctophilin has Tissue-Specific Functions and Regulates Neurotransmission with Extended-Synaptotagmin

RIM and RIM-binding protein localize synaptic CaV2 channels to differentially regulate transmission in neuronal circuits

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