Cloning of astacus leptodactylus ryanodine receptor gene

Cytoplasmic Ca2+ concentration plays an essential role in many types of cellular function including electro-mechanical coupling in striated muscle fibers. Ryanodine receptor channels (RyR), mediating Ca2+ release from sarcoplasmic reticulum (SR), has a homotetrameric structure. It is the largest ion channel with a size of 2.2 MDa. Vertebrate and invertebrate RyR channels are structurally and functionally similar. Although Astacus leptodactylus, narrow-clawed crayfish, is a widely used model animal in neuroscience, information about genetic properties of the animal is rather limited. The present study is focused onto de novo cloning of the mRNA of the crayfish RyR channel which encodes the largest ion channel. A hybrid cloning method has been used, referring to the homology between RyR mRNA molecules and the computational assembly of the next generation sequencing data. A mRNA molecule of 15236 bp in size has originally been cloned. The putative RyR protein, with 5042 amino acids, has a significant similarity to the sequences reported in other species. Furthermore, the putative sequence possessed many of the conserved domains specific to the RyR channel. Thus, it has been proposed that a mRNA coding RyR channel has originally been cloned in the present study. The 3D protein structure can also be determined by the help of this revealed genetic information, or future mutation studies can be designed.