L. N. Glushankova

Neuronal store-operated calcium entry pathway as a novel therapeutic target for Huntington's disease treatment

Huntingtons disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders.

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells.

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I(max) channels, while the properties of I(min) and I(NS) channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current-voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I(max) channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I(max) channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I(max) channels but is not an essential subunit for other store-operated channel types in HEK293 cells.

Attenuated presenilin‐1 endoproteolysis enhances store‐operated calcium currents in neuronal cells

Presenilins have been reported to regulate calcium homeostasis in the endoplasmic reticulum, and dysregulation of intracellular calcium has been implicated in the pathogenesis of Alzheimers disease (AD). Reduced endoproteolysis levels of presenilin‐1 (PS1) have been detected in postmortem brains of patients carrying familial Alzheimers disease PS1 mutations. This study deals with the effect of attenuated endoproteolysis of PS1 on store‐operated calcium (SOC) entry in neuronal cells and mouse fibroblasts with double knockouts of PS1 and PS2. Significant enhancement of SOC channel activation has been detected by electrophysiological measurements in cells with reduced PS1 endoproteolysis. The increase in SOC entry was not accompanied by any changes in protein levels of channels subunits or stromal interaction molecule. These data are important for understanding the role of PS1 in AD, apart from its involvement in γ‐secretase cleavage of amyloid precursor protein into Aβ. Taking into account that most of familial AD‐connected mutations in PS1 are loss‐of‐function, the observed effects may well be general for familial AD.

Store-operated calcium entry into SK-N-SH human neuroblastoma cells modeling huntington’s disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by expansion of polyglutamine at the N-terminus of the huntingtin protein. Striatal medium spiny neurons (MSN) are the primary targets of HD pathology. In our study, a cellular model of HD was based on the human neuroblastoma cells SK-N-SH transfected with plasmid for expression of the mutant huntingtin protein Htt138Q. Expression of Htt138Q increased store-dependent calcium entry into SK-N-SH cells. EVP4593 reversibly blocked the abnormal store-dependent response, probably generated by the channels incorporating TRPC1 ( transient receptor potential canonical 1) subunit.

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 role of STIM1 in the receptor- and store-operated calcium influx in HEK293 cells

The possible role of STIM1 protein in the regulation of activity of receptor- and store-operated Ca2+ channels in non-excitable cells has been studied. Receptor- and store-operated Ca2+ influxes have been measured using the fluorescent method of detection of cytosolic Ca2+ concentration and the electrophysiological methods of whole-cell and single-channel current recordings in the control HEK293 cells and in HEK293 cells with suppressed expression of STIM1. The experiments have shown that STIM1 suppression results in a reduction of the amplitudes of both receptor- and store-operated inward calcium currents. The decrease of total Ca2+ influx of in response to an agonist or to passive depletion of calcium stores upon STIM1 suppression was due to the decrease or total absence of the activity of high-conductance channels Imax and non-selective channels Ins in HEK293 cells. A decrease in the STIM1 amount also altered the activity regulation of low-conductance Imin channels that changed from exclusively agonist-operated into store-dependent channels in HEK293 cells.

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.

The study of a role of TRPC1 protein in formation of molecular structure of the receptor- and store-operated calcium ionic channels

Ca 2+ influx was estimated using the intracellular calcium level measurements by fluorescent calcium-selective probes. It was shown that in these cells there is a huge decrease (~70%) in Ca 2+ entry either in response to UTP application and to passive store depletion by thapsigargin application (thapsigargin is a specific inhibitor of the calcium ATPase of endoplasmic reticulum). The whole-cell experiments have shown similar data: receptorand store-operated Ca 2+ influx in HEK293T with suppressed TRPC1 expression was significantly lower than in control cells. The obtained data suggest that TRPC1 protein suppression considerably changes calcium-transferring ways in the cells. The work was supported by the Russian Foundation for Basic Research (project nos. 07-04-01107a and 0704-01224), the Molecular and Cellular Biology RAS program, the SS-1135.2008.4. The Study of a Role of TRPC1 Protein in Formation of Molecular Structure of the Receptorand Store-Operated Calcium Ionic Channels

Role of STIM1 Protein in the Regulation of Store-Operated Channels in Non-Excitable Cells

329 The role of STIM1 protein in activity modulation of receptor- and store-operated channels in HEK293T cells was investigated. In native conditions transmembrane protein STIM1 is localized both in plasma membrane (smaller part of STIM1 population) and in ER membrane (most part of STIM1 population). For an exception of STIM1 from signaling cascades, the methods of small interfering RNA (siRNA) was used. Stable line of HEK293T cells with expression of STIM1 suppressed up to 50% was created. This clone was named S4. For the investigation of the role of STIM1 protein in store-operated Ca 2+ influx we compared Ca 2+ influx in response to thapsigargin-induced passive store depletion in control HEK293T and in S4 cells (thapsigargin is a specific inhibitor of the calcium ATPase of endo

Role of TRPC3 in the formation of receptor-and store-operated calcium channels in carcinoma A431 cells

Activation of phospholipase C (PLC)-linked signaling cascades in nonexcitable cells stimulates Ca2+ release from inositol-1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ entry via plasma membrane Ca2+ channels. The attention of investigators is currently focused on the properties and molecular basis of channels involved in Ca2+ entry into nonexcitable cells. According to current views, mammalian TRP proteins are involved in receptor-and store-dependent influx of Ca2+; however, little is known about the linkage between specific TRP proteins and endogenous channels responsible for Ca2+ entry. The aim of the present study was to elucidate the role of TRPC3 in the formation of store-dependent or receptor-operated pathways of Ca2+ entry into A431 cells. Registration of Ca2+ influx based on fluorescence measurements of intracellular Ca2+ concentrations and analysis of integral membrane currents revealed that partial inhibition of TRPC3 expression by small interfering RNA (siRNA) results in suppression of store-dependent Ca2+ entry without any effect on receptor-operated Ca2+ influx. In-depth studies of single channels revealed that TRPC3 suppression in A431 cells results in the disappearance of one type of store-operated channels and formation of a novel type of store-independent Ca2+-permeable channels. This, in turn, testifies to the crucial role of TRPC3 in normal functioning of store-operated Ca2+ channels in A431 cells.