Cloning andin silicoinvestigation of a putative voltage-gated calcium channel gene and protein inAstacus leptodactylus

Abstract

Objectives Voltage-gated calcium channels are essential elements in development of many cellular processes like electrical signaling, contraction secretion and gene expression. There has been a fair amount of information about the functional and structural properties of the calcium channels in mammalian species. Crayfish serves as a model animal for many types of experiments. However, there has been no information related to the molecular and genetic properties of the calcium channels in the crayfish. Methods Conventional cloning methods, three-dimensional structural calculations, docking experiments have been conducted. Results An mRNA 7,791 bp in size has been cloned. The coding region has been translated into an alpha peptide with 1,942 residues. The cloned protein sequence has similarity to other L-type voltage-gated calcium channel sequences from the neighboring species. Three-dimensional structure, in reference to human L-type voltage-gated calcium channel, has been calculated. Known calcium channel blockers, nifedipine, verapamil and diltiazem have been successfully docked on the calculated three-dimensional model. Conclusions Considering the similarity assay in the National Center for Biotechnology Information (NCBI) platform, the three-dimensional structural calculations and the docking experiments it was concluded that the cloned mRNA codes an alpha peptide for a putative voltage-gated calcium channel protein in the crayfish. In the present work by using the conventional molecular biology methods a complete mRNA coding a putative calcium channel has been de novo cloned. Three-dimensional structure of the related protein has been calculated and several pharmacological agents blocking the channel have been docked to the identified receptor sites.