Activation of T lymphocytes is critical for a successful immune response. Pathogens are broken down into fragments that are recognized 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
Methods and results
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 manual. 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.
Conclusions and future work
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