A Novel Modulator of STIM2-Dependent Store-Operated Ca2+ Channel Activity.

Store-operated Ca2+ entry is one of the main pathways of calcium influx into non-excitable cells, which entails the initiation of many intracellular processes. The endoplasmic reticulum Ca2+ sensors STIM1 and STIM2 are the key components of store-operated Ca2+ entry in mammalian cells. Under physiological conditions, STIM proteins are responsible for store-operated Ca2+ entry activation. The STIM1 and STIM2 proteins differ in their potency for activating different store-operated channels. At the moment, there are no selective modulators of the STIM protein activity. We screened a library of small molecules and found the 4-MPTC compound, which selectively inhibited STIM2-dependent store-operated Ca2+ entry (IC50 = 1 µM) and had almost no effect on the STIM1-dependent activation of store-operated channels.

Store-Operated Calcium Entry in Mouse Cardiomyocytes.

The fluorescent dye fura-2 AM was employed to record activation of Ca2+ entry in response to a decrease in Ca2+ concentration in the endoplasmic reticulum. Using whole-cell voltage clamp technique, we revealed Ca2+ currents with an amplitude of 0.46±0.13 pA/pF that passed through selective channels with current-voltage characteristics similar to those of classical store-operated CRAC channels. These currents were sensitive to 2-APB (50 µM), an inhibitor of store-operated channels. The data suggest that store-operated calcium entry is a characteristic feature of mature ventricular cardiomyocytes. Pathological alterations in store-operated Ca2+ entry can be implicated in the development of heart diseases.

Homer 1a Induces Calcium Channel Activation, but Does Not Change Their Properties in A431 Cells.

Store-operated channels activated in response to intracellular calcium store depletion represent the main pathway of calcium entry from the extracellular space in nonelectroexcitable cells. Adapter proteins organize the components of this system into integral complex. We studied the influence of adapter proteins of the Homer family on endogenous store-operated calcium Imin channels in A431 cells. Monomeric Homer 1a proteins increase activity of Imin channels, but did not modulate their electrophysiological properties. Recombinant Homer 1c protein did not block the induced calcium currents.

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown.

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

STIM1 Protein Activates Store-Operated Calcium Channels in Cellular Model of Huntington's Disease.

We have shown that the expression of full-length mutated huntingtin in human neuroblastoma cells (SK-N-SH) leads to an abnormal increase in calcium entry through store-operated channels. In this paper, the expression of the N-terminal fragment of mutated huntingtin (Htt138Q-1exon) is shown to be enough to provide an actual model for Huntington's disease. We have shown that Htt138Q-1exon expression causes increased store-operated calcium entry, which is mediated by at least two types of channels in SK-N-SH cells with different reversal potentials. Calcium sensor, STIM1, is required for activation of store-operated calcium entry in these cells. The results provide grounds for considering the proteins responsible for the activation and maintenance of the store-operated calcium entry as promising targets for developing novel therapeutics for neurodegenerative diseases.

Regulation of store-operated channels by scaffold proteins in a431 cells.

Store-operated channels are major calcium influx pathways in nonexitable cells. Homer scaffold proteins are well known for their role in regulating calcium signaling. Here we report on a detailed single-channel level characterization of native store-operated channels regulated by Homer scaffold proteins in A431 carcinoma cells. By applying the single-channel patch-clamp technique, we found that different types of store-operated calcium channels have different sensitivities to Homer proteins.

[Store-operated cationic channels in human myeloid leukaemia K562 cells]. / Depozavisimye kationnye kanaly v kletkakh mieloidnoi leikemii cheloveka K562.

Using the whole-cell patch clamp technique, single channels operated by intracellular Ca(2+)-store depletion were first revealed in human myeloid leukaemia cells K562. A single store-operated channel could be detected in divalent-free extracellular solutions with Na+ as a permeant ion, and intracellular solutions with strong Ca(2+)-helating agent with some delay after whole-cell formation. Addition of inositol-1,4,5-triphosphate to the pipette solution resulted in a significant decrease of this latency. These channels had a conductance of 29 pS, and were inhibited by low concentration of external Ca2+. Our results enable us to assume that the revealed channels are calcium release-activated calcium channels, operated by Ca2+ depletion of endoplasmic reticulum.

[Study of "group" expression of mRNA of the ion transporters ATP1B1, NHE1 and NKCC1, beta-actin, glycerophosphosphate dehydrogenase, proteins regulating proliferation and apoptosis of p53, Bcl-2, IL-2 and hSGK kinase at the prereplicative stage of human lymphocyte activation]. / Issledovanie "gruppovoi" ékspressii mRNK ionnykh transporterov ATP1B1, NHE1 i NKCC1, beta-aktina, glitserofosfatdegidrogenazy, belkov--reguliatorov proliferatsii i apoptoza p53, Bcl-2, IL-2 i kinazy hSGK na predreplikativnoi stadii aktivatsii limfotsitov cheloveka.

Previously, we found no segregation in F2 obtained from crosses between two Dileptus anser clones differing (under the same culture conditions) in their serotypes, i.e. in their immobilization antigens (i-antigens); indeed, all the F2 clones had mixed, i.e. hybrid serotype, being immobilized simultaneously with both immune sera developed against either parental clone (Uspenskaya, Yudin, 2000). Presently, experiments were carried out to see if this unusual phenotype would be re-expressed after a temporary switching off. To switch off both expressed i-antigens, serotype transformation was induced in the F2 clones by shifting the culture temperature from 25 to 17 degrees C. Two weeks later, when the clones returned to the initial temperature conditions, each of them was seen to re-express both parental i-antigens. This result is discussed with reference to the role of i-antigens in regulation of their own expression as has been suggested by some authors.

Differential transcription of ion transporters, NHE1, ATP1B1, NKCC1 in human peripheral blood lymphocytes activated to proliferation.

This work, using RT PCR, studied expression of mRNAs encoding ion transporters, the Na/H antiporter (NHE1), the beta subunit of the Na,K-ATPase pump (ATP1B1), the NaK2Cl symporter (NKCC1), and some proteins unrelated to ion transport: the serum and glucocorticoid dependent kinase (hSGK), beta-actin, a glycolytic enzyme (GAPDH), and regulators of proliferation and apoptosis (p53, Bcl-2) during activation of human lymphocytes with phytohemagglutinin for 4-24 h. Within 24 hours the mRNA levels of NHE1, beta-actin, Bcl-2, and p53 increased by more than 100%, the mRNA levels of ATP1B1, GAPDH, and hSGK, by about 50%, while the mRNA levels of NKCC1 decreased transiently. These results indicate a differential transcriptional control of NHE1, ATP1B1, and NKCC1 following a proliferative stimulus of human lymphocytes.

[The effect of NaK2Cl symport and chloride channel permeability on ion flux balance and on transmembrane ion distribution in different types of animal cells]. / Vliianie simporta NaK2Cl i pronitsaemosti khlornykh kanalov na balans ionnykh potokov i transmembrannoe raspredelenie ionov u zhivotnykh kletok raznogo tipa.

The relationships between monovalent ion fluxes via major cell membrane pathways (Na/K pump, NaK2Cl symporter, electroconductive sodium, potassium and chloride channels) and steady state transmembrane ion distribution, membrane potential and cell water content were calculated for the high potassium animal cells with high and low membrane potential. It is found that variation in NaK2Cl symport or chloride electroconductive permeability causing changes in cell water content of high magnitude do not lead to significant changes in the intracellular Na/K ratio or membrane potential, in contrast to the effects caused by variation in the Na/K pump fluxes or permeability of the Na and K channels. It is shown that water content in cells with a high membrane potential, e.g. of about 70 mV, cannot be increased due to an increase in NaK2Cl symport by more than 1.6 times. In cells with a low membrane potential an increase in symport leads to a decrease in water content, which is also limited. In cells with membrane potential of about 10 mV the water content cannot be decreased more than by 1.8 times. When NaK2Cl symporter is operating, the effect of chloride channel permeability on the ion and water balance is quite opposite to the symport and is limited by the same boundaries. It is shown that effects caused by changes in symport and in chloride permeability can be differentiated only by the analysis of kinetic (fluxes, transport rate constants etc.) but not "static" characteristics of ion distribution. It is shown that under some circumstances the influence of NaK2Cl symport and chloride channel permeability on ion and water balance can be strong even at a very small symport share in the overall flux.

[The role of ionic transporters in the long-term regulation of the water content in animal cells. The mathematical model and real lymphoid cells]. / Rol' ionnykh transporterov v dolgovremennoi reguliatsii soderzhaniia vody v zhivotnykh kletkakh. Matematicheskaia model' i real'nye limfoidnye kletki.

Ion and water balance as well as flux balance in the animal cell, equilibrated to anisosmotic solutions, are computed for a cell model supplied by Na,K-ATPase pump, electroconductive ion channels and symporters Na-Cl, K-Cl and NaK2Cl. It is shown how the potency of each of the principal transporters to regulate cell volume depends on such conditions as the state of other ion transporters and channels, initial ion distribution and membrane potential. The obtained data are applied to studying changes in cation and water balance in the Jurkat lymphoid cells equilibrated to hyposmotic solutions. It is concluded that a steady state volume regulation in Jurkat cells, maintained in hyposmotic media 180 mosM for up to 24 h, is achieved due mainly to non-ionic mechanisms. Under the lower osmolarity of the media, 155 mosM, the ionic cell volume regulation occurs. The increase in PCl and/or the decrease in Na-Cl symport (or its equivalent, e.g. Na/H and Cl/HCO3 exchange) are considered as the most probable cause of the long-term volume decrease in lymphoid cells.

[The relationship between the cation and protein content, cationic transport and the rate of protein synthesis detected in cells in the presence and in the absence of ouabain]. / Sviaz' mezhdu soderzhaniem kationov, belka, kationnym transportom i skorost'iu sinteza belka, obnaruzhennaia na kletkakh v prisutstvii i v otsutstvii uabaina.

The measurements were made of monovalent cation (K+ + Rb+, Na+) and protein contents, of leucin incorporation into the protein (protein synthesis), and of ouabain-sensitive K+ influx during the growth of the Jurkat culture cells in the presence or in the absence of ouabain. All the data were calculated per cell. The time dependences of these parameters are nonlinear [correction of unline] curves. The results of three independent measurements are not reproduced. The problem was to find out an intracellular "main" parameter, so that the dependences between this and the rest of parameters be of line character. It has been found that it is the protein synthesis rate that may be taken as the main parameter, because between the protein synthesis rate and all the other parameters there is the line connection. Comparative results obtained with or without ouabain showed that K+ and Na+ concentrations in the cell were different, although the constants of the rate of protein synthesis were the same. This points to the same type of change of the protein synthesis process in the cells growing with or without ouabain.