Publications by authors named "E V Kaznacheyeva"

38 Publications

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

Cell Calcium 2021 Jun 8;97:102432. Epub 2021 Jun 8.

Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ceca.2021.102432DOI Listing
June 2021

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

Acta Naturae 2021 Jan-Mar;13(1):140-146

Institute of Cytology of Russian Academy of Sciences, St. Petersburg, 194064 Russia.

Store-operated Ca 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 Ca sensors STIM1 and STIM2 are the key components of store-operated Ca entry in mammalian cells. Under physiological conditions, STIM proteins are responsible for store-operated Ca 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 Ca entry (50 = 1 μM) and had almost no effect on the STIM1-dependent activation of store-operated channels.
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http://dx.doi.org/10.32607/actanaturae.11269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084296PMC
May 2021

Electrophysiological Properties of Endogenous Single Ca Activated Cl Channels Induced by Local Ca Entry in HEK293.

Int J Mol Sci 2021 Apr 30;22(9). Epub 2021 Apr 30.

Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia.

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca entry (SOCE). Ca release through inositol 1,4,5-trisphosphate receptor (IPR) and subsequent Ca store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca, 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 Ca entry through TRPC1 channels activated endogenous Ca-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 Ca concentration and/or transmembrane potential, conversely lowering of intracellular Ca concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca concentration. Experiments with Ca buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca-activated chloride channels are involved in Ca entry microdomains.
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http://dx.doi.org/10.3390/ijms22094767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124839PMC
April 2021

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes.

Int J Mol Sci 2021 Apr 22;22(9). Epub 2021 Apr 22.

Institute of Cytology, Russian Academy of Sciences, 194064 Saint-Petersburg, Russia.

The actin cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canonical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migration properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported that mutation in ACTN4, which encodes α-actinin-4, causes focal segmental glomerulosclerosis and solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.
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http://dx.doi.org/10.3390/ijms22094396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122765PMC
April 2021

Withaferin A Induces Heat Shock Response and Ameliorates Disease Progression in a Mouse Model of Huntington's Disease.

Mol Neurobiol 2021 Aug 26;58(8):3992-4006. Epub 2021 Apr 26.

Cellular and Molecular Neuroscience Laboratory, National Brain Research Centre, Manesar, Gurgaon, 122 051, India.

Impairment of proteostasis network is one of the characteristic features of many age-related neurodegenerative disorders including autosomal dominantly inherited Huntington's disease (HD). In HD, N-terminal portion of mutant huntingtin protein containing expanded polyglutamine repeats accumulates as inclusion bodies and leads to progressive deterioration of various cellular functioning including proteostasis network. Here we report that Withaferin A (a small bioactive molecule derived from Indian medicinal plant, Withania somnifera) partially rescues defective proteostasis by activating heat shock response (HSR) and delays the disease progression in a HD mouse model. Exposure of Withaferin A activates HSF1 and induces the expression of HSP70 chaperones in an in vitro cell culture system and also suppresses mutant huntingtin aggregation in a cellular model of HD. Withaferin A treatment to HD mice considerably increased their lifespan as well as restored progressive motor behavioral deficits and declined body weight. Biochemical studies confirmed the activation of HSR and global decrease in mutant huntingtin aggregates load accompanied with improvement of striatal function in Withaferin A-treated HD mouse brain. Withaferin A-treated HD mice also exhibit significant decrease in inflammatory processes as evident from the decreased microglial activation. These results indicate immense potential of Withaferin A for the treatment of HD and related neurodegenerative disorders involving protein misfolding and aggregation.
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http://dx.doi.org/10.1007/s12035-021-02397-8DOI Listing
August 2021
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