Andreas Krieger

Altered Seizure Susceptibility in Mice Lacking the Cav2.3 E‐type Ca2+ Channel

Summary:  Purpose: Recently the Cav2.3 (E/R‐type) voltage‐gated calcium channel (VGCC) has turned out to be not only a potential target for different antiepileptic drugs (e.g., lamotrigine, topiramate) but also a crucial component in the pathogenesis of absence epilepsy, human juvenile myoclonic epilepsy (JME), and epileptiform activity in CA1 neurons. The aim of our study was to perform an electroencephalographic analysis, seizure‐susceptibility testing, and histomorphologic characterization of Cav2.3−/− mice to unravel the functional relevance of Cav2.3 in ictogenesis.

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.

Presynaptic ‘Cav2.3‐containing’ E‐type Ca2+ channels share dual roles during neurotransmitter release

Ca2+ influx into excitable cells is a prerequisite for neurotransmitter release and regulated exocytosis. Within the group of ten cloned voltage‐gated Ca2+ channels, the Cav2.3‐containing E‐type Ca2+ channels are involved in various physiological processes, such as neurotransmitter release and exocytosis together with other voltage‐gated Ca2+ channels of the Cav1, Cav2 and Cav3 subfamily. However, E‐type Ca2+ channels also exhibit several subunit‐specific features, most of which still remain poorly understood. Cav2.3‐containing R‐type channels (here called ‘E‐type channels’) are also located in presynaptic terminals and interact with some synaptic vesicle proteins, the so‐called SNARE proteins, although lacking the classical synprint interaction site. E‐type channels trigger exocytosis and are also involved in long‐term potentiation. Recently, it was shown that the interaction of Cav2.3 with the EF‐hand motif containing protein EFHC1 is involved in the aetiology and pathogenesis of juvenile myoclonic epilepsy.

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.

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.

APLP1 and Rab5A interact with the II-III loop of the voltage-gated Ca-channel Ca(v)2.3 and modulate its internalization differently.

Background: Voltage gated calcium channels (VGCCs) regulate cellular activity in response to membrane depolarization by altering calcium homeostasis. Because calcium is the most versatile second messenger, regulation of the amount of VGCCs at the plasma membrane is highly critical for several essential cellular processes. Among the different types of VGCCs, the Cav2.3 calcium channel and its regulation mechanisms are least understood due to Cav2.3’s resistance to most pharmacological agents. Methods: In order to study regulation and surface expression of Cav2.3, a yeast two hybrid (Y2H) screen with the II-III loop of human Cav2.3 as bait, was performed. APLP1, a member of the APP gene family and Rab5A, an endocytotic catalyst were identified as putative interaction partners. The interaction were confirmed by immunoprecipitation. To study the functional importance of the interaction, patch-clamp recordings in Cav2.3 stably transfected HEK-293 cells (2C6) and surface biotin endocytosis assays were performed. Results: We are able to show that the II-III loop of the Cav2.3 calcium channel binds APLP1 and that this binding promotes internalization of the channel. In addition, Rab5A also binds to the same loop of the channel and exerts an inhibitory effect on APLP1 mediated channel internalization. Conclusions: This study identifies a regulation mechanism of Cav2.3’s surface expression, which implicates APLP1 as a regulator of calcium homeostasis. Thus APLP1 may play a crucial role in neuropathological mechanisms, which involve modulation of surface expression of voltage-gated Ca2+ channels.

The C-terminus of human Cav2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.

Interaction of recombinant and native Ca v 2.3 E-/R-type voltage-gated Ca 2+ channels with the molecular chaperone Hsp70

Correspondence: Toni schneider institute of neurophysiology, University of Cologne, Robert-Koch-str 39, D-50931 Köln, germany Tel +49 22 1478 6946 Fax +49 22 1478 6965 email toni.schneider@uni-koeln.de Abstract: Cytosolic segments of membrane-bound voltage-gated Ca2+ channels are targets for specific signaling complexes which regulate important physiological processes via soluble protein interaction partners. The molecular chaperone heat shock 70 kDa protein 1A (Hsp70) was identified as a binding partner for the II-III loop of the ion-conducting α1 subunit of the E-type voltage-gated Ca2+ channel (Ca v 2.3) by mass spectrometry. To substantiate this finding further and to test its functional significance in vivo, two approaches were chosen. HEK-293 cells stably transfected with Ca v 2.3 were treated with a cell-permeable form of Hsp70 (Tat-Hsp70, Tat being a protein transduction domain of the “transactivator of transcription” from the human immunodeficiency virus) and analyzed by whole cell Ca2+ current recordings. Tat-Hsp70 1 μM shifted the voltage-dependence of the inward currents to more hyperpolarized potentials. Further, the inactivation of transient inward Ca2+ currents carried by Ca v 2.3 was slowed down. After isolation of hippocampal microsomes from control mice by ultracentrifugation, about 0.09% of total Hsp70 protein was bound to the microsomal fraction. Hsp70 binding to membranes was increased when kainate 20 mg/kg was injected intraperitoneally at concentrations which induced seizures and neurodegeneration in control mice. In Ca v 2.3-deficient mice, only mild seizures were observed after kainate injection, with no hippocampal neurodegeneration. Moreover, we observed no increase in Hsp70 binding to the membrane fraction of the isolated hippocampal microsomes, indicating that Hsp70 may be an important intermediate signaling partner for hippocampal neurodegeneration in Ca v 2.3(+/+) mice. Our results suggest that the Ca v 2.3 interaction partner, Hsp70, and its membrane association are important for understanding the molecular events in kainate-induced hippocampal neurodegeneration.

Protein Interaction Partners of Cav2.3 R-Type Voltage-Gated Calcium Channels

The Cav2.3 voltage-gated calcium channel represents the most enigmatic of all voltage-gated calcium channels due to its pharmacological inertness and to its mixed characteristics of HVA and LVA calcium channels. Protein interaction partners of the cytosolic II-III linker of Cav2.3 contribute to calcium homeostasis by regulating the channels surface expression and activation. Specific regulation of Cav2.3 by proteins interacting with the carboxy terminal region plays an important role in exocytosis and presynaptic plasticity, linking channel function to long-term potentiation. Modulation of Cav2.3 by its interaction partners thus contributes to several physiologic processes such as signal transduction in the retina, insulin secretion and generation of rhythmic activity in the heart and in the brain.