Background:The composition of voltage-gated calcium channels is important for function. Results: In C. elegans, calcium currents are altered in mutants of one main subunit (EGL-19) or the accessory subunits UNC-36 and CCB-1. Conclusion: Voltage-gated calcium channels of C. elegans striated muscles are composed of EGL-19, UNC-36, and CCB-1. Significance: C. elegans could be a new model to study the roles of accessory subunits.
Several human diseases, including hypokalemic periodic paralysis and Timothy syndrome, are caused by mutations in voltage-gated calcium channels. The effects of these mutations are not always well understood, partially because of difficulties in expressing these channels in heterologous systems. The use of Caenorhabditis elegans could be an alternative approach to determine the effects of mutations on voltage-gated calcium channel function because all the main types of voltage-gated calcium channels are found in C. elegans, a large panel of mutations already exists and efficient genetic tools are available to engineer customized mutations in any gene. In this study, we characterize the effects of two gain-of-function mutations in egl-19, which encodes the L-type calcium channel α 1 subunit. One of these mutations, ad695, leads to the replacement of a hydrophobic residue in the IIIS4 segment. The other mutation, n2368, changes a conserved glycine of IS6 segment; this mutation has been identified in patients with Timothy syndrome. We show that both egl-19 (gain-of-function) mutants have defects in locomotion and morphology that are linked to higher muscle tone. Using in situ electrophysiological approaches in striated muscle cells, we provide evidence that this high muscle tone is due to a shift of the voltage dependency towards negative potentials, associated with a decrease of the inactivation rate of the L-type Ca 2+ current. Moreover, we show that the maximal conductance of the Ca 2+ current is decreased in the strongest mutant egl-19(n2368), and that this decrease is correlated with a mislocalization of the channel.
Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of
Caenorhabditis elegans
levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification in vivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at −60 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering–defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for
C. elegans
mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.