Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca2+ entry (SOCE). Ca2+ release through inositol 1,4,5-trisphosphate receptor (IP3R) and subsequent Ca2+ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca2+, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca2+ entry through TRPC1 channels activated endogenous Ca2+-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca2+ concentration and/or transmembrane potential, conversely lowering of intracellular Ca2+ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca2+ concentration. Experiments with Ca2+ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca2+-activated chloride channels are involved in Ca2+ entry microdomains.
Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca2+ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca2+ imaging indicated the involvement of ORAI and sodium–calcium exchanger in Ca2+ entry induced upon store depletion. In male rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of a store-operated Ca2+ entry (SOCE) in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca2+ influx such that TRPC6 channels lost their sensitivity to Ca2+ store depletion and ORAI-mediated Ca2+ entry was suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account when developing pharmacological treatment of the early stages of diabetic nephropathy.
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.
Type 2 diabetes mellitus (DM2) is a widespread metabolic disorder that results in podocyte damage and diabetic nephropathy. Previous studies demonstrated that TRPC6 channels play a pivotal role in podocyte function and their dysregulation is associated with development of different kidney diseases including nephropathy. It was assumed that activation of TRPC6 channels leads directly to the Ca2+ influx into cells. Another Ca2+ influx pathway, a store-operated Ca2+ entry (SOCE) via ORAI channels, was also shown to be disturbed during the diabetes development. Here, using single channel patch clamp technique, we demonstrated that non-selective cationic TRPC6 channels are sensitive to the Ca2+ store depletion in human podocyte cell line Ab8/13 and in freshly isolated rat glomerular podocytes. Ca2+ imaging indicated the involvement of ORAI and sodium-calcium exchanger (NCX) in Ca2+ entry induced upon store depletion. Ca2+ inflow by NCX is a result of pronounced TRPC6-mediated Na+ influx leading moreover to the membrane depolarization and ORAI suppression. In rats fed a high-fat diet combined with a low-dose streptozotocin injection, which leads to DM2 development, we observed the reduction of SOCE in rat glomerular podocytes. This was accompanied by a reorganization of store-operated Ca2+ influx such that TRPC6 channels lost their sensitivity to Ca2+ store depletion and ORAI-mediated Ca2+ entry is suppressed in TRPC6-independent manner. Altogether our data provide new insights into the mechanism of SOCE organization in podocytes in the norm and in pathology, which should be taken into account in the pharmacological treatment of the early stages of diabetic nephropathy.
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.
Store‐operated calcium entry is significant pathway of calcium concentration rise in cytoplasm. The main players in this process are Orai store‐operated channels, which are activated directly by STIM calcium sensors. Data on participation of TRPC channels in store‐operated calcium entry is controversial. Study was focused on interaction between STIM and Orai proteins with endogenous TRPC and its role in pathology. We investigated activity of TRPC channels in a model object — HEK293 cells and in the podocytes of the rat model of diabetic nephropathy, using single channel recording patch camp technique. To eliminate Orai channels activity in HEK293 cells we used several experimental approaches: overexpression of dominant‐negative Orai mutant, Orai genes knockout and knockdown via CRISPR‐Cas 9 system and shRNA. Endogenous TRPC1 channels remain active in cells without functional Orai proteins and become insensitive to store depletion. In experiments with different extracellular calcium concentration we confirmed that calcium entry is needed for TRPC1 activation by store depletion. In experiments with calcium, barium or sodium as a charge carrier we observed similarities in dependence of open channel probability versus membrane potential between TRPC1 and Orai channels. The role of STIM calcium sensors in TRPC1 channels activation was investigated in cells with different expression level of STIM1 and STIM2 proteins. The findings allow us to propose that STIM proteins don’t activate the endogenous TRPC channels directly. TRPC channels are activated downstream to calcium entry through the Orai channels. Support or Funding Information The study was supported by grant of Russian science foundation № 19‐14‐00114 (Glushankova L., Skopin A., Kaznacheyeva E.), and by grant of Russian foundation for basic research № 19‐315‐90065 (Shalygin A., Kolesnikov D.).
Cell survival and normal cell function requires tight control of basal cytosolic Ca 2þ concentration. Fine-tuned regulation of cytosolic Ca 2þ concentrations is essential for various signaling pathways, controlling among others gene transcription, proliferation and cell migration. My studies focus especially on the understanding of the structural requirements triggering the co-regulation of the molecular key players of the CRAC channel STIM1 and Orai1 with small conductance Ca 2þ activated K þ channels (SK channels). Several publications have already provided evidence, that the interplay of Orai1 with SK3 is essential in the control of cancer cell growth, as their downregulation reduces cancer cell proliferation and migration. SK3 channels are activated by intracellular Ca 2þ in the range of 0.1-0.25 mM. I discovered in electrophysiological studies in the absence of intracellular Ca 2þ that a co-expression of Orai1 and SK3 in HEK cells initiates the activation of SK3-like currents, in contrast to SK3 alone which remains inactive under these conditions. A combined approach of Ca 2þ imaging, electrophysiological and fluorescence microscopy will uncover the so far unresolved molecular key determinants regulating the interplay and co-localization of the channels, potentially providing more specific target sites for cancer treatment. (Supported by Austrian Science Fund FWF P25210 and P27641 to I.D. and P27263 to C.R.).
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