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The endoplasmic reticulum Ca(2+)-sensing STIM proteins mediate Ca(2+) entry signals by coupling to activate plasma membrane Orai channels. We reveal that STIM-Orai coupling is rapidly blocked by hypoxia and the ensuing decrease in cytosolic pH. In smooth muscle cells or HEK293 cells coexpressing STIM1 and Orai1, acute hypoxic conditions rapidly blocked store-operated Ca(2+) entry and the Orai1-mediated Ca(2+) release-activated Ca(2+) current (I(CRAC)). Hypoxia-induced blockade of Ca(2+) entry and I(CRAC) was reversed by NH(4)(+)-induced cytosolic alkalinization. Hypoxia and acidification both blocked I(CRAC) induced by the short STIM1 Orai-activating region. Although hypoxia induced STIM1 translocation into junctions, it did not dissociate the STIM1-Orai1 complex. However, both hypoxia and cytosolic acidosis rapidly decreased Förster resonance energy transfer (FRET) between STIM1-YFP and Orai1-CFP. Thus, although hypoxia promotes STIM1 junctional accumulation, the ensuing acidification functionally uncouples the STIM1-Orai1 complex providing an important mechanism protecting cells from Ca(2+) overload under hypoxic stress conditions.

Fetal cardiac gene reactivation is a hallmark of pathological cardiac hypertrophy (PCH) driven by cardiac transcription factors (TFs) such as nuclear factor of activated T-cells (NFATs). Nuclear import of dephosphorylated NFATs catalyzed by calcineurin (CaN) is a well-established hypertrophic mechanism. Here we report that NFATc4 expression is also up-regulated by newly expressed protein kinase D3 (PKD3) to induce PCH. In both in vitro and in vivo cardiac hypertrophic models, the normally undetectable PKD3 was profoundly up-regulated by isoproterenol followed by overt expression of cardiac TFs including NFATc4, NK family of transcription factor 2.5 (Nkx2.5), GATA4 and myocyte enhancer factor 2 (MEF2). Using gene silencing approaches, we demonstrate PKD3 is required for increasing the expression of NFATc4, Nkx2.5, and GATA4 while PKD1 is required for the increase in MEF2D expression. Upstream induction of PKD3 is driven by nuclear entry of CaN-activated NFATc1 and c3 but not c4. Therefore, PKD3 is a pivotal mediator of the CaN-NFATc1/c3-PKD3-NFATc4 hypertrophic signaling cascade and a potential new drug target for the PCH.

Female Eker rats harboring an insertional deletion in one copy of the tuberous sclerosis complex 2 (Tsc2) gene develop uterine leiomyoma, but the underlying mechanism of human uterine leiomyoma is not completely understood. To examine whether down-regulation of tuberin, a TSC2 gene product, is present in human uterine leiomyoma, we analyzed leiomyoma and matched myometrium tissues from 22 Chinese patients with Western blotting and real-time polymerase chain reaction analyses, and found that the expression of tuberin was significantly increased in leiomyoma tissues compared with matched myometrium tissues with inhibition of both the mammalian target of rapacmycin pathway and mitogen-activated protein kinase pathways.

Loss-of-function mutations in the inward rectifier potassium channel, Kir2.1, cause Andersen-Tawil syndrome (ATS-1), an inherited disorder of periodic paralysis and ventricular arrhythmias. Here, we explore the mechanism by which a specific ATS-1 mutation (V302M) alters channel function. Val-302 is located in the G-loop, a structure that is believed to form a flexible barrier for potassium permeation at the apex of the cytoplasmic pore. Consistent with a role in stabilizing the G-loop in an open conformation, we found the V302M mutation specifically renders the channel unable to conduct potassium without altering subunit assembly or attenuating cell surface expression. As predicted by the position of the Val-302 side chain in the crystal structure, amino acid substitution analysis revealed that channel activity and phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity are profoundly sensitive to alterations in the size, shape, and hydrophobicity of side chains at the Val-302 position. The observations establish that the Val-302 side chain is a critical determinant of potassium conduction through the G-loop. Based on our functional studies and the cytoplasmic domain crystal structure, we suggest that Val-302 may influence PIP2 gating indirectly by translating PIP2 binding to conformational changes in the G-loop pore.

The two membrane proteins, STIM1 and Orai1, have each been shown to be essential for the activation of store-operated channels (SOC). Yet, how these proteins functionally interact is not known. Here, we reveal that STIM1 and Orai1 expressed together reconstitute functional SOCs. Expressed alone, Orai1 strongly reduces store-operated Ca(2+) entry (SOCE) in human embryonic kidney 293 cells and the Ca(2+) release-activated Ca(2+) current (I(CRAC)) in rat basophilic leukemia cells. However, expressed along with the store-sensing STIM1 protein, Orai1 causes a massive increase in SOCE, enhancing the rate of Ca(2+)entry by up to 103-fold. This entry is entirely store-dependent since the same coexpression causes no measurable store-independent Ca(2+) entry. The entry is completely blocked by the SOC blocker, 2-aminoethoxydiphenylborate. Orai1 and STIM1 coexpression also caused a large gain in CRAC channel function in rat basophilic leukemia cells. The close STIM1 homologue, STIM2, inhibited SOCE when expressed alone but coexpressed with Orai1 caused substantial constitutive (store-independent) Ca(2+) entry. STIM proteins are known to mediate Ca(2+) store-sensing and endoplasmic reticulum-plasma membrane coupling with no intrinsic channel properties. Our results revealing a powerful gain in SOC function dependent on the presence of both Orai1 and STIM1 strongly suggest that Orai1 contributes the PM channel component responsible for Ca(2+) entry. The suppression of SOC function by Orai1 overexpression likely reflects a required stoichiometry between STIM1 and Orai1.

The guard cell K + channel KAT1, cloned from Arabidopsis thaliana , is activated by hyperpolarization and regulated by a variety of physiological factors. Low internal pH accelerated the activation kinetics of the KAT1 channel expressed in Xenopus oocytes with a pK of approximately 6, similar to guard cells in vivo. Mutations of histidine-118 located in the putative cytoplasmic linker between the S2 and S3 segments profoundly affected the gating behavior and pH dependence. At pH 7.2, substitution with a negatively charged amino acid (glutamate, aspartate) specifically slowed the activation time course, whereas that with a positively charged amino acid (lysine, arginine) accelerated. These mutations did not alter the channel's deactivation time course or the gating behavior after the first opening. Introducing an uncharged amino acid (alanine, asparagine) at position 118 did not have any obvious effect on the activation kinetics at pH 7.2. The charged substitutions markedly decreased the sensitivity of the KAT1 channel to internal pH in the physiological range. We propose a linear kinetic scheme to account for the KAT1 activation time course at the voltages where the opening transitions dominate. Changes in one forward rate constant in the model adequately account for the effects of the mutations at position 118 in the S2–S3 linker segment. These results provide a molecular and biophysical basis for the diversity in the activation kinetics of inward rectifiers among different plant species.

We report results of systematic numerical simulations of collisions between a soliton and an attractive defect described by the perturbing term in the nonlinear Schrodinger equation proportional to the δ-function. This model has applications in nonlinear optics and other physical systems. In the parametric range where the defect's strength is of the same order of magnitude as the soliton's amplitude, i.e., the interaction is essentially nonlinear, only two outcomes of the collision are possible: transmission and capture. We have found a border between the corresponding parametric regions. The border is represented in a universal form which does not depend on any free parameter. In this essentially nonlinear regime of the interaction, the soliton keeps its integrity, being transmitted or captured as a whole, practically without emission of radiation. At larger values of the defect's strength, the interaction becomes nearly linear. In this case, the soliton is split into reflected, transmitted, and trapped wave packets. Using the corresponding linear Schrodinger equation, we calculate analytically the shares of the reflected and transmitted energy in the limiting case when the defect's strength and the soliton's velocity are essentially larger than its amplitude.

Objective To determine different effects of epidermal growth factor (EGF) on cultured smooth muscle cells (SMCs) derived from human myometrium and leiomyoma. Design EGF effects on DNA synthesis and intracellular signal transduction were studied in cultured SMCs from leiomyoma and its matched myometrium. Setting Research laboratories. Patient(s) Patients 35–50 years old with uterine leiomyomas. Intervention(s) Hysterectomy. Main Outcome Measure(s) Signal transduction from EGF receptor. Result(s) As analyzed by laser scanning cytometry (LSC), EGF treatment stimulated DNA synthesis and induced polyploidization of leiomyomal, but not myometrial, SMCs. EGF stimulation was inhibited by AG1478, an EGF receptor (EGFR) inhibitor and PD98059, a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor. Both leiomyomal and myometrial SMCs had similar expression levels of EGFR, but EGF treatment induced transient phosphorylation activation of EGFR and Akt in leiomyomal SMCs. Consequently, EGF triggered transient phosphorylation activation of p44/42 MAPK in leiomyomal SMCs, followed by down-regulation of p27. In myometrial SMCs, however, EGF induced sustained activation of EGFR, Akt, and p44/42 MAPK with up-regulation of p27. Conclusion(s) EGF stimulates DNA synthesis and polyploidization in leiomyomal SMCs through transient activation of the EGFR-MAPK pathway. Given that polyploidization plays a role in tumorigenesis, our results shed new light on the pathogenesis of human uterine leiomyoma.