Tamara M. Stawicki, Kim Schuske, Erik M. Jorgensen, Yishi Jin, H. Robert Horvitz, Jean-Louis Bessereau, Yingchuan Qi, Maelle Jospin, Thomas Boulin, Department of Biology [Utah], University of Utah, Physiologie intégrative, cellulaire et moléculaire (PICM), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Division of Biological Sciences [San Diego], University of California [San Diego] (UC San Diego), University of California-University of California, Biologie Cellulaire de la Synapse Normale et Pathologique, Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Biology, Howard Hughes Medical Institute (HHMI)-Massachusetts Institute of Technology (MIT), This work was supported initially by a Jane Coffin Childs postdoctoral fellowship to YJ. Further support was from National Institute of Health grants NS035546 to YJ, NS034307 to EJ, GM024663 to HRH, INSERM Junior Contract to TB, Association Française Contre les Myopathies and ANR-07-NEURO-032-01 grants to J-LB, and Human Frontier Science Program long term fellowship to MJ. YBQ is an associate of, and YJ, EJ, and HRH are investigators of the Howard Hughes Medical Institute., Massachusetts Institute of Technology. Department of Biology, McGovern Institute for Brain Research at MIT, Horvitz, H. Robert, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), School of Biological Sciences [Univ California San Diego] (UC San Diego), University of California (UC)-University of California (UC), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), and Autard, Delphine
The role of a heterotrimeric neuronal acetylcholine receptor in regulating a Caenorhabditis elegans locomotion circuit are revealed down to the level of identifying all five subunits involved., In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three α-subunits, UNC-38, UNC-63 and ACR-12, and two non–α-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf) mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement., Author Summary Neuronal acetylcholine receptors modulate a wide range of activities in vertebrates and invertebrates. The activity and sensitivity of these receptors to particular pharmacological agents is determined by the subunit composition of the receptors. A rich diversity of acetylcholine receptors are expressed in the nervous system of the nematode C. elegans, and like their mammalian counterparts, their subunit compositions are not understood. Here, we identify an activating mutation in a neuronal acetylcholine receptor subunit that causes convulsive body muscle contractions. By isolating suppressors of the convulsive phenotype, we are able to identify the genes required for the assembly and function of this acetylcholine receptor. Reconstitution studies in oocytes demonstrate that this acetylcholine receptor is composed of five different subunits. The contraction and relaxation of body muscles are coordinated by the neurotransmitters acetylcholine and GABA, respectively. In vivo recordings reveal that loss of this ion channel leads to a decrease in the activation state of the cholinergic motor neurons. By contrast, hyperactivation of the ion channel leads indirectly to the silencing of GABAergic motor neurons. The resulting imbalance in the locomotory circuit causes convulsions of the body muscle. This imbalance in excitation and inhibition of the locomotion circuit mimics the neurological features observed in epilepsy.