Stable activation of single Ca2+ release-activated Ca2+ channels in divalent cation-free solutions

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca(2+), Mg(2+), and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca(2+) stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca(2+), and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg(2+) present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836-839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion.     

Stimulation of 45Ca efflux from rat lacrimal gland slices by carbachol and epinephrine

The effects of carbachol (10^?5M) and epinephrine (10^?5M) on efflux of ⁴⁵Ca from rat exorbital lacrimal gland slices were examined. Both carbachol and epinephrine stimulated a transient release of ⁴⁵Ca from the tissue. The quantity of Ca released was estimated to be of the order of 0.5 μmol/g. Release of ⁴⁵Ca by one agonist prevented subsequent release of ⁴⁵Ca by a different agonist. These data support the hypothesis put forth previously that in the lacrimal gland muscarinic or α-adrenergic receptor activation causes a transient increase in membrane permeability to K by triggering the release of a sizable intracellular pool of Ca common to both receptors.     

Phosphoregulation of STIM1 Leads to Exclusion of the Endoplasmic Reticulum from the Mitotic Spindle

The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown [1]. Stromal interaction molecule 1 (STIM1) is an ER Ca(2+) sensor that activates store-operated Ca(2+) entry (SOCE) [2, 3] and also functions in ER morphogenesis through its interaction with the microtubule?+TIP protein end binding 1 (EB1) [4]. We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE [5]. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM1(10A)) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM1(10A), demonstrating that aberrant association of STIM1(10A) with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired?+TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca(2+) signaling during mitosis require multimodal STIM1 regulation by phosphorylation.     

Calcium signaling: deciphering the calcium-NFAT pathway

Rapid cellular calcium oscillations activate gene expression hours later. How this temporal response amplification is achieved has until now been largely a mystery. An elegant combination of experimental strategies and a model that encompasses non-linear inputs and outputs now sheds new light on this long-standing problem.     

Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.     

Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian beta-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression.     

A calmodulin/inositol 1,4,5-trisphosphate (IP3) receptor-binding region targets TRPC3 to the plasma membrane in a calmodulin/IP3 receptor-independent process

Conformational coupling with the inositol 1,4,5-trisphosphate (IP3) receptor has been suggested as a possible mechanism of activation of TRPC3 channels and a region in the C terminus of TRPC3 has been shown to interact with the IP3 receptor as well as calmodulin (calmodulin/IP3 receptor-binding (CIRB) region). Here we show that internal deletion of 20 amino acids corresponding to the highly conserved CIRB region results in the loss of diacylglycerol and agonist-mediated channel activation in HEK293 cells. By using confocal microscopy to examine the cellular localization of Topaz fluorescent protein fusion constructs, we demonstrate that this loss in activity is caused by faulty targeting of CIRB-deleted mutants to intracellular compartments. Wild type TRPC3 and mutants lacking a C-terminal predicted coiled coil region downstream of CIRB were targeted to the plasma membrane correctly in HEK293 cells and exhibited TRPC3-mediated calcium entry in response to agonist activation. Mutation of conserved YQ and MKR motifs to alanine within the CIRB region in TRPC3-Topaz, which would be expected to interfere with IP3 receptor and/or calmodulin binding, had no effect on channel function or targeting. Additionally, TRPC3 targets to the plasma membrane of DT40 cells lacking all three IP3 receptors and forms functional ion channels. These findings indicate that the previously identified CIRB region of TRPC3 is involved in its targeting to the plasma membrane by a mechanism that does not involve interaction with IP3 receptors.