measuring-caenorhabditis-elegans-sensitivity-to-acetylcholine

Measuring <em>Caenorhabditis elegans</em> Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

University of Delaware

Calcium homeostasis modulator protein-1 (CALHM1) and its  (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca (Ca) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca, deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH-terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca In 2 mM Ca, ceCLHM-1 NH-terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels.

The NH terminus regulates voltage-dependent gating of CALHM ion channels.

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.

CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes.

With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in  Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a noncomplementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude  lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.

A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in .

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole-hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR-dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder with no effective treatment. Diet, as a modifiable risk factor for AD, could potentially be targeted to slow disease onset and progression. However, complexity of the human diet and indirect effects of the microbiome make it challenging to identify protective nutrients. Multiple factors contribute to AD pathogenesis, including amyloid beta (Aβ) deposition, energy crisis, and oxidative stress. Here, we use Caenorhabditis elegans to define the impact of diet on Aβ proteotoxicity. We discover that dietary vitamin B alleviates mitochondrial fragmentation, bioenergetic defects, and oxidative stress, delaying Aβ-induced paralysis without affecting Aβ accumulation. Vitamin B has this protective effect by acting as a cofactor for methionine synthase, impacting the methionine/S-adenosylmethionine (SAMe) cycle. Vitamin B supplementation of B-deficient adult Aβ animals is beneficial, demonstrating potential for vitamin B as a therapy to target pathogenic features of AD triggered by proteotoxic stress.

Article	Year
The NH terminus regulates voltage-dependent gating of CALHM ion channels. American journal of physiology. Cell physiology\| PubMed ID: 28515089	2017
CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes. Neuron\| PubMed ID: 29681531	2018
A Rapid, SuperSelective Method for Detection of Single Nucleotide Variants in . Genetics\| PubMed ID: 32817008	2020
A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function. G3 (Bethesda, Md.)\| PubMed ID: 33713125	2021
Vitamin B impacts amyloid beta-induced proteotoxicity by regulating the methionine/S-adenosylmethionine cycle. Cell reports\| PubMed ID: 34592146	2021

Jessica E. Tanis

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