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Updated: Aug 8, 2026
Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes
Published on: December 21, 2010
Intracellular calcium dependence of gene expression in single T lymphocytes
P A Negulescu1, N Shastri, M D Cahalan
1Department of Physiology and Biophysics, University of California, Irvine 92717.
Insights
Intracellular calcium (Ca2+) spikes in T cells regulate interleukin 2 gene expression. This study quantifies calcium
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- T-cell activation involves a rapid rise in intracellular calcium concentration ([Ca2+]i).
- This calcium increase is critical for initiating the immune response through interleukin 2 production.
- Understanding the precise relationship between calcium levels and gene expression is vital.
Purpose of the Study:
- To quantitatively link intracellular calcium ([Ca2+]i) dynamics to NF-AT-regulated gene expression in individual T cells.
- To determine the calcium dependency and sensitivity of gene expression.
- To investigate the modulatory effects of protein kinase C and A on calcium-mediated gene expression.
Main Methods:
- Utilized video-imaging techniques to track [Ca2+]i signals in single T cells.
- Measured beta-galactosidase reporter gene (lacZ) expression controlled by the NF-AT element.
- Employed thapsigargin, ionomycin, and controlled extracellular calcium/potassium levels to "clamp" [Ca2+]i.
Main Results:
- A positive correlation was observed between [Ca2+]i spikes and lacZ expression, though with significant cell-to-cell variability.
- Increasing [Ca2+]i from 70 nM to 200 nM–1.6 microM dose-dependently increased the fraction of cells expressing lacZ (Kd ≈ 1 microM).
- Protein kinase C activation enhanced calcium sensitivity (Kd = 210 nM), while protein kinase A inhibited it.
Conclusions:
- Established a quantitative, single-cell link between intracellular calcium levels and NF-AT-driven gene expression.
- Demonstrated that calcium concentration directly influences the probability of T-cell cytokine gene induction.
- Revealed differential modulation of calcium-dependent gene expression by key signaling pathways.
Abstract:
In T lymphocytes, intracellular Ca2+ concentration ([Ca2+]i) rises within seconds of T-cell antigen-receptor stimulation and initiates the synthesis and secretion of interleukin 2, a cytokine essential for T-cell proliferation and the immune response. Using video-imaging techniques, we tracked [Ca2+]i signals in individual T cells and measured subsequent expression of a beta-galactosidase reporter gene (lacZ) controlled by the NF-AT element of the interleukin 2 enhancer. [Ca2+]i spikes elicited by monoclonal antibody binding to the CD3 epsilon subunit of the T-cell receptor were positively correlated with gene expression, but varied widely between individual cells and were therefore difficult to relate quantitatively to lacZ expression. The [Ca2+]i dependence of NF-AT-regulated gene expression was determined by elevating [Ca2+]i with either thapsigargin or ionomycin and then "clamping" [Ca2+]i to various, stable levels by altering either extracellular [Ca2+] or extracellular [K+]. Raising [Ca2+]i from resting levels of 70 nM to between 200 nM and 1.6 microM increased the fraction of cells expressing lacZ, with Kd approximately 1 microM. Activation of protein kinase C enhanced the [Ca2+]i sensitivity of gene expression (Kd = 210 nM), whereas stimulation of protein kinase A inhibited [Ca2+]i-dependent gene expression. The experiments described here provide single-cell measurements linking a second messenger to gene expression in individual cells.
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