Kayalvizhi Radhakrishnan

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.

The Cav2.3 voltage-gated calcium channel in epileptogenesis—Shedding new light on an enigmatic channel

The Ca(v)2.3 encoded Ca2+ channel is probably one of the least well-understood voltage-gated calcium channels in terms of physiology, pharmacology and clinical relevance. Here we provide a detailed insight into the functional involvement of Ca(v)2.3 in etiology and pathogenesis of both convulsive and non-convulsive seizures. In the CNS, Ca(v)2.3 containing E/R-type Ca2+ channels are involved in triggering epileptiform discharges by significantly contributing to plateau potentials and afterdepolarisations. Pharmacological analysis further revealed that various antiepileptic drugs specifically target Ca(v)2.3 VGCCs capable of blocking epileptiform burst activity. Whereas electroencephalographic recordings in Ca(v)2.3-/- mice did not reveal any ictal-like discharges, seizure susceptibility was dramatically reduced in Ca(v)2.3-/- animals compared to controls, further supporting the observation that Ca(v)2.3 is an important factor in triggering epileptiform activity in neuronal populations. Although some aspects of its relationship to epilepsy have been uncovered, further functional characterization of Ca(v)2.3 in etiology and pathogenesis of human epileptic syndromes as well as development of new antiepileptic drugs specifically targeting Ca(v)2.3 turns out to become indispensable.

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.

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.

Ca(v)2.3 Ca2+ channel interacts with the G1-subunit of V-ATPase.

Background: Calcium channels are essential in coupling action potential to signal transduction in cells. There are several types of calcium channels, which can be pharmacologically classified as L-, N-, P/Q-, R- and T-type. But molecular basis of R-type channels is less clearly understood compared the other channel types. Therefore the current study aims at understanding the molecular functions of R-type calcium channels by identifying interaction partners of the channel. Methods: In order to do so, a yeast two hybrid (Y2H) screen, with carboxy terminus of α1 subunit of the channel, as the bait, was performed. G1 subunit of v-ATPase was identified as a putative interaction partner of human Cav2.3 by using the Y2H screening. The interaction was confirmed by immunoprecipitation. To study the functional importance of the interaction, bafilomycin A1, a potent and specific inhibitor of v-ATPase was used in patch-clamp recordings in Cav2.3 stably-transfected HEK-293 cells (2C6) as well as in electroretinography of the isolated bovine retina expressing R-type Ca2+ channels. Results: G1 subunit of v-ATPase interacts with C-terminal tail of Cav2.3 and bafilomycin A1 reduces Cav2.3 mediated calcium currents. Additionally peak ICa is inhibited in retinal signal transduction when recorded as ERG b-wave. Conclusions: The results suggest that v-ATPase interacts physically and also functionally with Cav2.3. This is the first demonstration of association of Cav2.3 C-terminus with a protein complex which is involved in transmembrane signalling.

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.

Heat Shock Proteins and Ion Channels. Functional Interactions and Therapeutic Consequences

Screening for protein interaction partners of ion channels helps to elucidate signaling cascades to cellular targets and processes for a better understanding of the origin of diseases. Most important are the cytosolic segments of membrane-bound voltage- and ligand-gated ion channels or from ion channel regulators, which may connect to specific signaling complexes. So far, not much is known about those interactions. Molecular chaperones are proteins, which support the biosynthesis of proteins during maturation without being part of the final protein complex or which support the degradation of targeted proteins within the cellular protein quality control.