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Jörg Striessnig - One of the best experts on this subject based on the ideXlab platform.

  • Enhancing VTA Cav1.3 L-type Ca2+ channel activity promotes cocaine and mood-related behaviors via overlapping AMPA receptor mechanisms in the nucleus accumbens.
    Molecular psychiatry, 2017
    Co-Authors: Arlene Martinez-rivera, Jörg Striessnig, Amy S. Lee, Jin Hao, Thomas F. Tropea, Thomas Giordano, Maria Kosovsky, Richard C. Rice, Richard L. Huganir, N. A. Addy
    Abstract:

    Genetic factors significantly influence susceptibility for substance abuse and mood disorders. Rodent studies have begun to elucidate a role of Cav1.3 L-type Ca2+ channels in neuropsychiatric-related behaviors, such as addictive and depressive-like behaviors. Human studies have also linked the CACNA1D gene, which codes for the Cav1.3 protein, with bipolar disorder. However, the neurocircuitry and the molecular mechanisms underlying the role of Cav1.3 in neuropsychiatric phenotypes are not well established. In the present study, we directly manipulated Cav1.3 channels in Cav1.2 dihydropyridine insensitive mutant mice and found that ventral tegmental area (VTA) Cav1.3 channels mediate cocaine-related and depressive-like behavior through a common nucleus accumbens (NAc) shell calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (CP-AMPAR) mechanism that requires GluA1 phosphorylation at S831. Selective activation of VTA Cav1.3 with (±)-BayK-8644 (BayK) enhanced cocaine conditioned place preference and cocaine psychomotor activity while inducing depressive-like behavior, an effect not observed in S831A phospho-mutant mice. Infusion of the CP-AMPAR-specific blocker Naspm into the NAc shell reversed the cocaine and depressive-like phenotypes. In addition, activation of VTA Cav1.3 channels resulted in social behavioral deficits. In contrast to the cocaine- and depression-related phenotypes, GluA1/A2 AMPARs in the NAc core mediated social deficits, independent of S831-GluA1 phosphorylation. Using a candidate gene analysis approach, we also identified single-nucleotide polymorphisms in the CACNA1D gene associated with cocaine dependence in human subjects. Together, our findings reveal novel, overlapping mechanisms through which VTA Cav1.3 mediates cocaine-related, depressive-like and social phenotypes, suggesting that Cav1.3 may serve as a target for the treatment of neuropsychiatric symptoms.

  • splice variants of the ca v 1 3 l type calcium channel regulate dendritic spine morphology
    Scientific Reports, 2016
    Co-Authors: Ruslan Stanika, Jörg Striessnig, Alexandra Pinggera, Amy S. Lee, Marta Campiglio, Bernhard E Flucher, Gerald J Obermair
    Abstract:

    Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson's disease. The L-type calcium channel Cav1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length Cav1.3L and two C-terminally truncated splice variants (Cav1.342A and Cav1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged Cav1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in Cav1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with Cav1.3L and correlated with increased Cav1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of Cav1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of Cav1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

  • CaV1.2 and Cav1.3 channel hyperactivation in mouse islet β cells exposed to type 1 diabetic serum.
    Cellular and molecular life sciences : CMLS, 2014
    Co-Authors: Guang Yang, Andrea Welling, Franz Hofmann, Jörg Striessnig, Yue Shi, Lisa Juntti-berggren, Perolof Berggren
    Abstract:

    The voltage-gated Ca2+ (CaV) channel acts as a key player in β cell physiology and pathophysiology. β cell CaV channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca2+ concentration and thereby Ca2+-triggered β cell apoptosis. The present study was aimed at revealing the subtypes of CaV1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of β cell CaV1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic β cells from CaV1 channel knockout and corresponding control mice. We now show that functional Cav1.3 channels are expressed in a subgroup of islet β cells from CaV1.2 knockout mice (CaV1.2−/−). T1D serum enhanced whole-cell CaV currents in islet β cells from Cav1.3 knockout mice (Cav1.3−/−). T1D serum increased the open probability and number of functional unitary CaV1 channels in CaV1.2−/− and Cav1.3−/− β cells. These data demonstrate that T1D serum hyperactivates both CaV1.2 and Cav1.3 channels by increasing their conductivity and number. These findings suggest CaV1.2 and Cav1.3 channels as potential targets for anti-diabetes therapy.

  • C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca2+ Channels
    Biophysical journal, 2014
    Co-Authors: Andreas Lieb, Nadine J Ortner, Jörg Striessnig
    Abstract:

    Activity of voltage-gated Cav1.3 L-type Ca 2þ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Cav1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (QON-V) and of current activation (ICa-V) of the long (Cav1.3L) and a short Cav1.3 splice variant (Cav1.342A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (QON )o f Cav1.3L displayed a much steeper voltage-dependence and a more negative half- maximal activation voltage than Cav1.2 and Cav3.1. However, a significantly higher fraction of the total charge had to move for activation of Cav1.3 half-maximal conductance (Cav1.3: 68%; Cav1.2: 52%; Cav3.1: 22%). This indicated a weaker coupling of Cav1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Cav1.342A, thereby shifting ICa-V by 7.2 mV to potentials that were more negative without changing QON-V.W e independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of QON-V and a more negative activation of ICa-V of all three channels. These findings illustrate that the voltage sensors of Cav1.3 channels respond more sensitively to depolarization than those of Cav1.2 or Cav3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Cav1.342A splice variants to activate at lower voltages without affecting QON-V.

  • Gating Properties of Cav1.3 Calcium Channels: Insight from Alternative Splicing and Human Mutations
    Biophysical Journal, 2014
    Co-Authors: Andreas Lieb, Nadine J Ortner, Petronel Tuluc, Alexandra Pinggera, Elena A.b. Azizan, Morris J. Brown, Jörg Striessnig
    Abstract:

    Cav1.3 voltage-gated L-type calcium channels can activate at more negative membrane potentials than other high-voltage activated calcium channels. This allows them to contribute to specific physiological functions, such as cardiac pacemaking and hearing. Cav1.3 α1-subunits are regulated by a C-terminal modulatory domain (CTM) serving an auto-inhibitory function. Alternative splicing removes the CTM in C-terminally short variants (e.g. Cav1.342A) thereby stabilizing an even more negative activation voltage-range than in the long variant (Cav1.3L). It is unknown if the CTM affects gating of the voltage-sensor, its coupling to pore opening or both. We therefore investigated CTM effects on Cav1.3 voltage-sensor function (ON-gating charge). We compared Cav1.3 gating properties with low voltage-gated Cav3.1 T-type channels and with somatic Cav1.3 mutants recently discovered in human adrenal aldosterone-producing adenomas (APA).We expressed Cav3.1, Cav1.3 and Cav1.2 α1 subunits (the latter with α2-δ1 and β3 subunits) in tsA-201 cells. ON-gating charge (QON) and inward calcium currents (ICa) were measured using whole cell patch-clamp.Cav3.1 ICa activated about 25mV more negative than Cav1.3L despite a much lower overall voltage-sensitivity of Cav3.1 voltage-sensor movements (QON-V). Half-maximal QON-V of Cav1.3L was also more negative than of Cav1.2. Although a proportionally higher fraction of QON had to be moved to activate Cav1.3L ICa, Cav1.3L channel activated at lower voltages than Cav1.2. Removal of the Cav1.3L CTM lowered Cav1.342A ICa half-maximal activation-voltage without affecting QON-V. The CTM therefore must enhance ICa at lower potentials by facilitating pore-opening upon voltage-sensor movements. APA mutations V259D (IS4-S5), I750M (IIS6) and P1336R (IVS4-S5) induced about 15 mV negative shifts in activation-voltage and/or slowed inactivation. Gain of Cav1.3 channel function can therefore explain enhanced calcium-dependent aldosterone synthesis in these tumors.(Support: Austrian Science Fund F44020)

Geoffrey G. Murphy - One of the best experts on this subject based on the ideXlab platform.

  • the l type voltage gated calcium channel cav1 2 mediates fear extinction and modulates synaptic tone in the lateral amygdala
    Learning & Memory, 2017
    Co-Authors: Stephanie J Temme, Geoffrey G. Murphy
    Abstract:

    : L-type voltage-gated calcium channels (LVGCCs) have been implicated in both the formation and the reduction of fear through Pavlovian fear conditioning and extinction. Despite the implication of LVGCCs in fear learning and extinction, studies of the individual LVGCC subtypes, CaV1.2 and Cav1.3, using transgenic mice have failed to find a role of either subtype in fear extinction. This discontinuity between the pharmacological studies of LVGCCs and the studies investigating individual subtype contributions could be due to the limited neuronal deletion pattern of the CaV1.2 conditional knockout mice previously studied to excitatory neurons in the forebrain. To investigate the effects of deletion of CaV1.2 in all neuronal populations, we generated CaV1.2 conditional knockout mice using the synapsin1 promoter to drive Cre recombinase expression. Pan-neuronal deletion of CaV1.2 did not alter basal anxiety or fear learning. However, pan-neuronal deletion of CaV1.2 resulted in a significant deficit in extinction of contextual fear, implicating LVGCCs, specifically CaV1.2, in extinction learning. Further exploration on the effects of deletion of CaV1.2 on inhibitory and excitatory input onto the principle neurons of the lateral amygdala revealed a significant shift in inhibitory/excitatory balance. Together these data illustrate an important role of CaV1.2 in fear extinction and the synaptic regulation of activity within the amygdala.

  • Iron overload decreases Cav1.3-dependent L-type Ca2+ currents leading to bradycardia, altered electrical conduction, and atrial fibrillation.
    Circulation. Arrhythmia and electrophysiology, 2011
    Co-Authors: Robert A. Rose, Michael Sellan, Jeremy A. Simpson, Farzad Izaddoustdar, Carlo Cifelli, Brian K. Panama, Mark Davis, Dongling Zhao, Moniba Markhani, Geoffrey G. Murphy
    Abstract:

    Background— Chronic iron overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy characterized by contractile dysfunction and electrical disturbances, including slow heart rate (bradycardia) and heart block. Methods and Results— We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca2+ current (ICa,L) densities and positive (rightward) voltage shifts in ICa,L activation. Pacemaker current (If) was not affected by CIO. Because ICa,L in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of Cav1.3 channels (in addition to CaV1.2 channels), our data suggest that elevated iron preferentially suppresses Cav1.3 channel function. Consistent with this suggestion, CIO reduced Cav1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in Cav1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in Cav1.3 knockout mice. Conclusions— Our results demonstrate that CIO selectively reduces Cav1.3-mediated ICa,L, leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.

  • Deletion of the L-type Calcium Channel Cav1.3 but not CaV1.2 Results in a Diminished sAHP in Mouse CA1 Pyramidal Neurons
    Hippocampus, 2011
    Co-Authors: Amy E. Gamelli, Brandon C. Mckinney, Jessica A. White, Geoffrey G. Murphy
    Abstract:

    Trains of action potentials in CA1 pyramidal neurons are followed by a prolonged calcium-dependent postburst afterhyperpolari- zation (AHP) that serves to limit further firing to a sustained depolariz- ing input. A reduction in the AHP accompanies acquisition of several types of learning and increases in the AHP are correlated with age- related cognitive impairment. The AHP develops primarily as the result of activation of outward calcium-activated potassium currents; however, the precise source of calcium for activation of the AHP remains unclear. There is substantial experimental evidence suggesting that calcium influx via voltage-gated L-type calcium channels (L-VGCCs) contributes to the generation of the AHP. Two L-VGCC subtypes are predominately expressed in the hippocampus, CaV1.2 and Cav1.3; however, it is not known which L-VGCC subtype is involved in generation of the AHP. This ambiguity is due in large part to the fact that at present there are no subunit-specific agonists or antagonists. Therefore, using mice in which the gene encoding CaV1.2 or Cav1.3 was deleted, we sought to determine the impact of alterations in levels of these two L-VCGG sub- types on neuronal excitability. No differences in any AHP measure were seen between neurons from CaV1.2 knockout mice and controls. How- ever, the total area of the AHP was significantly smaller in neurons from Cav1.3 knockout mice as compared with neurons from wild-type controls. A significant reduction in the amplitude of the AHP was also seen at the 1 s time point in neurons from Cav1.3 knockout mice as compared with those from controls. Reductions in both the area and 1 s amplitude suggest the involvement of calcium influx via Cav1.3 in the slow AHP (sAHP). Thus, the results of our study demonstrate that dele- tion of Cav1.3, but not CaV1.2, significantly impacts the generation of the sAHP. V C 2009 Wiley-Liss, Inc.

  • The L-Type voltage-gated calcium channel Cav1.3 mediates consolidation, but not extinction, of contextually conditioned fear in mice.
    Learning & memory (Cold Spring Harbor N.Y.), 2006
    Co-Authors: Brandon C. Mckinney, Geoffrey G. Murphy
    Abstract:

    Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the specific LVGCC isoform involved. Both of the major LVGCC subtypes found in the brain (Cav1.2 and Cav1.3) are targets of the pharmacological manipulations used in earlier work. In this study, we used mice in which the gene for the pore-forming subunit (alpha1D) Cav1.3 was deleted (Cav1.3 knockout mice) to elucidate its contribution to consolidation and extinction of conditioned fear. We find that Cav1.3 knockout mice exhibit significant impairments in consolidation of contextual fear conditioning. However, once sufficiently overtrained, the Cav1.3 knockout mice exhibit rates of extinction that are identical to that observed in wild-type mice. We also find that Cav1.3 knockout mice perform as well as wild-type mice on the hidden platform version of the Morris water maze, suggesting that the consolidation deficit in conditioned fear observed in the Cav1.3 knockout mice is not likely the result of an inability to encode the context, but may reflect an inability to make the association between the context and the unconditioned stimulus.

Alexandra Koschak - One of the best experts on this subject based on the ideXlab platform.

  • C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells
    Biophysical Journal, 2014
    Co-Authors: Stephanie Eckrich, Amy S. Lee, Alexandra Koschak, Anja Scharinger, Kai Schönig, Dusan Bartsch, Martina J. Sinnegger-brauns, Stefan Muenkner, Dietmar Hecker, Bernhard Schick
    Abstract:

    The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)

  • A Novel Animal Model to Study the In Vivo Role of a C-Terminal Regulatory Domain in Cav1.3 L-Type Calcium Channels
    Biophysical Journal, 2013
    Co-Authors: Anja Scharinger, Amy S. Lee, Alexandra Koschak, Anupam Sah, Mathias Gebhart, Kai Schönig, Dusan Bartsch, Gurjot Kaur, Nicolas Singewald, Martina J. Sinnegger-brauns
    Abstract:

    Cav1.3 channels-mediated calcium-signals are crucial for hearing, cardiac pacemaking, and for shaping activity patterns in neurons and endocrine cells. We previously found that Cav1.3 activity is strongly modulated by alternative splicing of their pore-forming α1-subunit.α1-variants with a long C-terminus can form a C-terminal modulatory domain (CTM) that reduces open probability, slows inactivation and decreases sensitivity to activation voltage. These modulatory properties are absent in short splice variants, which results in different dynamics of calcium inward current. Long and short splice variants are expressed together in brain and other tissues. However, the (patho-)physiological role of this CTM is unknown.We therefore generated a mutant mouse strain in which CTM function is disrupted by an HA-tag in one of the putative a-helices (DCRD) forming the CTM. Homozygous mutants (Cav1.3-DCRD-HA/HA mice) are viable and reproduce normally. Heterozygous mice show no overt differences in locomotive activity. As predicted, HA-immunoreactivity in Western blots of mutant mouse brains was only associated with the long Cav1.3 splice variant (230 kDa), and the mutation did not interfere with its protein expression level. Anti-Cav1.3 alpha1-antibodies recognizing all C-terminal splice variants revealed also the presence of short variants (180 kDa). These may arise from alternative splicing and/or from C-terminal post-translational proteolytic processing as described for Cav1.1 and Cav1.2 channels. Proteolytic processing would generate an HA-tagged low molecular mass fragment in Cav1.3-DCRD-HA/HA tissues, a possibility which we currently evaluate. using these animals we will also study the physiological role of CTM function in vivo. Furthermore, the HA-tagged α1-subunit will present an excellent target for specific detection with anti-HA antibodies in mouse tissues.Support: Austrian Science Fund (SFB F4402), EC-project MRTN-CT-2006-35367 ("CavNet"), University of Innsbruck

  • channelopathies in cav1 1 Cav1.3 and cav1 4 voltage gated l type ca2 channels
    Pflügers Archiv: European Journal of Physiology, 2010
    Co-Authors: Jörg Striessnig, Hanno J Bolz, Alexandra Koschak
    Abstract:

    Voltage-gated Ca2+ channels couple membrane depolarization to Ca2+-dependent intracellular signaling events. This is achieved by mediating Ca2+ ion influx or by direct conformational coupling to intracellular Ca2+ release channels. The family of Cav1 channels, also termed L-type Ca2+ channels (LTCCs), is uniquely sensitive to organic Ca2+ channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming α1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Cav1.1 α1), incomplete congenital stationary night blindness (CSNB2; Cav1.4 α1), and Timothy syndrome (Cav1.2 α1; reviewed separately in this issue). Cav1.3 α1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Cav2.1 α1 in tottering mice. Ca2+ channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function.

  • Modulation of Voltage- and Ca2+-dependent Gating of Cav1.3 L-type Calcium Channels by Alternative Splicing of a C-terminal Regulatory Domain
    The Journal of biological chemistry, 2008
    Co-Authors: Anamika Singh, Jutta Engel, Jörg Striessnig, Mathias Gebhart, Chiara Poggiani, Martina J. Sinnegger-brauns, Reinhard Fritsch, Jean-charles Hoda, Christoph Romanin, Alexandra Koschak
    Abstract:

    Low voltage activation of Cav1.3 L-type Ca2+ channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Cav1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in CaV1.4 L-type Ca2+ channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Cav1.3 α1 subunit C terminus gives rise to a long (Cav1.342) and a short form (Cav1.342A), we investigated if a C-terminal modulatory mechanism also controls Cav1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with β3 and α2δ1 subunits in HEK-293 cells. Activation of calcium current through Cav1.342A channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Cav1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Cav1.3ΔC116 channels showed gating properties similar to Cav1.342A that were reverted by co-expression of the corresponding C-terminal peptide C116. Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Cav1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Cav1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Cav1.3 channel activation at lower voltages expected to favor Cav1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.

Anja Scharinger - One of the best experts on this subject based on the ideXlab platform.

  • C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells
    Biophysical Journal, 2014
    Co-Authors: Stephanie Eckrich, Amy S. Lee, Alexandra Koschak, Anja Scharinger, Kai Schönig, Dusan Bartsch, Martina J. Sinnegger-brauns, Stefan Muenkner, Dietmar Hecker, Bernhard Schick
    Abstract:

    The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)

  • Is There a Contribution of Both Cav1.4 and Cav1.3 L-Type Calcium Channels to Retinal Synaptic Transmission?
    Biophysical Journal, 2014
    Co-Authors: Verena Burtscher, Jörg Striessnig, Anja Scharinger, Gerald J Obermair, Dagmar Knoflach, Christof Kugler, Martin Glösmann, Georgios Blatsios, Andreas R. Janecke, Klaus Schicker
    Abstract:

    Cav1.3 and Cav1.4 L-type calcium channels were previously both shown to be expressed in the retina. Whereas Cav1.4 channels are predominantly expressed in the outer plexiform layer (OPL) at photoreceptor ribbon synapses, reports on the distribution pattern of Cav1.3 channels in the retina are controversial. One study reported the uniform expression across all retinal cell layers, and others showed accumulation in photoreceptor inner segments or the OPL or the inner nuclear layer or the ganglion cell (GC) layer of the retina. Mutations in the pore-forming α1-subunit, found in patients diagnosed with Congenital Stationary Night Blindness type 2 (CSNB2), result in impaired signaling between photoreceptor cells and second-order neurons. Exemplary, we report the functional consequences of the novel CaV1.4 mutation GV found in an Austrian family. Biophysical analysis of GV channels in whole-cell patch-clamp experiments revealed a reduced current density ([pA/pF]: wt: 12.8±1.4, n=18; GV: 3.7±1.0, n=7; p

  • A Novel Animal Model to Study the In Vivo Role of a C-Terminal Regulatory Domain in Cav1.3 L-Type Calcium Channels
    Biophysical Journal, 2013
    Co-Authors: Anja Scharinger, Amy S. Lee, Alexandra Koschak, Anupam Sah, Mathias Gebhart, Kai Schönig, Dusan Bartsch, Gurjot Kaur, Nicolas Singewald, Martina J. Sinnegger-brauns
    Abstract:

    Cav1.3 channels-mediated calcium-signals are crucial for hearing, cardiac pacemaking, and for shaping activity patterns in neurons and endocrine cells. We previously found that Cav1.3 activity is strongly modulated by alternative splicing of their pore-forming α1-subunit.α1-variants with a long C-terminus can form a C-terminal modulatory domain (CTM) that reduces open probability, slows inactivation and decreases sensitivity to activation voltage. These modulatory properties are absent in short splice variants, which results in different dynamics of calcium inward current. Long and short splice variants are expressed together in brain and other tissues. However, the (patho-)physiological role of this CTM is unknown.We therefore generated a mutant mouse strain in which CTM function is disrupted by an HA-tag in one of the putative a-helices (DCRD) forming the CTM. Homozygous mutants (Cav1.3-DCRD-HA/HA mice) are viable and reproduce normally. Heterozygous mice show no overt differences in locomotive activity. As predicted, HA-immunoreactivity in Western blots of mutant mouse brains was only associated with the long Cav1.3 splice variant (230 kDa), and the mutation did not interfere with its protein expression level. Anti-Cav1.3 alpha1-antibodies recognizing all C-terminal splice variants revealed also the presence of short variants (180 kDa). These may arise from alternative splicing and/or from C-terminal post-translational proteolytic processing as described for Cav1.1 and Cav1.2 channels. Proteolytic processing would generate an HA-tagged low molecular mass fragment in Cav1.3-DCRD-HA/HA tissues, a possibility which we currently evaluate. using these animals we will also study the physiological role of CTM function in vivo. Furthermore, the HA-tagged α1-subunit will present an excellent target for specific detection with anti-HA antibodies in mouse tissues.Support: Austrian Science Fund (SFB F4402), EC-project MRTN-CT-2006-35367 ("CavNet"), University of Innsbruck

  • Structural determinants of Cav1.3 L-type calcium channel gating
    Channels (Austin Tex.), 2012
    Co-Authors: Andreas Lieb, Anja Scharinger, Simone B Sartori, Martina J. Sinnegger-brauns, Jörg Striessnig
    Abstract:

    A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Cav1.3 L-type Ca2+ channels, in particular the voltage dependence of activation (V0.5) and Ca2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Cav1.3 (Cav1.3L), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCav1.3scg). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCav1.3scg with a rat cDNA isolated from rat pancreas (rCav1.3L). When expressed in tsA-201 cells under identical experimental conditions rCav1.3L exhibited Ca2+ current properties indistinguishable from human and mouse Cav1.3L, compatible with the presence of a functional CTM. In contrast, rCav1.3scg showed gating properties similar to human short splice variants lacking a CTM. rCav1.3scg differs from rCav1.3L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-te...

  • functional properties of a newly identified c terminal splice variant of Cav1.3 l type ca2 channels
    Journal of Biological Chemistry, 2011
    Co-Authors: Gabriella Bock, Mathias Gebhart, Anja Scharinger, Wanchana Jangsangthong, Perrine Busquet, Chiara Poggiani, Simone B Sartori, Matteo E Mangoni, Martina J Sinneggerbrauns, Stefan Herzig
    Abstract:

    An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Cav1.3 L-type Ca2+ channels (Cav1.3L) is a major determinant of their voltage- and Ca2+-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Cav1.342A channels that activate at a more negative voltage range and exhibit more pronounced Ca2+-dependent inactivation. Here we describe the discovery of a novel short splice variant (Cav1.343S) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Cav1.342A, still contains PCRD. When expressed together with α2δ1 and β3 subunits in tsA-201 cells, Cav1.343S also activated at more negative voltages like Cav1.342A but Ca2+-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Cav1.3L. The presence of the proximal C terminus in Cav1.343S channels preserved their modulation by distal C terminus-containing Cav1.3- and Cav1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca2+ influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Cav1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca2+ channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca2+ accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca2+-induced neurodegenerative processes.

Martina J. Sinnegger-brauns - One of the best experts on this subject based on the ideXlab platform.

  • C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells
    Biophysical Journal, 2014
    Co-Authors: Stephanie Eckrich, Amy S. Lee, Alexandra Koschak, Anja Scharinger, Kai Schönig, Dusan Bartsch, Martina J. Sinnegger-brauns, Stefan Muenkner, Dietmar Hecker, Bernhard Schick
    Abstract:

    The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)

  • A Novel Animal Model to Study the In Vivo Role of a C-Terminal Regulatory Domain in Cav1.3 L-Type Calcium Channels
    Biophysical Journal, 2013
    Co-Authors: Anja Scharinger, Amy S. Lee, Alexandra Koschak, Anupam Sah, Mathias Gebhart, Kai Schönig, Dusan Bartsch, Gurjot Kaur, Nicolas Singewald, Martina J. Sinnegger-brauns
    Abstract:

    Cav1.3 channels-mediated calcium-signals are crucial for hearing, cardiac pacemaking, and for shaping activity patterns in neurons and endocrine cells. We previously found that Cav1.3 activity is strongly modulated by alternative splicing of their pore-forming α1-subunit.α1-variants with a long C-terminus can form a C-terminal modulatory domain (CTM) that reduces open probability, slows inactivation and decreases sensitivity to activation voltage. These modulatory properties are absent in short splice variants, which results in different dynamics of calcium inward current. Long and short splice variants are expressed together in brain and other tissues. However, the (patho-)physiological role of this CTM is unknown.We therefore generated a mutant mouse strain in which CTM function is disrupted by an HA-tag in one of the putative a-helices (DCRD) forming the CTM. Homozygous mutants (Cav1.3-DCRD-HA/HA mice) are viable and reproduce normally. Heterozygous mice show no overt differences in locomotive activity. As predicted, HA-immunoreactivity in Western blots of mutant mouse brains was only associated with the long Cav1.3 splice variant (230 kDa), and the mutation did not interfere with its protein expression level. Anti-Cav1.3 alpha1-antibodies recognizing all C-terminal splice variants revealed also the presence of short variants (180 kDa). These may arise from alternative splicing and/or from C-terminal post-translational proteolytic processing as described for Cav1.1 and Cav1.2 channels. Proteolytic processing would generate an HA-tagged low molecular mass fragment in Cav1.3-DCRD-HA/HA tissues, a possibility which we currently evaluate. using these animals we will also study the physiological role of CTM function in vivo. Furthermore, the HA-tagged α1-subunit will present an excellent target for specific detection with anti-HA antibodies in mouse tissues.Support: Austrian Science Fund (SFB F4402), EC-project MRTN-CT-2006-35367 ("CavNet"), University of Innsbruck

  • Structural determinants of Cav1.3 L-type calcium channel gating
    Channels (Austin Tex.), 2012
    Co-Authors: Andreas Lieb, Anja Scharinger, Simone B Sartori, Martina J. Sinnegger-brauns, Jörg Striessnig
    Abstract:

    A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Cav1.3 L-type Ca2+ channels, in particular the voltage dependence of activation (V0.5) and Ca2+ dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Cav1.3 (Cav1.3L), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCav1.3scg). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCav1.3scg with a rat cDNA isolated from rat pancreas (rCav1.3L). When expressed in tsA-201 cells under identical experimental conditions rCav1.3L exhibited Ca2+ current properties indistinguishable from human and mouse Cav1.3L, compatible with the presence of a functional CTM. In contrast, rCav1.3scg showed gating properties similar to human short splice variants lacking a CTM. rCav1.3scg differs from rCav1.3L at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-te...

  • Modulation of Voltage- and Ca2+-dependent Gating of Cav1.3 L-type Calcium Channels by Alternative Splicing of a C-terminal Regulatory Domain
    The Journal of biological chemistry, 2008
    Co-Authors: Anamika Singh, Jutta Engel, Jörg Striessnig, Mathias Gebhart, Chiara Poggiani, Martina J. Sinnegger-brauns, Reinhard Fritsch, Jean-charles Hoda, Christoph Romanin, Alexandra Koschak
    Abstract:

    Low voltage activation of Cav1.3 L-type Ca2+ channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Cav1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in CaV1.4 L-type Ca2+ channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Cav1.3 α1 subunit C terminus gives rise to a long (Cav1.342) and a short form (Cav1.342A), we investigated if a C-terminal modulatory mechanism also controls Cav1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with β3 and α2δ1 subunits in HEK-293 cells. Activation of calcium current through Cav1.342A channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Cav1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Cav1.3ΔC116 channels showed gating properties similar to Cav1.342A that were reverted by co-expression of the corresponding C-terminal peptide C116. Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Cav1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Cav1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Cav1.3 channel activation at lower voltages expected to favor Cav1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.