MATLAB CODE

Matlab code used to readout the SPAD array

Data from: Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid

TRPV1 channels support the detection of noxious and nociceptive input. Currently available functional and structural data suggest that TRPV1 channels have two gates within their permeation pathway: one formed by a ′bundle-crossing′ at the intracellular entrance and a second constriction at the selectivity filter. To describe conformational changes associated with channel gating, the fluorescent non-canonical amino acid coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. Total internal reflection fluorescence microscopy was performed to image the conformational dynamics of the channels in live cells. Photon counts and optical fluctuations from coumarin encoded within TRPV1 tetramers correlates with channel activation by capsaicin, providing an optical marker of conformational dynamics at the selectivity filter. In agreement with the fluorescence data, molecular dynamics simulations display alternating solvent exposure of Y671 in the closed and open states. Overall, the data point to a dynamic selectivity filter that may serve as a gate for permeation

Figure 1-Steinberg et al

Figure 1 - Source data

Figure 3-Steinberg et al

Figure 3 - Source dat

Figure 2-Steinberg et al

Figue 2 - Source dat

Figure 4-Steinberg et al

Figure 4 - Source dat

Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry

Abstract T lymphocytes enlarge (blast) and proliferate in response to antigens in a multistep program that involves obligatory cytosolic calcium elevations. Store-operated calcium entry (SOCE) pathway is the primary source of Ca2+ in these cells. Here, we describe a novel modulator of blastogenesis, proliferation and SOCE: the TRPM7 channel kinase. TRPM7 kinase-dead (KD) K1646R knock-in mice exhibited splenomegaly and impaired blastogenic responses elicited by PMA/ionomycin or anti-CD3/CD28 antibodies. Splenic T-cell proliferation in vitro was weaker in the mutant compared to wildtype littermates. TRPM7 current magnitudes in WT and KD mouse T cells were, however, similar. We tested the dependence of T-cell proliferation on external Ca2+ and Mg2+ concentrations. At a fixed [Mg2+ o] of ~0.4 mM, Ca2+ o stimulated proliferation with a steep concentration dependence and vice versa, at a fixed [Ca2+ o] of ~0.4 mM, Mg2+ o positively regulated proliferation but with a shallower dependence. Proliferation was significantly lower in KD mouse than in wildtype at all Ca2+ and Mg2+ concentrations. Ca2+ elevations elicited by anti-CD3 antibody were diminished in KD mutant T cells and SOCE measured in activated KD splenocytes was reduced. These results demonstrate that a functional TRPM7 kinase supports robust SOCE, blastogenesis and proliferation, whereas its inactivation suppresses these cellular events