Macoilin: an ER Protein Regulating Neuron Activity Figure 7. Loss of MACO-1 causes failure of Ca2+ transients in O2-sensing neurons. Average traces and scatter plots of Ca2+ transients in PQR neurons responding to a 21 – 7 – 21 – 7% O2 cycle as measured by cameleon YC3.60. (A) maco-1 (db9); npr-1 (ad609) worms (n = 13) usually fail to respond to O2 changes, as opposed to npr-1 (ad609) worms which respond consistently (n = 11; p,0.001). (B) egl-19(ad1006); npr-1 (ad609) worms (n = 11) are also unresponsive to O2, as compared with npr-1 (ad609) worms (n = 11; p,0.001). (C) nca-1 (gk9); nca-2 (gk5); npr-1(ad609) worms (n = 10) do not significantly (p.0.05) differ in their responses from npr-1(ad609) worms (n = 12). Gray error bars represent s.e.m. The data in Panel C were obtained on a different imaging set-up from those in panels A and B, and so cannot be directly compared. doi:10.1371/journal.pgen.1001341.g007 a C-terminal region that has coiled coil domains; the transmem- brane domains are the most highly conserved parts of the protein. This combination of structural motifs is reminiscent of that of RIC-3 and its orthologues, which are implicated in assembly and traffic of nicotinic acetylcholine receptors in C. elegans [45] and of nicotinic acetylcholine receptors and 5-HT3 receptors in verte- brates [46–47]. Like macoilin, RIC-3 is expressed broadly in the nervous system and is an ER membrane protein with a coiled-coil region towards the C-terminus. Macoilin mutants exhibit defects in cell intrinsic neuronal excitability, not only in PQR (this study) but also in other neurons (see associated paper by Miyara et al.). Previous work has reported that neural activity levels regulate the morphology of certain synaptic connections in C. elegans [33]; the synaptic morphology defects of maco-1 mutants could therefore reflect loss of neuronal excitability. A simple hypothesis is that macoilin acts in the endoplasmic reticulum of neurons to promote the folding, assembly, or traffic of ion channels or ion channel regulators that control excitability of neurons. What might these targets be? Since baseline Ca2+ levels are normal in maco-1 mutants we do not think loss of macoilin disrupts function of ion pumps that keep neurons hyperpolarized. Instead our data point towards compromised signal transduction or signal amplification down- stream of the GCY-35/GCY-36 O2-sensing soluble guanylate cyclases. As far as we can tell the cGMP-gated ion channel that transduces the O2-evoked cGMP rise in PQR, and which includes the TAX-4 a subunit, is appropriately expressed and localized in maco-1 mutants, although we cannot exclude the possibility that its function is somehow compromised. cGMP channels are expressed PLoS Genetics | www.plosgenetics.org 9 March 2011 | Volume 7 | Issue 3 | e1001341