Presynaptic CaV2 calcium channel traffic requires CALF-1 and the alpha(2)delta subunit UNC-36.
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ABSTRACT:
SUBMITTER: Saheki Y
PROVIDER: S-EPMC2805665 | biostudies-literature | 2009 Oct
REPOSITORIES: biostudies-literature
Publications
Presynaptic CaV2 calcium channel traffic requires CALF-1 and the alpha(2)delta subunit UNC-36.
Saheki Yasunori Y Bargmann Cornelia I CI
Nature neuroscience 20090830 10
Presynaptic voltage-gated calcium channels provide calcium for synaptic vesicle exocytosis. We show here that a green fluorescent protein-tagged alpha(1) subunit of the Caenorhabditis elegans CaV2 channel, UNC-2, is localized to presynaptic active zones of sensory and motor neurons. Synaptic localization of CaV2 requires the alpha(2)delta subunit UNC-36 and CALF-1 (Calcium Channel Localization Factor-1), a neuronal transmembrane protein that localizes to the endoplasmic reticulum. In calf-1 muta ...[more]
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Project description:Presynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel localization by active zone proteins is not completely understood. In a Caenorhabditis elegans (C. elegans) forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic localization 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 puncta at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel localization, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that although 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 localization by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel localization. In addition, we find that core active zone proteins are unequal in their abundance. Although 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 localization in redundant yet distinct manners.SIGNIFICANCE STATEMENT Precise 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, our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, a core 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.
| S-EPMC8260173 | biostudies-literature
Project description:The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α1 subunit and two auxiliary β and α2δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans, we show that UNC-2/α1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α2δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes.SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α1 subunit and two auxiliary β and α2δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α1 and aid trafficking, they are recruited to α1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels.
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Project description:The ? subunit of high voltage-activated Ca(2+) (Ca(v)) channels targets the pore-forming ?(1) subunit to the plasma membrane and tunes the biophysical phenotype of the Ca(v) channel complex. We used a combination of molecular biology and whole-cell patch clamp to investigate the functional role of a long N-terminal polyacidic motif (NPAM) in a Ca(v)? subunit of the human parasite Schistosoma mansoni (?(Sm)), a motif that does not occur in other known Ca(v)? subunits. When expressed in human embryonic kidney cells stably expressing Ca(v)2.3, ?(Sm) accelerates Ca(2+)/calmodulin-independent inactivation of Ca(v)2.3. Deleting the first 44 amino acids of ?(Sm), a region that includes NPAM, significantly slows the predominant time constant of inactivation (?(fast)) under conditions that prevent Ca(2+)/CaM-dependent inactivation (?(Sm): ?(fast) = 66 ms; ?(Sm?2-44): ?(fast) = 111 ms, p < 0.01). Interestingly, deleting the amino acids that are N-terminal to NPAM (2-24 or 2-17) results in faster inactivation than with an intact N terminus (?(fast) = 42 ms with ?(Sm?2-17); ?(fast) = 40 ms with ?(Sm?2-24), p < 0.01). This suggests that NPAM is the structural determinant for accelerating Ca(2+)/calmodulin-independent inactivation. We also created three chimeric subunits that contain the first 44 amino acids of ?(Sm) attached to mammalian ?(1b), ?(2a), and ?(3) subunits. For any given mammalian ? subunit, inactivation was faster if it contained the N terminus of ?(Sm) than if it did not. Co-expression of the mammalian ?(2)?-1 subunit resulted in doubling of the inactivation rate, but the effects of NPAM persisted. Thus, it appears that the schistosome Ca(v) channel complex has acquired a new function that likely contributes to reducing the amount of Ca(2+) that enters the cells in vivo. This feature is of potential interest as a target for new antihelminthics.
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Project description:Cerebellar granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta) and assemble various pentameric receptor subtypes with unknown subunit compositions; however, the rules guiding receptor subunit assembly are unclear. Here, removal of intact alpha6 protein from cerebellar granule cells allowed perturbations in other subunit levels to be studied. Exon 8 of the mouse alpha6 subunit gene was disrupted by homologous recombination. In alpha6 -/- granule cells, the delta subunit was selectively degraded as seen by immunoprecipitation, immunocytochemistry, and immunoblot analysis with delta subunit-specific antibodies. The delta subunit mRNA was present at wild-type levels in the mutant granule cells, indicating a post-translational loss of the delta subunit. These results provide genetic evidence for a specific association between the alpha6 and delta subunits. Because in alpha6 -/- neurons the remaining alpha1, beta2/3, and gamma2 subunits cannot rescue the delta subunit, certain potential subunit combinations may not be found in wild-type cells.
| S-EPMC6793744 | biostudies-literature
Project description:Presynaptic active zones (AZs) contain many molecules essential for neurotransmitter release and are assembled in a highly organized manner. A network of adaptor proteins known as cytomatrix at the AZ (CAZ) is important for shaping the structural characteristics of AZ. Rab3-interacting molecule (RIM)-binding protein (RBP) family are binding partners of the CAZ protein RIM and also bind the voltage-gated calcium channels (VGCCs) in mice and flies. Here, we investigated the physiological roles of RIMB-1, the homolog of RBPs in the nematode Caenorhabditis elegans RIMB-1 is expressed broadly in neurons and predominantly localized at presynaptic sites. Loss-of-function animals of rimb-1 displayed slight defects in motility and response to pharmacological inhibition of synaptic transmission, suggesting a modest involvement of rimb-1 in synapse function. We analyzed genetic interactions of rimb-1 by testing candidate genes and by an unbiased forward genetic screen for rimb-1 enhancer. Both analyses identified the RIM homolog UNC-10 that acts together with RIMB-1 to regulate presynaptic localization of the P/Q-type VGCC UNC-2/Cav2. We also find that the precise localization of RIMB-1 to presynaptic sites requires presynaptic UNC-2/Cav2. RIMB-1 has multiple FN3 and SH3 domains. Our transgenic rescue analysis with RIMB-1 deletion constructs revealed a functional requirement of a C-terminal SH3 in regulating UNC-2/Cav2 localization. Together, these findings suggest a redundant role of RIMB-1/RBP and UNC-10/RIM to regulate the abundance of UNC-2/Cav2 at the presynaptic AZ in C. elegans, depending on the bidirectional interplay between CAZ adaptor and channel proteins.SIGNIFICANCE STATEMENT Presynaptic active zones (AZs) are highly organized structures for synaptic transmission with characteristic networks of adaptor proteins called cytomatrix at the AZ (CAZ). In this study, we characterized a CAZ protein RIMB-1, named for RIM-binding protein (RBP), in the nematode Caenorhabditis elegans Through systematic analyses of genetic interactions and an unbiased genetic enhancer screen of rimb-1, we revealed a redundant role of two CAZ proteins RIMB-1/RBP and UNC-10/RIM in regulating presynaptic localization of UNC-2/Cav2, a voltage-gated calcium channel (VGCC) critical for proper neurotransmitter release. Additionally, the precise localization of RIMB-1/RBP requires presynaptic UNC-2/Cav2. These findings provide new mechanistic insight about how the interplay among multiple CAZ adaptor proteins and VGCC contributes to the organization of presynaptic AZ.
| S-EPMC6820209 | biostudies-literature
Project description:The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (alphabetagamma). In humans an additional delta-subunit (delta-hENaC) exists but little is known about its function. Using the Xenopus laevis oocyte expression system, we compared the functional properties of alphabetagamma- and deltabetagamma-hENaC and investigated whether deltabetagamma-hENaC can be proteolytically activated. The amiloride-sensitive ENaC whole-cell current (DeltaI(ami)) was about 11-fold larger in oocytes expressing deltabetagamma-hENaC than in oocytes expressing alphabetagamma-hENaC. The 2-fold larger single-channel Na(+) conductance of deltabetagamma-hENaC cannot explain this difference. Using a chemiluminescence assay, we demonstrated that an increased channel surface expression is also not the cause. Thus, overall channel activity of deltabetagamma-hENaC must be higher than that of alphabetagamma-hENaC. Experiments exploiting the properties of the known betaS520C mutant ENaC confirmed this conclusion. Moreover, chymotrypsin had a reduced stimulatory effect on deltabetagamma-hENaC whole-cell currents compared with its effect on alphabetagamma-hENaC whole-cell currents (2-fold versus 5-fold). This suggests that the cell surface pool of so-called near-silent channels that can be proteolytically activated is smaller for deltabetagamma-hENaC than for alphabetagamma-hENaC. Proteolytic activation of deltabetagamma-hENaC was associated with the appearance of a delta-hENaC cleavage product at the cell surface. Finally, we demonstrated that a short inhibitory 13-mer peptide corresponding to a region of the extracellular loop of human alpha-ENaC inhibited DeltaI(ami) in oocytes expressing alphabetagamma-hENaC but not in those expressing deltabetagamma-hENaC. We conclude that the delta-subunit of ENaC alters proteolytic channel activation and enhances base-line channel activity.
| S-EPMC2781449 | biostudies-literature
Project description:ObjectiveTo characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS).MethodsIntracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure.ResultsAmong 24 variants reported to cause SCCMS only two appear in the AChR δ-subunit. We here report a 16-year-old patient harboring a novel δL273F mutation (δL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR δ subunit. Kinetic analyses with ACh and the weak agonist choline indicate that δL273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified εL269F mutation (εL289F in HGVS nomenclature) at an equivalent location in the AChR ε-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the δ and ε subunit and nearby M2 domain residues in neighboring α subunits contribute to structural stability of the open relative to the closed channel states.InterpretationThe greater increase in gating efficiency by δL273F than by εL269F explains why δL273F has more severe clinical effects. Both δL273F and εL269F impair channel gating by disrupting hydrophobic interactions with neighboring α-subunits. Differences in the extent of impairment of channel gating in δ and ε mutant receptors suggest unequal contributions of ε/α and δ/α subunit pairs to gating efficiency.
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