De Novo Cloning and Functional Characterization of a Mechanosensitive Piezo-Like Ion Channel in the Crayfish.

BACKGROUND/AIMS: Mechanosensitive ion channels are the principal elements in the transduction of mechanical force to neural activity. To date, considerably fewer studies have been published about the molecular and structural properties of mechanosensitive channels. Piezo channels are the only ion channel family in eukaryotes which is selectively gated by the membrane tension. Piezo channels have been described in mammals and some other eukaryotes. However, not much information is available for the crustaceans. METHODS: Conventional cloning methods were used to clone the putative PIEZO channel mRNA in crayfish ganglia samples. HEK293T cells were transfected by the plasmid of the cloned gene for functional studies. The CDS of the mRNA translated into the protein sequence and three-dimensional structure of the channel has been calculated. RESULTS: An mRNA, 9378 bp, was firstly cloned from crayfish which codes a 2674 residues protein. The cloned sequence is similar to the piezo channel mRNAs reported in the other species. The sequence of the coded protein has been analyzed, and some functional domains have been identified. A three-dimensional structure of the coded protein was successfully calculated in reference to mouse piezo 1 channel protein data. A plasmid with a fluorescent protein indicator was synthesized for heterologous expression in HEK293T cells. The evoked calcium response to mechanical stimulation was not different from those observed in the control cells. However, the transfected cells were more sensitive to the gating modifier YODA-1. CONCLUSION: Based on the apparent similarity in sequence, structure and functional properties to other known piezo channels, it has been proposed that cloned mRNA may code a piezo-like ion channel in crayfish.

Knockout of zebrafish desmin genes does not cause skeletal muscle degeneration but alters calcium flux.

Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon.

Cloning of a putative sodium/calcium exchanger gene in the crayfish.

Crayfish is a common model animal for different experimental purposes. However, the lack of information about the genetic properties of the animal limits its use in comparison to other model animals. In the present study, a putative crayfish sodium/calcium exchanger gene has firstly been cloned in ganglia cDNA samples by conducting a series of PCR experiments, where a set of degenerate and specific primers and RACE method were used. The complete sequence is 2955 bp, and the ORF is 2718 bp in length. Molecular properties of the calculated peptide were similar to the sodium/calcium exchangers reported in the other species. Analysis of the qPCR data indicated that the putative gene has the highest expression level in the ganglia. However, an apparently elevated level of expression is observed in highly active tissues like heart, muscle and intestine, while the least expression level was observed in the stomach samples. It was proposed that the cloned gene may code the sodium/calcium exchanger protein in the crayfish.