Cloning of a T Cell Receptor-Activated Ca2+ Channel
Activation of T lymphocytes is critical for a successful immune response. Pathogens are broken down into fragments that are recognised by individual T cells through their unique T cell receptor (TCR). This recognition initiates T cell activation and expansion, ultimately leading to destruction of the pathogen. These events depend on signals through the TCR that increase cytosolic free Ca2+. The Ca2+ is first released from intracellular stores, followed by Ca2+ influx. For successful T cell activation, Ca2+ influx needs to be sustained for at least two hours. This is thought to occur through Ca2+-release activated Ca2+ (CRAC) channels.
The molecular identity of CRAC channels is still unknown, although TRP channels and CaT1 are candidates in some cells. In an attempt to clone the T lymphocyte CRAC channel we have focused on conserved regions of Ca2+ channels. Previous work has shown that classic L-type Ca2+ channel modulators affect the TCR-mediated Ca2+ response.
Given the estimate of only a few hundred CRAC channels per T lymphocyte we anticipated a low abundance of mRNA for the protein. In order to clone the channel a pure, full-length sample of mRNA was essential together with a quick and sensitive isolation procedure. Dynabeads® provide such a procedure and is also economical since the Dynabeads® can be regenerated and reused
Magnetic mRNA isolation
Jurkat T lymphocytes (20 x 106) were first washed with PBS, pelleted and lysed with 5 ml Lysis/Binding Buffer. The lysate was passed several times through a 21-gauge needle to shear the DNA released from the cells and generate a lysate with reduced viscosity. By using a Dynal MPC® magnet, mRNA was isolated according to the instructions following the Dynabeads® product. After elution of the mRNA, the beads were regenerated and reused for further mRNA isolations.
Rapid Amplification of cDNA Ends (RACE) is a method for amplification of a mRNA template between a defined internal site and an unknown sequence at either the 3’ or 5’ end. 3’ RACE takes advantage of the natural polyadenylated tail of mRNA as a generic priming site and uses an adaptor primer targeted to this region. From previous work using pharmacological tools to study the lymphocyte Ca2+ channel, PCR primers were designed to highly conserved regions of this class of Ca2+ channel. Sequencing of the PCR product revealed it to be an L-type Ca2+ channel, and gene specific primers were designed for RACE-PCR. 1 microgram of the isolated mRNA was used to create 3' RACE first strand cDNA, and two rounds of 3' RACE were performed. PCR products were analysed by gel-electrophoresis (Figure 1). Prominent bands of DNA were excised from the gel and purified. Cloning was performed using pGEM-T Easy vectos system ii (Promega) and plasmids sent for sequencing (MWG Biotech).
Figure 1. Shows the presence of an L-type Ca2+ channel transcript in B (lane 2) and T (lane 3) lymphocytes. Lane 1 is a DNA marker. A gene specific primer (GSP) is used together with the adaptor primer to produce a gene-specific product.
Figure 2. The 3’RACE product (>900bp) was cloned into a pGEM-T Easy vector. Lane 3 shows the pGEM-T Easy vector and cloned insert after digestion with EcoR1. Uncut plasmid is shown in lane 2 and a DNA marker in lane 1.
Analysis of the sequence revealed the Ca2+ channel to be an alternatively spliced form of the cardiac L-type channel (Cav1.2). Future work will involve studying the expression of the Ca2+ channel in a range of hematopoietic cells, the electrophysiological properties of the Ca2+ channel, and the signalling pathways that regulate its expression.
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Article written by Leanne Thwaite, Gillian Grafton and John Gordon. MRC Centre for Immune Regulation, University of Birmingham, The Medical School, Vincent Drive, Birmingham B15 2TT, UK. E-mail: LMT860@bham.ac.uk