RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle - PubMed (original) (raw)
RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle
Gracia Safdie et al. Mol Biol Cell. 2016.
Abstract
Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons. Here we show that phosphorylation of the ER-resident chaperone of nicotinic acetylcholine receptors, RIC-3, leads to increased muscle excitability. RIC-3 phosphorylation at Ser-164 depends on opposing functions of the phosphatase calcineurin (TAX-6), and of the casein kinase II homologue KIN-10. Effects of calcineurin down-regulation and of phosphorylated RIC-3 on muscle excitability are mediated by GABAA receptor inhibition. Thus RIC-3 phosphorylation enables effects of this chaperone on GABAA receptors in addition to nAChRs. This dual effect provides coordinated regulation of excitation and inhibition and enables fine-tuning of the excitation-inhibition balance. Moreover, regulation of inhibitory GABAA signaling by calcineurin, a calcium- and calmodulin-dependent phosphatase, enables homeostatic balancing of excitation and inhibition.
© 2016 Safdie et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Figures
FIGURE 1:
Sequence of the C. elegans RIC-3. Highlighted in gray is the sequence of the minimal _ric-3_–rescuing fragment. White letters on gray indicate sequence of the alternatively spliced intron missing in the minimal fragment. The transmembrane domains are single underlined, and the coiled-coil domains are double underlined. Residues shown to be phosphorylated by Zielinska et al. (2009) are italicized and indicated by dots. Arrow, Ser-164.
FIGURE 2:
Phosphorylation of Ser-164 enhances muscle excitability and reduces RIC-3 quantity. (A) Levamisole sensitivity of animals expressing the minimal RIC-3 fragment (diamonds) or the same fragment having the S164A mutation (triangles) or the S164E mutation (squares), all under a ric-3 promoter in a ric-3(md1181) (loss-of-function) background. Percentage of animals paralyzed at different time points after being placed on plates containing levamisole (0.2 mM). Significance is relative to minimal RIC-3–expressing animals at the same time point; eight plates and four independent experiments. (B) Levamisole sensitivity of animals expressing minimal RIC-3 (dark gray) or minimal RIC-3 S164E (light gray) expressed from a muscle-specific promoter, myo-3p, and in a wild-type background. Percentage of animals paralyzed at different time points, as described in A. Significance is relative to minimal RIC-3–expressing animals at the same time point; six or seven plates and three independent experiments. (C) Quantity and distribution of RIC-3::GFP and RIC-3(S164E)::GFP expressed from a muscle-specific promoter. Top, representative images of GFP-tag fluorescence; scale bar, 5 μm. Bottom left, average intensity; n = 12 or 13, N = 2. GFP intensity is normalized to the average GFP intensity in S164E animals imaged in the same experiment/day. Bottom right, coefficient of variation of fluorescence intensity from the same images. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 3:
TAX-6 knockdown enhances muscle excitability and reduces RIC-3 quantity. (A) Levamisole sensitivity of wild-type animals fed with empty vector– (control; dark gray) or tax-6 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed at different time points after being placed on plates containing levamisole (0.2 mM). Significance is relative to animals fed with control dsRNA at the same time point; four plates and two independent experiments. (B) Quantity of RIC-3 as seen using immunohistochemistry in animals fed with control or tax-6 dsRNA. Left, representative images of muscles and neurons after each treatment; scale bar, 5 μm. Right, average intensity relative to intensity of neurons from the same animal. two independent stainings and 9–13 animals from each treatment. (C, D) Effects of TAX-6 knockdown are suppressed by Ser164 mutations. Animals expressing full-length, wild-type (WT) RIC-3, minimal RIC-3 (Minimal), minimal RIC-3 S164E (S164E), or minimal RIC-3 S164A (S164A) in a ric-3(md1181) background and treated with control (dark gray) or tax-6 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed at 15 min after being placed on levamisole (C; 8–12 plates and four to seven independent experiments each) or 30 min (D; 8–12 plates and four to seven independent experiments each). Significance is relative to the same strain fed with control dsRNA (empty vector) at the same time point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
FIGURE 4:
Effects of TAX-6 knockdown or loss of function are mediated by GABAA inhibition. (A) Effects of tax-6 knockdown on synaptic abundance of UNC-63. YFP intensity in synaptic puncta of animals fed with empty vector (Control) or tax-6 dsRNA–expressing bacteria. Left, representative images; right, average intensity; n = 35–36 each. YFP intensity is normalized to the average YFP intensity in animals fed with control dsRNA, which were imaged in the same experiment/day. (B, C) Currents recorded from patch-clamped body-wall muscle of wild-type or tax-6(lf) animals after application of levamisole (100 μM; B) or GABA (100 μM; C). Left, representative traces; right, average peak amplitudes; n = 7–9 each. ***p < 0.001.
FIGURE 5:
Effects of the S164E mutation are mediated by GABAA inhibition. (A) Effects of unc-49 knockdown are suppressed by the S164E mutation. Animals expressing minimal RIC-3 (Minimal) or minimal RIC-3 S164E (S164E) specifically in muscle in a wild-type background and treated with empty vector– (Control; dark gray) or unc-49 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed 60 min after being placed on levamisole. Significance is relative to the same strain fed with control dsRNA (empty vector) at the same time point; seven plates and N = 3 each. (B) Currents recorded from oocytes expressing UNC49 B/C alone (UNC-49), with RIC-3 minimal S164A (S164A), or with RIC-3 minimal S164E (S164E). Left, representative traces; right, average peak amplitudes normalized relative to average peak current amplitudes of receptor alone from the same experiment; n = 26–31 and N = 3–4. (C) Western analysis showing quantity of RIC-3 S164E and S164A mutants expressed in X. laevis oocytes with UNC-49 B/C. Left, representative Western analysis; right, average intensity (arbitrary units), three experiments. **p < 0.01, ***p < 0.001.
FIGURE 6:
KIN-10, but not UNC-43, functions opposite to TAX-6. (A) Levamisole sensitivity of wild-type animals fed with empty vector– (Control; dark gray) or unc-43 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed at different time points after being placed on plates containing levamisole (0.2 mM). Significance is relative to animals fed with control dsRNA at the same time point; 11 plates and four independent experiments. (B) Effects of unc-43 knockdown are not suppressed by Ser-164 mutations. Animals expressing minimal RIC-3 (Minimal), minimal RIC-3 S164A (S164A), or minimal RIC-3 S164E (S164E) in a ric-3(md1181) background and fed with empty vector– (Control; dark gray) or unc-43 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed 15 min after being placed on levamisole. Significance is relative to the same strain fed with control dsRNA (empty vector) at the same time point; 8–11 plates, N = 4 each. (C) Levamisole sensitivity of wild-type animals fed with empty vector– (Control; dark gray) or kin-10 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed at different time points after being placed on plates containing levamisole (0.2 mM). Significance is relative to animals fed with control dsRNA at the same time point; three plates and N = 2. (D, E) Effects of kin-10 knockdown are suppressed by Ser-164 mutations. Animals expressing minimal RIC-3 (Minimal), minimal RIC-3 S164A (S164A), or minimal RIC-3 S164E (S164E) in a ric-3(md1181) background and treated with empty vector– (Control; dark gray) or kin-10 dsRNA–expressing (light gray) bacteria. Percentage of animals paralyzed 15 (D) or 30 (E) min after being placed on levamisole. Significance is relative to the same strain fed with control dsRNA (empty vector) at the same time point; four to eight plates and N = 3 each. *p < 0.05, **p < 0.01, ***p < 0.001.
References
- Baumgärtel K, Mansuy IM. Neural functions of calcineurin in synaptic plasticity and memory. Learn Mem. 2012;19:375–384. -PubMed
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