<i>De novo</i> cloning and functional characterization of potassium channel genes and proteins in the crayfish <i>Astacus leptodactylus</i> (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae)

Ergin, Bora; Saglam, Berk; Taşkıran, Ekim Z.; Baştuğ, Turgut; Puralı, Nuhan

doi:10.1093/jcbiol/ruac018

Cited by 4 publications

(3 citation statements)

References 39 publications

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“…The contig was very long, 195231 bp, as compared to the size of the cloned mRNA. By using an algorithm developed in our laboratory [22], possible exons along the contig were estimated (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

“…The ligated cDNA was amplified by using SensiPhi DNA Polymerase in an isothermal reaction for two hours (Repli-g WTA kit, Qiagen, Hilden, Germany). The final concentration of the cDNA library was 0.3-0.5 µg/µl [22].…”

Section: Methodsmentioning

confidence: 99%

“…The original plasmid, a gift from Dr. Alfred L. George Jr. from University of Feinberg School of Medicine Chicago USA [21], was modified to house cloned mRNA instead of SCN1A. The huge size of the cloned mRNA, 9378 bp, was the major challenge hindering the successful ligation of the insert to the plasmid backbone by using conventional ligation methods [22]. A set of primers (Table 1) was designed for the Gibson cloning method [23].…”

Section: Methodsmentioning

confidence: 99%

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

2023

Cell Physiol Biochem

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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. Materials: 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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

2023

Cell Physiol Biochem

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Structural, Functional and Molecular Dynamics Examination of a de novo cloned Otopetrin-like Proton Channel in crayfish

Sağsöz,

Sağlam,

Arslan

et al. 2024

Cell Biochem Biophys

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Cloning and functional characterization of a TMC-like channel gene and protein in the crayfish Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae)

Arslan,

Saglam,

Beyatli

et al. 2023

Journal of Crustacean Biology

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Ion channels gated selectively by mechanical stimulus are the key elements of mechanosensation. Several genes have been associated with putative mechanosensitive ion channels or mechanosensitive channel complexes. Transmembrane channel (TMC)-like protein is one of those candidate proteins that have been explored in mammals and several invertebrates. The presence and possible function of TMC related genes has not been investigated yet in crustaceans. In the present work an mRNA coding TMC-like protein was firstly cloned in Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae) and expressed in HEK293T cells. Three-dimensional structural calculations of the protein predicted a channel. Functional studies, however, indicated that the mechanosensitivity of the transfected HEK293T cells is similar to that in the control cells. It was concluded that a TMC-like protein is present in the crayfish but future studies are necessary to define its function.

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De novo cloning and functional characterization of potassium channel genes and proteins in the crayfish Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae)

Cited by 4 publications

References 39 publications

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

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

Structural, Functional and Molecular Dynamics Examination of a de novo cloned Otopetrin-like Proton Channel in crayfish

Cloning and functional characterization of a TMC-like channel gene and protein in the crayfish Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae)

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