Cloning and in silico investigation of a putative voltage-gated calcium channel gene and protein in Astacus leptodactylus

Introduction

Voltage-gated calcium channels are transmembrane proteins making transitions between open and closed states in response to membrane potential changes. Activation of the channel generates a calcium influx which may act as a secondary messenger in addition to the electrical signaling. Depending on the cell type the calcium influx could be related to many cellular processes such as excitability, contraction, secretion, protein phosphorylation, and gene expression [1], [2], [3]. Presently, a fair amount of information is available for the voltage-gated calcium channels [4, 5]. In 1973, a calcium current was first recorded in the skeletal muscle fibers of an invertebrate preparation [6, 7]. In the following years, calcium currents were discovered in many excitable cells [8]. Primary sequence of the calcium channel protein was first discovered in C. elegans [9] and the first purification was performed in rabbit skeletal fast muscle of T-tubule samples [10]. Rabbit skeletal muscle calcium channel was the first from which a three-dimensional structure of calcium channel was originally calculated [11].

Crayfish has been an essential model for neurophysiology experiments. Many reports related to the functional and pharmacological properties of the ion channels have already been reported [1, 4, 5, 12, 13]. Unlike the mammals, muscle action potentials triggering the contraction are generated by calcium fluxes in the crayfish [14, 15]. Therefore, calcium channels are a direct component of the excitability both in neurons and muscle fibres in the crayfish. However, not much information is available for the genetic and molecular properties of the voltage-gated calcium channels in crayfish. Recently, we have cloned several ion channel genes and assembled a transcriptome draft of our model animal, Astacus leptodactylus [16], [17], [18], [19], [20], [21]. In the present work, we have focused onto cloning the calcium channel gene and functional characterization of the related protein in A. leptodactylus. Conventional cloning methods, three dimensional structural analyses and docking experiments have been conducted.

Materials and methods

The animals were collected from lakes of Central Turkey, kept in 18–20 °C fresh water aquarium. The muscle tissue samples were dissected out and kept on ice until the RNA isolation. The total RNA was extracted from the tissues by using Qiazol Lysis Reagent (Qiagen, Hilden, Germany). cDNA library was constructed by using SMARTer RACE 5′/3′ Kit (Clontech, Mountain View, CA, USA) and samples were aliquoted and stored at −20 °C.

A set of reference sequences from a related species and our transcriptome draft was used for cloning of the initial segment. An algorithm developed in our laboratory was used to design primers for the following Polymerase Chain Reaction (PCR) experiments (MATLAB, MathWorks, USA). PCR experiments were done with cDNA samples as template and by using Onetaq DNA polymerase (New England Biolabs, MA, USA) for short amplicons and SuperFi II (Thermo Fisher Scientific, Massachusetts, USA) was used for longer amplicons. For revealing 5′ and 3′ ends, SeqAMP DNA polymerase (Takara Bio, Kasatsu, Japan) was used in 5′/3′ the rapid amplification of cDNA ends (RACE) PCR experiments. For observing the separated DNA products on agarose gel, AlphaImager EC device (Protein Simple, CA, USA) and AlphaImager EC software (Protein Simple, CA, USA) were used. The target band was extracted from gel by using Monarch DNA Gel Extraction Kit (New England Biolabs, MA, USA). The products without any non-specific bands were purified by using Monarch PCR&DNA Cleanup Kit (New England Biolabs, MA, USA). After the purification, the DNA samples’ concentrations were measured by Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, MA, USA) and samples were stored at −20 °C.

The purified DNA samples, up to 450 bp, were Sanger sequenced. Longer amplicons were sequenced by using a Next Generation Sequencing (NGS) method. To prepare the samples for NGS, DNA samples were fragmented and tagged by using Nextera XT DNA Library Preparation Kit (Illumina, CA, USA). The short reads were obtained and sequenced in Illumina Miseq platform. Transcripts were de novo assembled in DNASTAR Software (Madison, USA). The obtained sequences were analyzed in NCBI BLAST platform and also examined by our algorithms in the MATLAB platform.

Once the whole transcript was revealed, it was translated to amino acid sequence and submitted to Swiss-Model platform (Biozentrum University of Basel, Switzerland) for three-dimensional structure calculations [22]. The relevancy of the resulted calculations was evaluated by using the parameters GMQE=0.5 and QMEANDisCo Global=0.65± 0.05. VMD software was used to view and analyze the 3D structure [23].

In order to analyze the interactions between ligand and the channel, docking simulations were performed using AutoDock 4.2.6. AutoGrid program was used for the preparation of the grid map using a grid box. Autodock relies on two methods to predict binding modes: first, a semi-empirical force field to estimate the free energy of binding of the complex, a typically estimate based on a particular bound conformation, and second, a search approach to investigate the conformational space that the ligand and target can occupy [24]. The grid size was set to 126×98×126 xyz points with grid spacing of 0.375 Å and grid center was designated at dimensions (x, y, and z): 156.162, 168.813 and 161.227. The grid space was chosen to include the binding site and the selectivity filter of the protein. For each complex, 1,000 docking experiments were performed using the Lamarckian genetic algorithm with the default parameters except “maximum number for evals” which is set to “long”. During the docking procedure, both the protein and ligands are considered as rigid. The results less than 2.0 Å in positional root-mean-square deviation (RMSD) were clustered together and represented by the result with the most favorable free energy of binding and the highest number of conformations.

Results

A set of voltage-gated calcium channel nucleotide sequences was assembled by collecting 15 mRNA sequences from closely related species in the NCBI database. Those were aligned to obtain a common sequence to be used as a reference. The reference sequence was scanned in our transcriptome draft for similarity. A contig, numbered as 74313, was found to be similar. A primer pair was designed and the reference sequence has been amplified in a muscle template for confirmation. In order to obtain the complete sequence of desired mRNA, several PCR and RACE PCR reactions were conducted with the primers given Table 1.

Five overlapping parts of the target sequence were independently revealed in five different de novo assembly experiments. As illustrated in Figure 1, the complete sequence of the target mRNA was compiled. A RACE experiment to both 3′ and 5′ ends were conducted to reveal the flanking parts of the sequence. Finally, a complete mRNA with a size of 7,791 bp was revealed. The sequence was analyzed and the coding sequence (CDS) was identified between the 520 and 6,348 positions.

Figure 1:

Map of the complete sequence (black) with ORF (orange). Alignment of the fragments (grey).

Finally, a primer pair was designed to amplify the complete sequence of the mRNA in muscle templates (Table 2). An amplicon with a size of 6,440 bp was obtained (Figure 2). The revealed mRNA sequence was quality controlled in the MATLAB platform and submitted to NCBI nucleotide database with accession code ON661560.1. Blast analyses indicated that the cloned sequence was highly (%93.45) similar to L type voltage-gated calcium channels.

Figure 2:

Image of gel electrophoresis: Lane 1: 1 kb Ladder. Lane 2: the product of PCR with cavFk1 and cavRk1.

Complete mRNA sequence was translated to amino acid sequence and submitted to Swiss-Model platform for obtaining three-dimensional structure of the channel. Human L-type voltage-gated calcium channel CaV1.3 (7uhg: Electron Microscopy) was chosen as a reference which generated the best fit and the most accurate structure calculation for cloned protein (Figure 3). Three-dimensional structure for voltage sensor, ion selectivity filter and dihydropyridines (DHP) binding sites were marked in Figure 4.

Figure 3:

Calculated 3D dimensional structure of the cloned channel in cartoon mode (A) and estimated transmembrane parts in molecule mode (B). Blue line indicates extracellular layer of the bilayer (MEMEMBED prediction, PSIPRED, UCL).

Figure 4:

Voltage sensor (purple), selectivity filter (green) and DHP receptor (red) regions are shown in (A), (B) and (C), respectively.

In order to analyze the interactions between ligand and the channel, docking experiments were performed with blockers diltiazem, nifedipine and verapamil. The results of these docking experiments are shown in Figure 5.

Figure 5:

Docking of diltiazem, nifedipine and verapamil molecules (yellow) to the cloned channel protein, (A), (B) and (C) respectively. Green and red segments are the selectivity filters and DHP binding sites, respectively.

Discussion

Several conserved and functional domains were identified when the Open Reading Frame (ORF) region of the cloned mRNA was translated into amino acid sequence (Figure 4). The segments between 1,520–1,523 and 1,655–1,664 residues were similar to GPHH and IQ domains [25]. Both domains are specific to eukaryotic voltage-gated calcium channels and responsible for calcium induced conductance regulation [26].

The pore-forming segments, Segment 5 and Segment 6 (S5–S6), and the connecting P loop play a significant role in determining selectivity and conductance of a calcium channel. A cluster of negatively charged glutamate residues in each domain is responsible for Ca²⁺ selectivity [27]. A conserved aspartate residue in domain II of the selectivity filter is required for calcium-dependent inactivation. This is an important self-regulatory mechanism [28]. Amino acid residues, such as lysine (K) and arginine (R) in the S4, act as the voltage sensors of the channel [29]. In reference to rabbit and human Cav protein sequences, the segments for ion selectivity filter and voltage sensor of the cloned protein were identified [4, 30, 31] and illustrated in Figure 6. The putative alpha peptide was assigned into four domains with many transmembrane segments as described in a typical voltage-gated calcium channel. The four consecutive fragments along the putative alpha peptide “KALRAFRVLR”, “RLLRVFKVTK”, “VKILRVLR” and “FFRLFRVMRLVKLLS” were identified as the components of the voltage sensor of the channel. Similarly, ion selectivity filter was assigned onto four regions as “QCITMEGWTDMMY”, “QILTGEDWNVVMY”, “TVSTFEGWPGLLY” and “RSATGEAWQEIML”.

Figure 6:

The amino acid sequence of cloned voltage-gated calcium channel. Voltage sensors are highlighted in purple, selectivity filter in green, IQ and GPHH domains in blue and DHP receptor sites colored in red.

The L-type calcium channel has three main categories of blockers. The first group consists of DHP with nitrendipine, nifedipine and nimodipine. The second and third groups are the benzodiazepines (BTZ) including diltiazem and phenylalkylamines (PAA) including verapamil [32, 33]. In reference to docking experiments, binding site for the dihydropyridine was identified at several different parts of the cloned sequence [13] (Figure 6). However, both verapamil and diltiazem docked onto the selectivity filter which is in an agreement with the former reports [13]. Structural calculations indicated a successful fit to the reference structure. Four domains and the individual transmembrane segments could distinctly be identified.

The sequence analyses, structural calculations and the docking experiments have revealed many functional segments in the cloned alpha peptide. Presence of a typical calcium ion selectivity filter and voltage sensor strongly indicates that the peptide may be a voltage-gated calcium ion channel. Further, presence of DHP receptors and docking of other typical calcium blockers such as verapamil and diltiazem to directly selectivity filter supports the hypothesis.

In the present work, a novel mRNA was de novo cloned in the crayfish. Considering the similarity assay in the NCBI platform, the three-dimensional structural calculations and the docking experiments, it was concluded that the mRNA codes an alpha peptide for a putative voltage-gated calcium channel protein in the crayfish.