Figure 5. maco-1 mutants are resistant to aldicarb but sensitive to levamisole. (A) maco-1(db9) animals are resistant to aldicarb but (B) sensitive to levamisole, consistent with a pre-synaptic role for MACO-1. Also plotted are control responses of N2 animals and mutants in the nicotinic acetylcholine receptor subunit unc-29(e193) and the synapse defective gene syd-2(ju37), which encodes alpha liprin. doi:10.1371/journal.pgen.1001341.g005 ventral nerve cord. The average punctum area in the ventral nerve cord for wild-type animals was 0.5560.035 mm2, with an average of 35.1660.99 (n = 13) puncta per 100 mm. Average puncta size in maco-1 mutants was 0.8560.12 mm2, and the average number of puncta was 28.1262.29 (n = 12) per 100 mm (Figure 6E–6H). Together these data suggest that loss of maco-1 alters the structure of the synaptic active zone, but the effects are subtle. We also examined the periactive zone, using the marker RPM- 1::GFP [31]. This region just surrounds active zones and has been proposed to regulate synapse growth [32]. We found a slight increase in the size and number of puncta in maco-1 mutants (0.7560.068 mm2 and 37.2463.376, n = 10), compared to wild- type animals (0.6360.036 mm2 and 33.0962.7, n = 16). However, these differences were not significant (p = 0.1 and p = 0.34, respectively), suggesting that the periactive zone was not disorganized in maco-1 mutants. maco-1 mutants have defects in Ca2+ influx Synapse development can be influenced by neural activity [33]. This prompted us to explore if the subtle synaptic defects in maco-1 reflected altered neuronal excitability. We focused our analyses on the O2-sensing neuron PQR, since a subset of the phenotypes of maco-1 mutants resembled those associated with loss of O2 sensing neurons [34]. To image Ca2+ transients we used the cameleon reporter YC3.60 [35] expressed from the gcy-32 promoter [15]. Baseline Ca2+ levels in 7% O2 were similar in wild type and maco-1 mutants, suggesting that PQR neurons were not chronically depolarized in maco-1 mutants. However the Ca2+ rise seen in wild type when O2 is raised to 21% was absent in most maco-1 mutant animals (Figure 7A). These data suggest that maco-1 is required for efficient activation of PQR neurons in response to a rise in O2. To explore this further we first asked if pre-synaptic input was required for PQR neurons to respond to O2 stimuli. Null mutations in unc-13 or unc-31 CAPS, which disrupt release of synaptic vesicles and dense core vesicles respectively, did not significantly alter Ca2+ transients in PQR to a 7 – 21% O2 upstep or a 21 to 7% downstep (data not shown). This suggests that Ca2+ fluxes in PQR reflect cell-intrinsic responses to the O2 stimuli, and that loss of maco-1 disrupts either primary sensory transduction of ambient O2 or amplification of the sensory potential. O2- stimulated Ca2+ influx in PQR requires the atypical soluble guanylate cyclases GCY-35 and GCY-36. These soluble guanylate cyclases are themselves O2 sensors and activate a cGMP-gated ion channel [36-37]. Consistent with this, in a separate study we have Macoilin: an ER Protein Regulating Neuron Activity shown that a rise in O2 stimulates a rise in cGMP in PQR neurons (A.C. and M.dB, in preparation). Mutations in maco-1 did not alter PQR cGMP responses to an O2 stimulus, suggesting that O2 sensing by GCY-35/36 was unaffected (A.C. and M.dB, in preparation). Previous work has shown that tax-4, which encodes a cGMP- gated cation channel alpha subunit is required for the O2-evoked Ca2+ transients in PQR [15]. To explore how depolarization evoked by the cGMP channel is amplified and leads to Ca2+ influx in the cell body we imaged PQR responses to O2 stimuli in animals defective in various ion channels. The C. elegans genome does not appear to encode voltage-gated sodium channels. Instead, electrical signals are thought to propagate via voltage-gated Ca2+ channels and cation leak channels [38,39]. C. elegans encodes 3 voltage gated Ca2+ channel a1 subunits: egl-19 (CaV1, L-type), unc- 2 (CaV2, N-, P/Q, R-type) and cca-1 (CaV3, T-type) [40]. It also encodes 2 homologs of the vertebrate cation leak channel NALCN that regulates neuronal excitability [38]. Animals mutant for the UNC-2 P/Q-like voltage-gated Ca2+ channel (VGCC) [41], the T- type channel CCA-1 [42], or double mutant for the NCA-1and NCA-2 NALCN-like leak channels [38] showed overtly wild-type Ca2+ transients in the cell body of PQR in response to a 7 to 21% O2 shift (Figure 7C and data not shown). This is consistent with previous results in other neurons that suggest these channels contribute to Ca2+ signals at synapses and axons, but are not essential for Ca2+ changes in the cell body [38,39]. In contrast, animals with partial loss-of-function mutations in the EGL-19 L- type VGCC showed frequent failure of cell body Ca2+ transients (Figure 7B). L-type VGCCs have previously been shown to contribute to dendritic Ca2+ currents both in C. elegans [39] and vertebrates [43]. Consistent with these imaging results, egl- 19(ad1006); npr-1(ad609) double mutants animals failed to aggregate. Together, our Ca2+ imaging results suggest that MACO-1 acts in the endoplasmic reticulum to promote assembly and/or traffic of either a cGMP-gated cation channel that contains the TAX-4 alpha subunit, or of an L-type Ca2+ channel containing the EGL- 19 a1 subunit, or of another as yet unknown regulator that modulates O2-evoked Ca2+ entry into PQR. To investigate the first possibility we made transgenic animals that expressed a functional GFP-tagged TAX-4 protein in the AQR, PQR and URX neurons, and compared the localization of this channel subunit in npr-1 and maco-1; npr-1 mutant animals. We saw enrichment of TAX-4-GFP in the sensory endings of the O2- sensing neurons, as expected for a sensory transduction channel (Figure S8). We also observed TAX-4-GFP in the cell body and on axons and dendrites. However we found no effect of loss-of- function mutations in maco-1 on this distribution pattern (Figure S8 and data not shown). These data suggest maco-1 is not required for TAX-4 to be exported from the ER, although they do not rule out a potential role in the function of a TAX-4-containing channel. Next, we transgenically expressed EGL-19 protein that is N- terminally tagged with GFP from its endogenous promoter, and examined its localization in wild type and maco-1 mutant animals. As expected, GFP-EGL-19 was expressed very broadly, and both in muscles and neurons (Figure S9) [44]. In neurons GFP-EGL-19 was enriched in sensory endings and in cell bodies. However we did not see any striking defects in the EGL-19 localization pattern in maco-1 mutants (Figure S9). This does not rule out that MACO- 1 modulates the function of an EGL-19-containing channel, but it does suggest that if it has a role it involves only a subset of EGL- 19-containing channels; alternatively maco-1 regulates function of other, as yet unknown, ion channels. PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1001341