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. 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.

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K⁺ channel blockers, membrane depolarization elicited Ca²⁺ currents inhibited by nifedipine and exhibiting Ca²⁺-dependent inactivation. Our results give evidence that the Ca²⁺ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca²⁺ channel originally thought to be a member of this class on the basis of genomic data. Using Ca²⁺ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca²⁺ transients elicited by membrane depolarization are under the control of Ca²⁺ entry through L-type Ca²⁺ channels. In reduction of function egl-19 mutant muscle cells, Ca²⁺ currents displayed slower activation kinetics and provided a significantly smaller Ca²⁺ entry, whereas the threshold for Ca²⁺ transients was shifted toward positive membrane potentials.

Voltage-gated calcium channels, which play key roles in many physiological processes, are composed of a pore-forming α1 subunit associated with up to three auxiliary subunits. In vertebrates, the role of auxiliary subunits has mostly been studied in heterologous systems, mainly because of the severe phenotypes of knock-out animals. The genetic model Caenorhabditis elegans has all main types of voltage-gated calcium channels and strong loss-of-function mutations in all pore-forming and auxiliary subunits; it is therefore a useful model to investigate the roles of auxiliary subunits in their native context. By recording calcium currents from channel and auxiliary subunit mutants, we molecularly dissected the voltage-dependent calcium currents in striated muscle of C. elegans. We show that EGL-19 is the only α1 subunit that carries calcium currents in muscle cells. We then demonstrate that the α2/δ subunit UNC-36 modulates the voltage dependence, the activation kinetics, and the conductance of calcium currents, whereas another α2/δ subunit TAG-180 has no effect. Finally, we characterize mutants of the two β subunits, CCB-1 and CCB-2. CCB-1 is necessary for viability, and voltage-dependent calcium currents are abolished in the absence of CCB-1 whereas CCB-2 does not affect currents. Altogether these results show that EGL-19, UNC-36, and CCB-1 underlie voltage-dependent calcium currents in C. elegans striated muscle.

Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca²⁺ channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.