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IP3 receptors: some lessons from DT40 cells
Colin W Taylor1, Taufiq Rahman, Stephen C Tovey
1Department of Pharmacology, University of Cambridge, Cambridge, UK. cwt1000@cam.ac.uk
Immunological Reviews
|September 17, 2009
Summary
DT40-knockout cells provide a unique null background to study inositol-1,4,5-trisphosphate receptors (IP3Rs). These cells reveal how IP3Rs cluster and re-tune Ca2+ signaling, and their role in B-cell antigen receptor-evoked Ca2+ entry.
Area of Science:
- Cellular Biology
- Molecular Physiology
- Immunology
Background:
- Inositol-1,4,5-trisphosphate receptors (IP3Rs) are crucial intracellular Ca2+ channels involved in signal transduction.
- Their ubiquitous expression complicates the study of specific IP3R functions.
- DT40 cells, a pre-B-lymphocyte line, offer a powerful system for genetic manipulation due to high homologous recombination rates.
Purpose of the Study:
- To leverage DT40-knockout cells to investigate the precise mechanisms of IP3R activation and function.
- To analyze the role of IP3Rs in initiating and propagating Ca2+ signals.
- To determine the contribution of IP3Rs to Ca2+ entry in B lymphocytes.
Main Methods:
- Utilized DT40-knockout cell lines with disrupted IP3R genes to create a null background.
- Employed patch-clamp recording at the nuclear envelope to study IP3R activation.
- Measured Ca2+ release from intracellular stores using a low-affinity Ca2+ indicator.
- Quantified IP3R presence in the plasma membrane.
Main Results:
- Demonstrated that inositol trisphosphate (IP3) induces IP3R clustering, enhancing their responsiveness to IP3 and Ca2+ for regenerative signaling.
- Showed that IP3Rs are essential for initiating and propagating Ca2+ signals.
- Quantified IP3Rs in the plasma membrane of DT40 cells, revealing their significant role in mediating Ca2+ entry stimulated by the B-cell antigen receptor.
Conclusions:
- DT40-knockout cells are invaluable for dissecting the complex behavior of IP3Rs.
- IP3R clustering and altered sensitivity to IP3 and Ca2+ are key mechanisms for signal propagation.
- IP3Rs play a substantial role in B-cell calcium signaling and Ca2+ entry.
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