Role of STIM2 and Orai proteins in regulating TRPC1 channel activity upon calcium store depletion.

Store-operated calcium channels are the major player in calcium signaling in non-excitable cells. Store-operated calcium entry is associated with the Orai, stromal interaction molecule (STIM), and transient receptor potential canonical (TRPC) protein families. Researchers have provided conflicting data about TRPC1 channel regulation by Orai and STIM. To determine how Orai and STIM influence endogenous TRPC1 pore properties and regulation, we used single channel patch-clamp recordings. Here we showed that knockout or knockdown of Orai1 or Orai3 or overexpression of the dominant-negative mutant Orai1 E106Q did not change the conductance or selectivity of single TRPC1 channels. In addition, these TRPC1 channel properties did not depend on the amount of STIM1 and STIM2 proteins. To study STIM2-mediated regulation of TRPC1 channels, we utilized partial calcium store depletion induced by application of 10 nM thapsigargin (Tg). TRPC1 activation by endogenous STIM2 was greatly decreased in acute extracellular calcium-free experiments. STIM2 overexpression increased both the basal activity and number of silent TRPC1 channels in the plasma membrane. After calcium store depletion, overexpressed STIM2 directly activated TRPC1 in the plasma membrane even without calcium entry in acute experiments. However, this effect was abrogated by co-expression with the non-permeable Orai1 E106Q mutant protein. Taken together, our single-channel patch clamp experiments clearly demonstrated that endogenous TRPC1 forms a channel pore without involving Orai proteins. Calcium entry through Orai triggered TRPC1 channel activation in the plasma membrane, while subsequent STIM2-mediated TRPC1 activity regulation was not dependent on calcium entry.

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.

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.

[The reaction of the immune system to insulinlike growth factor-1 in postmenopausal women with breast cancer].

Taking into account the information about the role of insulin-like growth factor -1 (IGF-1) in breast cancer the main aim of the present study was to investigate the cellular immune response to IGF-1. There were examined 58 patients with breast cancer (61 +/- 1 years, BMI 31,2 +/- 0,9 kg/m2, all postmenopausal) and 10 with benign tumors of the breast (56 +/- 2 years, BMI 30,8 +/- 0,7 kg/m2 all menopausal) as well as 17 patients suffering from cancer of different sites. Sensibilization of lymphocytes to IGF-1 was measured in the reaction of blast transformation of peripheral blood lymphocytes. The majority of breast cancer patients (47/58) revealed the sensibilization of peripheral blood lymphocytes to IGF-1. Thus in a part of patients (29 /47) this reaction was suppressed and revealed after suppression of cyclooxygenase and/or blocking of the histamine H2-receptors by corresponding pharmaceutics drugs in vitro.

[Immunologic predictors of diabetes mellitus in menopausal breast cancer patients].

Although the relations between diabetes mellitus (DM) and breast cancer (BC) are lately widely discussed, the actual causes for cancer predisposition in patients with diabetes are currently unclear. This study was designed to define the frequency of DM immunological predictors occurrence and immune system function shifts in patients with breast cancer. Sixty four BC patients, 19 patients with benign breast conditions and 40 healthy individuals were included. The lymphocyte sensibilization with insulin suppressed by prostaglandin-synthesizing cells or cells with histamine receptor expression (DM predictor) is more common in BC patients than in control group (29 of 56 vs 5 of 37, p < 0.001). This is not a tumor marker, but rather is an objective factor reflecting higher occurrence of insulin resistance in this group. For BC patients is also characteristic the lower PHA-stimulated peripheral lymphocyte proliferation rate probably caused by increase in short-lived suppressor cell activity, a usual sign of the impairment of cell-mediated immunity. It is also possible, that the immunologic predictors of DM associated with insulin resistance, combined with the effects of short-lived suppressor cells, promote tumor cell proliferation.

[Comparative evaluation of efficacy of intracoronary shunts in minimally invasive myocardial revascularization].

Intracoronary shunts (IS) are devices for maintaining distal coronary blood flow when placing anastomoses with coronary arteries. The use of IS ensures functional and electrical stability of myocardium during its revascularization on the working heart. There are reports of endothelial damage by IS leading to dysfunction of coronary shunts. This study compares 208 patients with and without IS. Results of surgery were evaluated during hospitalization period. The groups were matched for initial clinical and instrumental characteristics and the number of shunt placements. It was shown that minimally invasive myocardial revascularization with the use of IS when placing distal anastomoses has a number of advantages over similar surgery without IS, viz. smaller degree of intraoperative myocardial lesion, blood loss, and frequency of postoperative complications along with enhanced stability of intraoperative hemodynamics.