Macoilin, a Conserved Nervous System–Specific ER Membrane Protein That Regulates Neuronal Excitability Fausto Arellano-Carbajal1.¤a, Luis Brisen˜ o-Roa1.¤b*, Africa Couto1, Benny H. H. Cheung 1 , Michel Labouesse2, Mario de Bono 1 * 1 Medical Research Council–Laboratory of Molecular Biology, Cambridge, United Kingdom, 2 Institute of Genetics and Molecular and Cellular Biology, Illkirch, France Abstract Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O2 responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca2+ transients, at least in some neurons: in maco-1 mutants the O2-sensing neuron PQR is unable to generate a Ca2+ response to a rise in O2. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O2, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca2+ channels, also fails to disrupt Ca2+ responses in the PQR cell body to O2 stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca2+ channel a1 subunit, recapitulate the Ca2+ response defect we see in maco- 1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators. Citation: Arellano-Carbajal F, Brisen˜ o-Roa L, Couto A, Cheung BHH, Labouesse M, et al. (2011) Macoilin, a Conserved Nervous System–Specific ER Membrane Protein That Regulates Neuronal Excitability. PLoS Genet 7(3): e1001341. doi:10.1371/journal.pgen.1001341 Editor: Andrew D. Chisholm, University of California San Diego, United States of America Received June 3, 2010; Accepted February 16, 2011; Published March 17, 2011 Copyright: 2011 Arellano-Carbajal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the UK Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: debono@mrc-lmb.cam.ac.uk (MdB); briseno@biologie.ens.fr (LB-R) . These authors contributed equally to this work. ¤a Current address: Facultad de Ciencias Naturales, Universidad Auto´ noma de Quere´taro, Juriquilla, Quere´taro, Me´xico ¤b Current address: INSERM U1024, E´cole Normale Supe´rieure, Paris, France Introduction One of the most striking innovations in Metazoa is a nervous system with specialized nerve cells, pre- and post-synaptic structures, and associated signaling molecules. Neuronal signaling depends on complexes of multipass transmembrane proteins such as ion channels and G-protein-coupled receptors. Over the past few years several studies have identified specialized molecular machines in the endoplasmic reticulum by which particular complexes are assembled with appropriate stoichiometries and trafficked to their destination [1]. The emerging picture is that neurons have a highly specialized endoplasmic reticulum (ER), allowing channels to undergo quality control prior to export. However the identity of such maturation complexes remains unclear except for a handful of channels. The extensive intracellular membrane system that makes up the ER varies, depending on cell type, but two domains, the rough and smooth ER (RER and SER), can usually be distinguished. The RER is studded with ribosomes and mediates translocation of secretory proteins across the membrane and insertion of membrane proteins. The SER is implicated in lipid synthesis and regulation of Ca2+ storage and signalling. Whereas most ER proteins are found in both RER and SER, a subset of proteins involved in translocation of newly synthesized proteins across the ER membrane is highly concentrated in the RER [2,3]. In C. elegans neurons, RER proteins are concentrated in the cell body and excluded from dendrites and axons, whereas general ER proteins are found in both cell body and neurites [4]. Electron microscopy confirms that ribosomes and RER are abundant in the cell body of C. elegans neurons but rare in neurites, whereas smooth ER-like structures can be seen in axons and dendrites as well as the cell body. Over the last decade, genome sequencing projects have provided gene catalogs for animals representing a spectrum of metazoan phyla, including Placozoa [5], Cnidaria [6], Echino- dermata [7], Annelida (http://genome.jgi-psf.org/Capca1/ Capca1.home.html) and Chordata [8]. Genome-wide compari- sons have identified human genes that are conserved across these PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1001341