Alexey Pereverzev

The CaV2.3 Ca2+ channel subunit contributes to R‐Type Ca2+ currents in murine hippocampal and neocortical neurones

Different subtypes of voltage‐dependent Ca2+ currents in native neurones are essential in coupling action potential firing to Ca2+ influx. For most of these currents, the underlying Ca2+ channel subunits have been identified on the basis of pharmacological and biophysical similarities. In contrast, the molecular basis of R‐type Ca2+ currents remains controversial. We have therefore examined the contribution of the CaV2.3 (α1E) subunits to R‐type currents in different types of central neurones using wild‐type mice and mice in which the CaV2.3 subunit gene was deleted. In hippocampal CA1 pyramidal cells and dentate granule neurones, as well as neocortical neurones of wild‐type mice, Ca2+ current components resistant to the combined application of ω‐conotoxin GVIA and MVIIC, ω‐agatoxin IVa and nifedipine (ICa,R) were detected that were composed of a large R‐type and a smaller T‐type component. In CaV2.3‐deficient mice, ICa,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 ICa,R is due to a selective reduction of the rapidly deactivating R‐type current component in CA1 and cortical neurones. In all cell types, ICa,R was highly sensitive to Ni2+ (100 μM: 71–86 % block). A selective antagonist of cloned CaV2.3 channels, the spider toxin SNX‐482, partially inhibited ICa,R at concentrations up to 300 nm in dentate granule cells and cortical neurones (50 and 57 % block, EC50 30 and 47 nm, respectively). ICa,R in CA1 neurones was significantly less sensitive to SNX‐482 (27 % block, 300 nm SNX‐482). Taken together, our results show clearly that CaV2.3 subunits underlie a significant fraction of ICa,R in different types of central neurones. They also indicate that CaV2.3 subunits may give rise to Ca2+ currents with differing pharmacological properties in native neurones.

Immunodetection of α1E Voltage-gated Ca2+ Channel in Chromogranin-positive Muscle Cells of Rat Heart, and in Distal Tubules of Human Kidney

The calcium channel α1E subunit was originally cloned from mammalian brain. A new splice variant was recently identified in rat islets of Langerhans and in human kidney by the polymerase chain reaction. The same isoform of α1E was detected in rat and guinea pig heart by amplifying indicative cDNA fragments and by immunostaining using peptide-specific antibodies. The apparent molecular size of cardiac α1E was determined by SDS-PAGE and immunoblotting (218 ± 6 kD; n = 3). Compared to α1E from stably transfected HEK-293 cells, this is smaller by 28 kD. The distribution of α1E in cardiac muscle cells of the conducting system and in the cardiomyoblast cell line H9c2 was compared to the distribution of chromogranin, a marker of neuroendocrine cells, and to the distribution of atrial natriuretic peptide (ANP). In serial sections from atrial and ventricular regions of rat heart, co-localization of α1E with ANP was detected in atrium and with chromogranin A/B in Purkinje fibers of the conducting system in both rat atrium and ventricle. The kidney is another organ in which natriuretic peptide hormones are secreted. The detection of α1E in the distal tubules of human kidney, where urodilatin is stored and secreted, led to the conclusion that the expression of α1E in rat heart and human kidney is linked to regions with endocrine functions and therefore is involved in the Ca2+-dependent secretion of peptide hormones such as ANP and urodilatin.

Receptor‐mediated modulation of recombinant neuronal class E calcium channels

The modulation of a cloned neuronal calcium channel was studied in a human embryonic kidney cell line (HEK293). The HEK293 cells were stably transfected with the α 1Ed cDNA, containing the pore forming subunit of a neuronal class E calcium channel. Inward currents of 25±1.9 pA/pF (n=79) were measured with the cloned α 1Ed‐subunit. The application of the peptide hormone somatostatin, carbachol, ATP or adenosine reduced the amplitude of Ca2+ and Ba2+ inward currents and exhibited a slowing of inactivation. This inhibitory effect by somatostatin was significantly impaired after pre‐incubating the transfected cell line with pertussis toxin (PTX). Internal perfusion of the cells with the G‐protein‐inactivating agent GDP‐β‐S or with the permanently activating agent GTP‐γ‐S also attenuated the somatostatin effect. The inhibition indicates that modulation of the α 1Ed‐mediated Ca2+ current involves pertussis toxin‐sensitive G‐proteins. The block of Ca2+ and Ba2+ inward currents by somatostatin is also found in cells expressing a truncated α 1Ed‐subunit which lacks a 129‐bp fragment in the C‐terminus. This fragment corresponds to the major structural difference between two native human α 1E splice variants. As somatostatin inhibits inward currents through both, the cloned α 1Ed‐ and the truncated α 1Ed‐DEL‐subunit, the hormone‐mediated modulation is independent from the presence of the 129‐bp insertion in the C‐terminus.

Alternate splicing in the cytosolic II-III loop and the carboxy terminus of human E-type voltage-gated Ca^ 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.

Structural diversity of the voltage‐dependent Ca2+ channel α1E‐subunit

Voltage‐operated Ca2+ channels are heteromultimeric proteins. Their structural diversity is caused by several genes encoding homologous subunits and by alternative splicing of single transcripts. Isoforms of α1 subunits, which contain the ion conducting pore, have been deduced from each of the six cDNA sequences cloned so far from different species. The isoforms predicted for the α1E subunit are structurally related to the primary sequence of the amino terminus, the centre of the subunit (II–III loop), and the carboxy terminus. Mouse and human α1E transcripts have been analysed by reverse transcription–polymerase chain reaction and by sequencing of amplified fragments. For the II–III loop three different α1E cDNA fragments are amplified from mouse and human brain, showing that isoforms originally predicted from sequence alignment of different species are expressed in a single one. Both predicted α1E cDNA fragments of the carboxy terminus are identified in vivo. Two different α1E constructs, referring to the major structural difference in the carboxy terminus, were stably transfected in HEK293 cells. The biophysical properties of these cells were compared in order to evaluate the importance in vitro of the carboxy terminal insertion found in vivo. The wild‐type α1E subunit showed properties, typical for a high‐voltage activated Ca2+ channel. The deletion of 43 amino acid residues at the carboxy terminus does not cause significant differences in the current density and the basic biophysical properties. However, a functional difference is suggested, as in embryonic stem cells, differentiated in vitro to neuronal cells, the pattern of transcripts indicative for different α1E isoforms changes during development. In human cerebellum the longer α1E isoform is expressed predominantly. Although, it has not been possible to assign functional differences to the two α1E constructs tested in vitro, the expression pattern of the structurally related isoforms may have functional importance in vivo.

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).

Arrhythmia in Isolated Prenatal Hearts after Ablation of the Cav2.3 (α1E) Subunit of Voltage-gated Ca2+ Channels

A voltage-gated calcium channel containing Cav2.3e (α1Ee) 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 Ca2+ 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.

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 µm 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.

The Molecular Chaperone hsp70 Interacts with the Cytosolic II-III Loop of the Cav2.3 E-type Voltagegated 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.

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 Ca2+ channels (Cav2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long‐term potentiation. The mechanism underlying a novel Ca2+ dependent stimulation of E‐type Ca2+ channels was investigated in the context of the recent finding that influx of Ca2+ through other voltage dependent Ca2+ channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba2+ as charge carrier through Cav2.3 channel α1 subunits expressed in HEK‐293 cells, activation of PKC by low concentrations of phorbol ester augmented peak IBa by approximately 60%. In addition, the non‐inactivating fraction of IBa was increased by more than three‐fold and recovery from short‐term inactivation was accelerated. The effect of phorbol ester on IBa was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca2+ as charge carrier, application of phorbol ester did not change the activity of Cav2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca2+ as charge carrier the incoming Ca2+ can activate PKC, thereby augmenting Ca2+ influx into the cytosol. No modulation of Cav2.3 channels by PKC was observed when an arginine rich region in the II–III loop of Cav2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Cav2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.