T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

Abstract

The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling.

Fig 1. Ca_v1.1 channel protein expression in CD4 T cells after stimulation.

(A) Purified unstimulated (day 0) naïve CD4 T cells were stimulated with plate-bound antibodies to CD3 and CD28 under either Th1 or Th2 conditions (see experimental procedures for detail) for the indicated period of time. Immunoblot detection of the Ca_v1.1 channel is shown. Actin was used as loading control. (B) Protein extracts from brain, undifferentiated (u) or differentiated (d) C2C12 skeletal muscle cell line, and from CD4 T cells were analyzed for the expression of Ca_v1.1 channels. Calnexin and actin were used as internal controls. Results are representative of at least three experiments.

Fig 2. Schematic representation of mRNA splice sites and putative protein topology of the Ca_v1.1 channel T cell variants.

(A) Five new Exons were detected by 5’-RACE (A-E). The ATG of Ca_v1.1 open reading frame is located in Exon C. Exons C-E replace the original Exons 1 and 2 (NM_000069). Alternative splice out of Exon 29 leads to the deletion of the IVS3-S4 linker. Exons encoding transmembrane segments are written below their respective boxes. Introns are represented as lines in between exon boxes. Below is a diagram of putative channel topology of the Ca_v1.1 channel variant. (B) Diagram of putative channel topology of the Ca_v1.1 variant. Transmembrane segments are white boxes, except the S4 voltage sensor domain, which is shown in gray. Sequence comparison between muscle and T cell Ca_v1.1 at the IVS3-S4 linker region is shown.

Fig 3. Calcium influx measurements through over expressed T cell Ca_v1.1 channels.

(A) Schematic representation of wild type T cell Ca_v1.1 channel, a mutated construct containing insertion of Exon 29 and a pore-mutant. (B) HEK cells transfected with T cell Ca_v1.1 construct described in A, were loaded with Fura-2 AM and Dual wavelength imaging was performed using InCyt Imaging system. A zoomed-in microscope picture montage is shown. Calcium concentration is shown in the left panel followed by the corresponding GFP and 380nm (the later is aimed at showing the entire population of cells in the picture). (C) HEK cells transfected with either of the constructs described in A, were loaded with Fura-2 AM and Dual wavelength imaging was performed using InCyt Imaging system. Calcium concentration is shown in the top panel and below are corresponding GFP and 380nm (the later is aimed at showing the entire population of cells in the picture). (D) Graphic representation of similar independent experiments as described in B and C. Results are representative of 5 independent experiments. *pValue = 0.003. 40X objective was used for all the recordings. Scale bar (white line located at the bottom right montage in B and C, denotes 50μ for all the montages presented.

Fig 4. Effects of Nifedipine on calcium flow through T cell Ca_v1.1 channels.

HEK cells transfected with T cell Ca_v1.1 channel plasmid construct, were loaded with Fura-2 AM and Dual wavelength imaging was performed using InCyt Imaging system. Nifedipine (10μM) was then added (indicated in A). Montages (B) or graphic representation (C) of calcium concentration is shown, before and after addition of nifedipine as described in Fig 3. *pValue = 0.0002 between GFP positive cells treated and untreated with nifedipine. 40X objective was used for all the recordings. The scale bar (white line located at the bottom right montage in B) denotes 50μ for all the montages presented.

Fig 5. Characterization of calcium influx in Ca_v1.1 knockdown T cells.

(A) DO11.10 cells were transduced with lentivirus encoding Ca_v1.1 1271 siRNA (1271), Ca_v1.1 2184 siRNA (2184), Ca_v1.1 3549 siRNA (3549), or with control virus containing pLL3.7 vector (empty vector). Three days later, GFP⁺ cells were sorted then Ca_v1.1 α1 expression was assessed by immunoblot. Densitometry of Ca_v1.1 α1 protein expression normalized to actin after background subtraction. Densitometry indicates mean + SD analysis of two independent experiments. (B) The expression of other genes was also tested to ensure specificity of siRNA activity. (C) A representative immunoblot used for Ca_v1.1 1271 siRNA (1271) densitometry is shown. (D) Population based intracellular free calcium measurement in control (black line) and Ca_v1.1 knockdown cells, 2184 (blue line), 3549 (red line) using ratiometric Fura2/AM calcium probe. Cells were stimulated by using a TCR cross-linking system with goat anti-hamster (GAH) Ab in calcium containing media. Calculation of the absolute calcium concentration was performed by normalizing to ionomycin response of each cell type. Results are representative of three independent experiments shown in S2 Fig. * = Statistically significant results. pValues are 1.0x10^-5 and 2.3x10^-9 for empty vector vs 2184 or 3549, respectively.