Introduction
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
mRNA Methods and Results
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. After elution of the mRNA, the beads were regenerated and reused for further mRNA isolations.

RACE-PCR

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).
RACE PCR Fig1
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.
RACE PCR Fig2
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.
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.


References

  1. Grafton G, Thwaite L, Wilkinson B, Toellner KM and Gordon J. A non-voltage-gated calcium channel with L-type characteristics regulates antigen receptor-signalling in Group I Burkitt lymphoma cells. In Prep. 2001.
  2. Fomina et al 2000, Single channel properties and regulated expression of Ca2+ -release activated Ca2+ (CRAC) channels in human T cells. J. Biol. Chem 150: 1435-1444.
  3. Frohman, M.A. 1990. PCR protocols: A guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J. eds.)
  4. Yue et al, 2001. CaT1 manifests the pore properties of the calcium-release-activated calcium channel. Nature 410: 705-709.

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