Biomedical subjects

Alexey Pereverzev

Publications and source records attributed to Alexey Pereverzev.

13 recordsLinked to original sources

PubMed2006

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.

Amino Acid Sequence↗

PubMed2005

The ablation of the Ca(v)2.3/E-type voltage-gated Ca2+ channel causes a mild phenotype despite an altered glucose induced glucagon response in isolated islets of Langerhans.

Glucagon release upon hypoglycemia is an important homeostatic mechanism utilized by vertebrates to restore blood glucose to normal. Glucagon secretion itself is triggered by Ca2+ influx through voltage-gated ion channels, and the gene inactivation of R-type Ca2+ channels, with Ca(v)2.3 as the ion conducting subunit, has been shown to disturb glucose homeostasis. To understand how glucagon release may be affected in Ca(v)2.3-deficient mice, carbachol, insulin and glucose induced glucagon response was investigated. While the rise of insulin and glucose induced by carbachol is normal, mutant mice show an impaired glucagon-response. Further, the effect of insulin injection on glucagon levels was altered by the loss of the Ca(v)2.3 subunit. Ca(v)2.3-deficient mice are characterized by an impaired glucose suppression of glucagon release. This was most obvious at the level of isolated islets suggesting that Ca(v)2.3 containing R-type voltage-gated Ca2+ channels are involved in the glucose-mediated signalling to glucagon release in mice.

Animals↗

PubMed2005

Convergent signaling by acidosis and receptor activator of NF-kappaB ligand (RANKL) on the calcium/calcineurin/NFAT pathway in osteoclasts.

Systemic acidosis has detrimental effects on the skeleton, and local acidosis coincides with bone destruction in inflammatory and metastatic diseases. Acidification dramatically enhances osteoclastic resorption, although the underlying mechanism has remained elusive. We investigated the effect of acidosis on the osteoclastogenic transcription factor NFATc1, which upon dephosphorylation translocates from the cytoplasm to nuclei. Lowering extracellular pH dramatically increased accumulation of NFATc1 in nuclei of rat and rabbit osteoclasts to levels comparable with those induced by the proresorptive cytokine receptor activator of NF-kappaB ligand (RANKL). Activation of NFATc1 by RANKL was mediated by means of prolonged stimulation of the Ca2+/calmodulin-dependent protein phosphatase, calcineurin. In contrast, NFATc1 activation by acidosis involved stimulation of calcineurin and suppression of NFATc1 inactivation. Acidosis, like RANKL, induced transient elevation of cytosolic free Ca2+ concentration ([Ca2+]i), which persisted in Ca2+-free media and was abolished by inhibition of phospholipase C or depletion of intracellular Ca2+ stores. Real-time-PCR of osteoclast-like cells generated from RAW 264.7 cells revealed high levels of expression of ovarian cancer G protein-coupled receptor 1, which links extracellular acidification to elevation of [Ca2+]i. In addition, the calcineurin inhibitor cyclosporin A suppressed the stimulatory effect of acidification on resorption, implicating NFAT in mediating the actions of acidosis on osteoclast activity. In summary, acidification and RANKL induce signals in osteoclasts that converge on the Ca2+/calcineurin/NFAT pathway. Acidosis acts directly on osteoclasts to activate NFATc1 and stimulate resorption.

Acidosis↗

PubMed2005

CaV2.3 calcium channels control second-phase insulin release.

Concerted activation of different voltage-gated Ca( (2+) ) channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca(V)2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca(V)2.3-knockout (Ca(V)2.3(-/-)) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca(2+) channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca(V)2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca(2+) signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca(V)2.3(-/-) beta cells. Ca(V)2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca(V)2.3(-/-) mouse. We propose a specific role for Ca(V)2.3 Ca(2+) channels in second-phase insulin release, that of mediating the Ca(2+) entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.

Animals↗

PubMed2004

The cytosolic II-III loop of Cav2.3 provides an essential determinant for the phorbol ester-mediated stimulation of E-type Ca2+ channel activity.

There is growing evidence that E-type voltage dependent Ca(2+) channels (Ca(v)2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long-term potentiation. The mechanism underlying a novel Ca(2+) dependent stimulation of E-type Ca(2+) channels was investigated in the context of the recent finding that influx of Ca(2+) through other voltage dependent Ca(2+) channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba(2+) as charge carrier through Ca(v)2.3 channel alpha(1) subunits expressed in HEK-293 cells, activation of PKC by low concentrations of phorbol ester augmented peak I(Ba) by approximately 60%. In addition, the non-inactivating fraction of I(Ba) was increased by more than three-fold and recovery from short-term inactivation was accelerated. The effect of phorbol ester on I(Ba) was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca(2+) as charge carrier, application of phorbol ester did not change the activity of Ca(v)2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca(2+) as charge carrier the incoming Ca(2+) can activate PKC, thereby augmenting Ca(2+) influx into the cytosol. No modulation of Ca(v)2.3 channels by PKC was observed when an arginine rich region in the II-III loop of Ca(v)2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Ca(v)2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2004

Arrhythmia in isolated prenatal hearts after ablation of the Cav2.3 (alpha1E) subunit of voltage-gated Ca2+ channels.

A voltage-gated calcium channel containing Cav2.3e (alpha1Ee) as the ion conducting pore has recently been detected in rat heart. Functional evidence for this Ca2+ channel to be involved in the regulation of heart beating, besides L- and T-type channels, was derived from murine embryos where the gene for Cav1.2 had been ablated. The remaining "L-type like" current component was not related to recombinant splice variants of Cav1.3 containing channels. As recombinant Cav2.3 channels from rat were reported to be weakly dihydropyridine sensitive, the spontaneous activity of the prenatal hearts from Cav2.3(-|-) mice was compared to that of Cav2.3(+|+) control animals to investigate if Cav2.3 could represent such a L-type like Ca(2+) channel. The spontaneous activity of murine embryonic hearts was recorded by using a multielectrode array. Between day 9.5 p.c. to 12.5 p.c., the beating frequency of isolated embryonic hearts from Cav2.3-deficient mice did not differ significantly from control mice but the coefficient of variation within individual episodes was more than four-fold increased in Cav2.3-deficient mice indicating arrhythmia. In isolated hearts from wild type mice, arrhythmia was induced by superfusion with a solution containing 200 nM SNX-482, a blocker of some R-type voltage gated Ca2+ channels, suggesting that R-type channels containing the splice variant Cav2.3e as ion conducting pore stabilize a more regular heart beat in prenatal mice.

Alternative Splicing↗

PubMed2003

Actions of sipatrigine, 202W92 and lamotrigine on R-type and T-type Ca2+ channel currents.

Relatively little has been published on the pharmacology of R-type and T-type Ca(2+) channels. Here, whole-cell Ca(2+) channel currents were recorded from human embryonic kidney 293 cell-lines transfected with either alpha1E subunits (R-type currents) or alpha1G or alpha1I subunits (T-type currents). R-type currents were inhibited by sipatrigine and the related compound 202W92 (R-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)pyrimidine) with IC(50) 10 and 56 microM, respectively. A therapeutic concentration of lamotrigine (10 microM) inhibited R-type currents (30%) but was without effect on alpha1I-mediated T-type currents. Lamotrigine was also a weak inhibitor of T-type currents mediated by alpha1G subunits (<10% inhibition by 100 microM).

Calcium Channel Blockers↗

PubMed2003

Ca2+-sensitive regulation of E-type Ca2+ channel activity depends on an arginine-rich region in the cytosolic II-III loop.

Ca2+-dependent regulation of L-type and P/Q-type Ca2+ channel activity is an important mechanism to control Ca2+ entry into excitable cells. Here we addressed the question whether the activity of E-type Ca2+ channels can also be controlled by Ca2+. Switching from Ba2+ to Ca2+ as charge carrier increased within 50 s, the density of currents observed in HEK-293 cells expressing a human Cav2.3d subunit and slowed down the inactivation kinetics. Furthermore, with Ca2+ as permeant ion, recovery from inactivation was accelerated, compared to the recovery process recorded under conditions where the accumulation of [Ca2+]i was prevented. In a Ba2+ containing bath solution the Ca2+-dependent changes of E-type channel activity could be induced by dialysing the cells with 1 micro m free [Ca2+]i suggesting that an elevation of [Ca2+]i is responsible for these effects. Deleting 19 amino acids in the intracellular II-III linker (exon 19) as part of an arginine-rich region, severely impairs the Ca2+ responsiveness of the expressed channels. Interestingly, deletion of an adjacent homologue arginine-rich region activates channel activity but now independently from [Ca2+]i. As a positive feedback-regulation of channel activity this novel activation mechanism might determine specific biological functions of E-type Ca2+ channels.

Arginine↗

PubMed2002

The Ca(V)2.3 Ca(2+) channel subunit contributes to R-type Ca(2+) currents in murine hippocampal and neocortical neurones.

Different subtypes of voltage-dependent Ca(2+) currents in native neurones are essential in coupling action potential firing to Ca(2+) influx. For most of these currents, the underlying Ca(2+) channel subunits have been identified on the basis of pharmacological and biophysical similarities. In contrast, the molecular basis of R-type Ca(2+) currents remains controversial. We have therefore examined the contribution of the Ca(V)2.3 (alpha(1E)) subunits to R-type currents in different types of central neurones using wild-type mice and mice in which the Ca(V)2.3 subunit gene was deleted. In hippocampal CA1 pyramidal cells and dentate granule neurones, as well as neocortical neurones of wild-type mice, Ca(2+) current components resistant to the combined application of omega-conotoxin GVIA and MVIIC, omega-agatoxin IVa and nifedipine (I(Ca,R)) were detected that were composed of a large R-type and a smaller T-type component. In Ca(V)2.3-deficient mice, I(Ca,R) was considerably reduced in CA1 neurones (79 %) and cortical neurones (87 %), with less reduction occurring in dentate granule neurones (47 %). Analysis of tail currents revealed that the reduction of I(Ca,R) is due to a selective reduction of the rapidly deactivating R-type current component in CA1 and cortical neurones. In all cell types, I(Ca,R) was highly sensitive to Ni(2+) (100 microM: 71-86 % block). A selective antagonist of cloned Ca(V)2.3 channels, the spider toxin SNX-482, partially inhibited I(Ca,R) at concentrations up to 300 nM in dentate granule cells and cortical neurones (50 and 57 % block, EC(50) 30 and 47 nM, respectively). I(Ca,R) in CA1 neurones was significantly less sensitive to SNX-482 (27 % block, 300 nM SNX-482). Taken together, our results show clearly that Ca(V)2.3 subunits underlie a significant fraction of I(Ca,R) in different types of central neurones. They also indicate that Ca(V)2.3 subunits may give rise to Ca(2+) currents with differing pharmacological properties in native neurones.

Animals↗

PubMed2002

Alternate splicing in the cytosolic II-III loop and the carboxy terminus of human E-type voltage-gated Ca(2+) channels: electrophysiological characterization of isoforms.

There is growing evidence that Ca(v)2.3 (alpha1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca(2+) channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca(2+) channels. To understand whether splice variation of Ca(v)2.3 contributes to the divergence of R-type channels, individual variants of Ca(v)2.3 were constructed and expressed in HEK-293 cells. With Ba(2+) as charge carrier, the tested biophysical properties were similar. In Ca(2+), the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca(v)2.3 Ca(2+) channel subunits. This different Ca(2+) sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca(v)2.3 splice variants.

Action Potentials↗

PubMed2002

Single-channel pharmacology of mibefradil in human native T-type and recombinant Ca(v)3.2 calcium channels.

To study the molecular pharmacology of low-voltage-activated calcium channels in biophysical detail, human medullary thyroid carcinoma (hMTC) cells were investigated using the single-channel technique. These cells had been reported to express T-type whole-cell currents and a Ca(v)3.2 (or alpha 1H) channel subunit. We observed two types of single-channel activity that were easily distinguished based on single-channel conductance, voltage dependence of activation, time course of inactivation, rapid gating kinetics, and the response to the calcium agonist (S)-Bay K 8644. Type II channels had biophysical properties (activation, inactivation, conductance) typical for high-voltage-activated calcium channels. They were markedly stimulated by 1 microM (S)-Bay K 8644, allowing to identify them as L-type channels. The channel termed type I is a low-voltage-activated, small-conductance (7.2 pS) channel that inactivates rapidly and is not modulated by (S)-Bay K 8644. Type I channels are therefore classified as T-type channels. They were strongly inhibited by 10 microM mibefradil. Mibefradil block was caused by changes in two gating parameters: a pronounced reduction in fraction of active sweeps and a slight shortening of the open-state duration. Single recombinant low-voltage-activated T-type calcium channels were studied in comparison, using human embryonic kidney 293 cells overexpressing the pore-forming Ca(v)3.2 subunit. Along all criteria examined (mechanisms of block, extent of block), recombinant Ca(v)3.2 interact with mibefradil in the same way as their native counterparts expressed in hMTC cells. In conclusion, the pharmacologic phenotype of these native human T-type channels--as probed by mibefradil--is similar to recombinant human Ca(v)3.2.

Calcium Channel Blockers↗

PubMed2002

Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels.

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.

Animals↗

PubMed2002

Reduction of insulin secretion in the insulinoma cell line INS-1 by overexpression of a Ca(v)2.3 (alpha1E) calcium channel antisense cassette.

OBJECTIVE: Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q and R type) coordinate a variety of Ca(2+)-dependent processes in neurons and neuroendocrine cells. In insulinoma cell lines as well as in endocrine tissues, the non-L-type alpha1E (Ca(v)2.3) subunit is expressed as the tissue-specific splice variant alpha1Ee. DESIGN AND METHODS: To understand the functional role of alpha1E-containing Ca(2+) channels, antisense alpha1E mRNA was overexpressed in INS-1 cells by stable transfection of an antisense alpha1E cassette cDNA. As controls, either a sense alpha1E cassette or a control vector containing enhanced green fluorescent protein as an unrelated gene was stably transfected. The overexpression of each transfected cassette cDNA was recorded by RT-PCR. RESULTS: In three independent antisense alpha1E INS-1 clones, the glucose-induced insulin release was significantly reduced as compared with wild-type INS-1 cells and with a sense alpha1E INS-1 clone. However, in the antisense INS-1 clones, the KCl-induced insulin release was less impaired by overexpressing the antisense alpha1E cassette than the glucose-induced insulin release, leading to the assumption that glucose (15 mmol/l) and KCl (25 mmol/l) finally depolarize the membrane potential to a different extent. CONCLUSION: alpha1E is involved in glucose-induced insulin secretion probably by influencing the excitability of INS-1 cells.

Blotting, Western↗