Selective photoinactivation of protein function through environment-sensitive switching of singlet oxygen generation by photosensitizer.

Chromophore-assisted light inactivation is a promising technique to inactivate selected proteins with high spatial and temporal resolution in living cells, but its use has been limited because of the lack of a methodology to prevent nonspecific photodamage in the cell owing to reactive oxygen species generated by the photosensitizer. Here we present a design strategy for photosensitizers with an environment-sensitive off/on switch for singlet oxygen ((1)O(2)) generation, which is switched on by binding to the target, to improve the specificity of protein photoinactivation. (1)O(2) generation in the unbound state is quenched by photoinduced electron transfer, whereas (1)O(2) generation can occur in the hydrophobic environment provided by the target protein, after specific binding. Inositol 1,4,5-trisphosphate receptor, which has been suggested to have a hydrophobic pocket around the ligand binding site, was specifically inactivated by an environment-sensitive photosensitizer-conjugated inositol 1,4,5-trisphosphate receptor ligand without (1)O(2) generation in the cytosol of the target cells, despite light illumination, demonstrating the potential of environment-sensitive photosensitizers to allow high-resolution control of generation of reactive oxygen species in the cell.

Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum.

Depletion of intracellular calcium (Ca(2+)) stores induces store-operated Ca(2+) (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca(2+) sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca(2+) store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile alpha motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile alpha motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels.

Functional and biochemical analysis of the type 1 inositol (1,4,5)-trisphosphate receptor calcium sensor.

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.

The high-affinity calcium[bond]calmodulin-binding site does not play a role in the modulation of type 1 inositol 1,4,5-trisphosphate receptor function by calcium and calmodulin.

Modulation of the inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)R) by cytosolic calcium (Ca(2+)) plays an essential role in Ca(2+) signalling, but structural determinants and mechanisms responsible for the InsP(3)R regulation by Ca(2+) are poorly understood. In the present study, we expressed rat InsP(3)R type 1 (InsP(3)R1) in Spodoptera frugiperda cells using a baculovirus-expression system and reconstituted the recombinant InsP(3)R1 into planar lipid bilayers for functional analysis. We observed only minor effects of 0.5 mM of calmodulin (CaM) antagonist W-7 on the Ca(2+) dependence of InsP(3)R1. Based on a previous analysis of mouse InsP(3)R1 [Yamada, Miyawaki, Saito, Nakajima, Yamamoto-Hino, Ryo, Furuichi and Mikoshiba (1995) Biochem J. 308, 83-88], we generated the Trp(1577)-->Ala (W1577A) mutant of rat InsP(3)R1 which lacks the high-affinity Ca(2+)[bond]CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Ca(2+) dependence similar to the wild-type InsP(3)R1 in planar lipid bilayers. Activation of B cell receptors resulted in identical Ca(2+) signals in intact DT40 cells lacking the endogenous InsP(3)R and transfected with the wild-type InsP(3)R1 or the W1577A mutant cDNA subcloned into a mammalian expression vector. In the planar lipid bilayer experiments, we showed that both wild-type InsP(3)R1 and W1577A mutant were equally sensitive to inhibition by exogenous CaM. From these results, we concluded that the interaction of CaM with the high-affinity Ca(2+)[bond]CaM-binding site in the coupling domain of the InsP(3)R1 does not play a direct role in biphasic modulation of InsP(3)R1 by cytosolic Ca(2+) or in InsP(3)R1 inhibition by CaM.

Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes.

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.