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NEW Genetically Encoded Calcium Indicators

GCaMPs and GECOs fused to Orai1 (e.g., Orai1-GCaMP6f). To examine calcium signaling in the nanodomain adjacent to the Orai1 channel, we tagged Orai1 at the N- or C-terminus with several new-generation GECI indicators, tuned for detection in the high nanomolar to low micromolar range of affinity. By Total Internal Reflectance Fluorescence (TIRF) microscopy, we are now able to image Orai1 channel activity across the cell and in multiple cells simultaneously. pdf
Constructs: https://www.addgene.org/search/advanced/?q=Cahalan

Salsa6f: a fusion of GCaMP6f and TdTomato, localizes to cytosol. This simple combination imparts powerful capabilities, which include tracking cells when the GCaMP6f fluorescence is very dim, ratiometric imaging to eliminate motility artifacts, and convenient single-wavelength (920 nm) femtosecond excitation for 2P microscopy. pdf
Salsa6f Mouse: https://www.jax.org/mouse-search?searchTerm=Salsa6f


Channel phenotype of T lymphocytes.

Using the patch clamp technique, we described a unique subset of ion channels expressed in T lymphocytes. The five major classes are: voltage-gated K+ (KV) channels, calcium-activated K+ (KCa) channels, calcium-release-activated Ca2+ (CRAC) channels, Mg2+ -inhibited cation (MIC) channels, and swelling-activated Cl- channels. During activation and differentiation changes in expression patterns alter functional responses and affect the sensitivity of T cells to channel blockade. 






Biophysical fingerprint.

The biophysical phenotype for each channel subtype includes quantitative and mechanistic studies of gating, ion selectivity, pharmacology, and modulation by intracellular signaling pathways. This essential information paves the way toward functional and structural investigation of channels.






Toxins and other channel blockers.

We discovered (by milking a scorpion and applying a highly dilute sample to a patch clamped T cell) that scorpion venom contains components that block the lymphocyte K+ channel. Channel blockers, including native scorpion and sea anemone peptide toxins with nanomolar to picomolar affinities, rationally designed peptide analogs, and small organic compounds, provide unique tools for high-throughput screening, functional evaluation, and possible development as immunosuppressive agents.





Molecular cloning and expression.

Homotetramers of Kv1.3, a mammalian Shaker-related gene, form the predominant lymphocyte voltage-gated K+ channel. IKCa1 encodes the lymphocyte KCa channel in human T cells; in Jurkat T cells SKCa2 is the KCa channel. Both KCa channels are gated by Ca2+ binding to calmodulin pre-bound to the carboxy terminus of the channel protein. TRPM7 protein that forms the MIC channel. Most recently, we used RNA interference screening to identify STIM and Orai proteins that together form the molecular basis of store-operated CRAC channel function.





[Ca2+]i imaging.

Video imaging revealed [Ca2+]i oscillations mediated by the activity of Ca2+ and K+ channels in single T lymphocytes. 






Reporter gene systems.

The [Ca2+]i requirements for calcineurin/NF-AT and other transcriptional regulatory pathways were decoded using a [Ca2+]i clamp method in combination with a reporter gene assay (lacZ) in the same individual T cells.






Dynamics of Ca2+ signaling and motility.

Antigen specific T cells and antigen presenting cells interact in a dynamic and stereotyped manner during antigen recognition, with Ca2+ regulating motility and morphology changes that anchor the activated T cell at the site of antigen presentation. Using laser tweezers and beads as surrogate antigen-presenting cells, we discovered that T cells are preferentially responsive to antigen at the leading edge.





In vivo immunosuppression based upon K+ channel blockade.

Specific K+ channel blockers are non-toxic and show efficacy in preventing and treating the symptoms in adoptive transfer EAE, a model for multiple sclerosis.




In vivo imaging of T cell dynamics.

Using two-photon microscopy, we are examining the behavior of living T and B lymphocytes in intact lymph nodes. T and B cells are highly motile in their native habitat and this plays an important role in locating and responding to antigen. More recently, we have extended this approach to surgically exposed lymph nodes in anesthetized mice, and to image T cell interactions with dendritic cells and with B cells during the initiation of an immune response. These approaches are revealing important insights into the choreography of an immune response in vivo.


Maintained by
M Cahalan
Physiology and Biophysics, University of California,Irvine, CA, USA, 92697-4561
Phone: (949) 824-6754 Fax: (949) 824-3143 Email:mcahalan@uci.edu