Dopamine negatively modulates the NCA ion channels in <i>C</i>. <i>elegans</i>

<div><p>The NALCN/NCA ion channel is a cation channel related to voltage-gated sodium and calcium channels. NALCN has been reported to be a sodium leak channel with a conserved role in establishing neuronal resting membrane potential, but its precise cellular role and regulation are unclear. The <i>Caenorhabditis elegans</i> orthologs of NALCN, NCA-1 and NCA-2, act in premotor interneurons to regulate motor circuit activity that sustains locomotion. Recently we found that NCA-1 and NCA-2 are activated by a signal transduction pathway acting downstream of the heterotrimeric G protein G_q and the small GTPase Rho. Through a forward genetic screen, here we identify the GPCR kinase GRK-2 as a new player affecting signaling through the G_q-Rho-NCA pathway. Using structure-function analysis, we find that the GPCR phosphorylation and membrane association domains of GRK-2 are required for its function. Genetic epistasis experiments suggest that GRK-2 acts on the D2-like dopamine receptor DOP-3 to inhibit G_o signaling and positively modulate NCA-1 and NCA-2 activity. Through cell-specific rescuing experiments, we find that GRK-2 and DOP-3 act in premotor interneurons to modulate NCA channel function. Finally, we demonstrate that dopamine, through DOP-3, negatively regulates NCA activity. Thus, this study identifies a pathway by which dopamine modulates the activity of the NCA channels.</p></div

Dopamine negatively modulates NCA-1 and NCA-2 channel activity.

<p>(A) The <i>cat-2(e1112)</i> mutation suppresses the weak forward fainting phenotype of the <i>nlf-1(tm3631)</i> mutant. (***, P<0.001. Error bars = SEM; n = 40). (B) The <i>dop-3(vs106)</i> mutation suppresses the weak forward fainting phenotype of the <i>nlf-1(tm3631)</i> mutant. (***, P<0.001. Error bars = SEM; n = 40). (C) The <i>dop-3(vs106)</i> mutation partially suppresses the strong forward fainting phenotype of the <i>grk-2(gk268)</i>; <i>nlf-1(tm3631)</i> double mutant. (***, P<0.001. Error bars = SEM; n = 40). (D) Exogenous dopamine causes the <i>grk-2(gk268)</i> mutant to faint in a <i>dop-3</i> dependent manner. Shown is the percentage of animals that faint within a period of ten body bends when moving backwards after exposure to 2 mM dopamine for 20 min. (***, P<0.001. Error bars = SEM; n = 2–5 trials of 14–25 animals each).</p

Model for GRK-2 and dopamine action in modulating activity of the NCA channels.

<p>Schematic representation of the dopamine, G_q and G_o signaling pathways [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007032#pgen.1007032.ref061" target="_blank">61</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007032#pgen.1007032.ref082" target="_blank">82</a>]. Solid arrows indicate direct actions or direct physical interactions. Dashed arrows indicate interactions that may be indirect. Our results suggest that dopamine decreases activity of the NCA-1 and NCA-2 channels (shown here collectively as “NCA”) by binding to DOP-3 and activating G_o signaling. GRK-2 acts as a kinase for the D2-like dopamine receptor DOP-3 to inhibit DOP-3, and thereby inhibit G_o, activate G_q, and positively regulate NCA-1 and NCA-2 channel activity.</p

GRK-2 acts in head acetylcholine neurons.

<p>(A) <i>grk-2</i> acts in head acetylcholine neurons to control locomotion. The <i>grk-2</i> cDNA was expressed in <i>grk-2(gk268)</i> mutants under a pan-neuronal promoter (<i>Prab-3</i>, transgene <i>yakEx44</i>), acetylcholine neuron promoter (<i>Punc-17</i>, transgene <i>yakEx45</i>), ventral cord acetylcholine motor neuron promoter (<i>Pacr-2</i>, transgene <i>yakEx47</i>), head acetylcholine neuron promoter (<i>Punc-17H</i>, transgene <i>yakEx51</i>), glutamate receptor promoter (<i>Pglr-1</i>, transgene <i>yakEx52</i>), and ciliated sensory neuron promoter (<i>Pxbx-1</i>, transgene <i>yakEx71</i>). Expression driven by the pan-neuronal, acetylcholine neuron, and head acetylcholine neuron promoters rescued the slow locomotion of <i>grk-2</i> mutants. (***, P<0.001. Error bars = SEM; n = 10–25). (B,C) GRK-2 acts in head acetylcholine neurons to positively regulate G_q signaling. A <i>grk-2(gk268)</i> mutant suppresses the loopy posture and hyperactive locomotion of the activated G_q mutant <i>egl-30(tg26)</i> (Gq*). Expression of the <i>grk-2</i> cDNA under a head acetylcholine neuron promoter (<i>Punc-17H</i>, transgene <i>yakEx51</i>) reverses the <i>grk-2</i> suppression of the loopy posture (B) and hyperactive locomotion (C) of activated G_q. (***, P<0.001. Error bars = SEM; n = 10). (D) <i>grk-2</i> is expressed in head acetylcholine neurons. Representative images of a Z-stack projection of the area around the nerve ring in the head of an animal coexpressing tagRFP fused to the GRK-2 ORF driven by the <i>grk-2</i> promoter (<i>grk-2</i>::tagRFP, integration <i>yakIs19</i>) and GFP under a head acetylcholine neuron promoter (<i>Punc-17H</i>::eGFP, transgene <i>yakEx94</i>). Anterior to the left. Because <i>Punc-17H</i>::GFP is highly expressed and diffuse throughout the cell but <i>grk-2</i>::tagRFP is dimmer and localized only in the cytoplasm, their coexpression is hard to see in the merged image. For this reason, we have circled the cells where there is coexpression. Scale bar: 10 μm.</p

GRK-2 is a positive modulator of NCA-1 and NCA-2 channel activity.

<p>(A) A <i>grk-2</i> mutation enhances the weak forward fainting phenotype of an <i>nlf-1</i> mutant. Representative images of wild-type, <i>nlf-1(tm3631)</i>, and <i>grk-2(gk268); nlf-1(tm3631)</i> mutant animals. The asterisk shows the anterior part of the worm that becomes straight when an animal faints. (B) A <i>grk-2</i> mutation enhances the weak forward fainting phenotype of an <i>nlf-1</i> mutant. The <i>nlf-1(tm3631)</i> mutant is a weak fainter. The <i>grk-2(gk268)</i> mutation enhances the <i>nlf-1</i> mutant so that the double is a strong fainter. (***, P<0.001. Error bars = SEM; n = 10–20). The number shown is the number of body bends before the animal faints. If the animal made ten body bends without fainting, the assay was stopped and we recorded ten as the number (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007032#sec012" target="_blank">Methods</a>). (C) The <i>grk-2(gk268)</i> mutation enhances the <i>unc-73(ox317)</i> mutant so that the double mutant is a strong fainter. The <i>grk-2(gk268)</i> mutation has no effect on an <i>egl-8(sa47)</i> mutant. (***, P<0.001. Error bars = SEM; n = 15). (D) The <i>egl-10(md176)</i> mutation enhances the <i>nlf-1(tm3631)</i> mutant so that the double mutant is a strong fainter. (***, P<0.001. Error bars = SEM; n = 25). (E) Expression of activated G_o in head acetylcholine neurons inhibits locomotion. Animals expressing an activated G_o mutant (GOA-1[Q205L]) under a head acetylcholine neuron promoter (<i>Punc-17H</i>::GOA-1*, transgene <i>yakEx103</i>) move more slowly than wild-type animals. (***, P<0.001. Error bars = SEM; n = 17). (F) Expression of activated G_o in head acetylcholine neurons enhances the weak forward fainting phenotype of an <i>nlf-1</i> mutant. The <i>nlf-1(tm3631)</i> mutant is a weak fainter in forward movement. The <i>nlf-1(tm3631)</i> mutant expressing an activated G_o mutant (GOA-1[Q205L]) under a head acetylcholine neuron promoter (<i>Punc-17H</i>::GOA-1*, transgene <i>yakEx103</i>) is a stronger fainter than the <i>nlf-1(tm3631)</i> mutant. (***, P<0.001. Error bars = SEM; n = 54).</p

Mutations in <i>dop-3</i> and <i>cat-2</i> suppress <i>grk-2</i>.

<p>(A) Mutations in <i>dop-3</i> and <i>cat-2</i> suppress the slow locomotion of <i>grk-2</i> mutants. The <i>grk-2(gk268)</i> mutant has a slow locomotion phenotype. The <i>dop-3(vs106)</i> mutation fully suppresses and the <i>cat-2(e1112)</i> mutation partially suppresses the slow locomotion of the <i>grk-2(gk268)</i> mutant (***, P<0.001. Error bars = SEM; n = 32–72). (B,C) A <i>dop-3</i> mutation reverses the <i>grk-2</i> mutant suppression of activated G_q. The <i>grk-2(gk268)</i> mutation suppresses the loopy posture and hyperactive locomotion of the activated G_q mutant <i>egl-30(tg26)</i> (Gq*). The <i>dop-3(vs106)</i> mutation reverses the <i>grk-2</i> suppression of the loopy posture (B) and hyperactive locomotion (C) of Gq*. (***, P<0.001. Error bars = SEM; n = 15–20). (D,E) <i>dop-3</i> and <i>cat-2</i> mutations reverse the <i>grk-2</i> mutant suppression of the loopy posture of activated G_q. The <i>grk-2(gk268)</i> mutation suppresses the loopy posture of the activated G_q mutant <i>egl-30(tg26)</i> (Gq*). The <i>dop-3 (vs106)</i> and <i>cat-2(e1112)</i> mutations reverse the <i>grk-2</i> suppression of the loopy posture of Gq*. (***, P<0.001. ns, P>0.05. Error bars = SEM; n = 5). (F) <i>grk-2</i> mutants are hypersensitive to dopamine in a <i>dop-3</i>-dependent manner. Shown is the percentage of wild-type, <i>dop-3(vs106)</i>, <i>grk-2(gk268)</i>, or <i>grk-2(gk268); dop-3(vs106)</i> animals that moved ten body bends after a 20 min exposure to the indicated concentrations of dopamine. Every data point represents the mean +/- SEM of three trials (15–20 animals per experiment and strain).</p

A <i>grk-2</i> mutation partially suppresses activated Rho but does not suppress activated NCA-1.

<p>(A,B) A <i>grk-2</i> mutation partially suppresses activated Rho. Animals expressing activated RHO-1 [RHO-1[G14V]) under an acetylcholine promoter (Rho*, transgene <i>nzIs29</i>) have slow locomotion and loopy posture. The <i>grk-2(gk268)</i> mutation partially suppresses both the loopy posture (A) and slow locomotion (B) of the Rho* animals. (***, P<0.001. Error bars = SEM; n = 10). (C,D) A <i>grk-2</i> mutation does not suppress activated NCA-1. The activated NCA-1 mutant (Nca*, <i>nca-1(ox352)</i>) has slow locomotion and loopy posture. The <i>grk-2(gk268)</i> mutation does not suppress the loopy posture (C) or the slow locomotion (D) of Nca*. To measure the locomotion of the slow moving Nca* animals, we used a radial locomotion assay in which we placed animals in the center of a 10 cm plate and measured how far the animals had moved in one hour. (ns, P>0.05. Error bars = SEM; n = 10).</p

GRK-2 regulation of locomotion requires GPCR-phosphorylation and membrane association.

<p>(A) Domain structure of GRK-2. GRK-2 is a 707 amino acid protein with three well-characterized domains: the RGS homology (RH) domain, the kinase domain, and the pleckstrin homology (PH) domain. The protein structure was drawn using DOG 1.0. (B-D) Residues required for GPCR phosphorylation are required for GRK-2 function in locomotion. The D3K (transgene <i>yakEx77</i>), L4K (transgene <i>yakEx78</i>), V7A/L8A (transgene <i>yakEx79</i>), and D10A (transgene <i>yakEx80</i>) mutations are predicted to block GPCR phosphorylation. The R195A mutation (transgene <i>yakEx95</i>) disrupts predicted intramolecular stabilizing interactions that are required for effective phosphorylation. In each case, expression of the mutant <i>grk-2</i> cDNA under the control of its own promoter did not rescue the slow locomotion of the <i>grk-2(gk268)</i> mutant (ns, P>0.05, each strain compared to <i>grk-2</i>. Error bars = SEM; n = 10–20). (E) Residues in the RH domain predicted to disrupt G_q binding are not required for GRK-2 function in locomotion. The R106A (transgene <i>yakEx57</i>), Y109I (transgene <i>yakEx55</i>), and D110A (transgene <i>yakEx56</i>) mutations are predicted to disrupt G_q binding. In each case, expression of the mutant <i>grk-2</i> cDNA under the control of the <i>grk-2</i> promoter significantly rescued the slow locomotion of the <i>grk-2(gk268)</i> mutant (**, P<0.01; ***, P<0.001. Error bars = SEM; n = 10). (F) Residues in the PH domain predicted to disrupt GRK-2 phospholipid binding or binding to Gβγ are required for GRK-2 function in locomotion. Mutation K567E (transgene <i>yakEx87</i>) is predicted to disrupt GRK-2 phospholipid binding, and mutation R587Q (transgene <i>yakEx88</i>) is predicted to disrupt binding to Gβγ. In both cases, expression of the mutant <i>grk-2</i> cDNA under the control of the <i>grk-2</i> promoter did not rescue the slow locomotion of the <i>grk-2(gk268)</i> mutant. (**, P<0.01. ns, P>0.05. Error bars = SEM; n = 10). (G) Verification of the expression of the mutant <i>grk-2</i> cDNAs used for the experiments shown in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007032#pgen.1007032.g001" target="_blank">1D</a> and 2B–2F. Western blot analysis of whole worm extracts from <i>grk-2(gk268)</i> mutants expressing the indicated <i>grk-2</i> mutant cDNAs as extrachromosomal arrays.</p

GRK-2 is expressed and acts in command interneurons to regulate locomotion.

<p>(A) <i>grk-2</i> is expressed in command interneurons. Representative images of a Z-stack projection of the area around the nerve ring (head) of an animal coexpressing tagRFP fused to the GRK-2 cDNA driven by the <i>grk-2</i> promoter (<i>grk-2</i>::<i>RFP</i>, transgene <i>yakIs19</i>) and a <i>cho-1</i> fosmid YFP reporter (<i>cho-1</i>^{<i>fosmid</i>}::SL2::YFP::H2B, transgene <i>otIs534</i>). For the <i>cho-1</i> fosmid reporter, an SL2-spliced, nuclear-localized <i>YFP</i>::<i>H2B</i> sequence was engineered right after the stop codon of the gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007032#pgen.1007032.ref093" target="_blank">93</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007032#pgen.1007032.ref110" target="_blank">110</a>]. As indicated in the figure, <i>grk-2</i>::<i>RFP</i> is expressed in the AVA, AVB, AVD, and AVE command interneurons; SMD and RMD head motor neurons; and in the AIN, AIY, SIA, SIB, and SAA interneurons. Scale bar: 10 μm. (B-D) <i>grk-2</i> cDNA expression in (B) SMD/RMD (<i>Pcho-1</i>, 3.3 to 2.6 kb upstream of the ATG, transgene <i>yakEx135</i>), (C) SIA/SIB (<i>Pceh-24</i>, transgene <i>yakEx149</i>), or (D) AIY (<i>Pttx-3</i>, transgene <i>yakEx138</i>) neurons does not rescue the slow locomotion of <i>grk-2(gk268)</i> mutants. (Error bars = SEM; n = 15). (E) <i>grk-2</i> cDNA expression in command interneurons (<i>Psra-11 + Pnmr-1</i>, transgene <i>yakEx141</i>) is sufficient to rescue the slow locomotion of <i>grk-2(gk268)</i> mutants. (***, P<0.001. Error bars = SEM; n = 25). (F) <i>grk-2</i> cDNA expression in command interneurons (<i>Psra-11 + Pnmr-1</i>, transgene <i>yakEx141</i>) is sufficient to rescue the strong fainting phenotype of <i>grk-2(gk268); nlf-1(tm3631)</i> mutants. (***, P<0.001. Error bars = SEM; n = 40). (G) <i>dop-3</i> cDNA expression in command interneurons (<i>Psra-11 + Pnmr-1</i>, transgene <i>yakEx148</i>) is sufficient to reverse the <i>dop-3(vs106)</i> mutant suppression of the slow locomotion of <i>grk-2(gk268)</i> mutant animals. (**, P<0.01. Error bars = SEM; n = 23).</p

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

<div><p>The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in <i>Caenorhabditis elegans</i> for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.</p></div