Publications by authors named "Marta Campiglio"

21 Publications

  • Page 1 of 1

Structural determinants of voltage-gating properties in calcium channels.

Elife 2021 Mar 30;10. Epub 2021 Mar 30.

Department of Physiology and Medical Physics, Medical University Innsbruck, Innsbruck, Austria.

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel Ca1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.
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http://dx.doi.org/10.7554/eLife.64087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099428PMC
March 2021

Presynaptic αδ subunits are key organizers of glutamatergic synapses.

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Institute of Physiology, Medical University of Innsbruck, A-6020 Innsbruck, Austria;

In nerve cells the genes encoding for αδ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that αδ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular αδ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of αδ isoforms as synaptic organizers is highly redundant, as each individual αδ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, αδ-2 and αδ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of αδ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on αδ implicates αδ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that αδ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.
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http://dx.doi.org/10.1073/pnas.1920827118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040823PMC
April 2021

CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders.

Brain 2021 Mar 11. Epub 2021 Mar 11.

Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria.

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analyzed possible effects on CaV3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with CaV3.3-I860N showing the strongest and CaV3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in CaV3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the CaV3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the CaV3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the CaV3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type CaV3.3 to slow oscillations with CaV3.3-I860N and an intermediate firing mode with CaV3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.
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http://dx.doi.org/10.1093/brain/awab101DOI Listing
March 2021

Multiple Sequence Variants in STAC3 Affect Interactions with Ca1.1 and Excitation-Contraction Coupling.

Structure 2020 08 2;28(8):922-932.e5. Epub 2020 Jun 2.

Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

STAC3 is a soluble protein essential for skeletal muscle excitation-contraction (EC) coupling. Through its tandem SH3 domains, it interacts with the cytosolic II-III loop of the skeletal muscle voltage-gated calcium channel. STAC3 is the target for a mutation (W284S) that causes Native American myopathy, but multiple other sequence variants have been reported. Here, we report a crystal structure of the human STAC3 tandem SH3 domains. We analyzed the effect of five disease-associated variants, spread over both SH3 domains, on their ability to bind to the Ca1.1 II-III loop and on muscle EC coupling. In addition to W284S, we find the F295L and K329N variants to affect both binding and EC coupling. The ability of the K329N variant, located in the second SH3 domain, to affect the interaction highlights the importance of both SH3 domains in association with Ca1.1. Our results suggest that multiple STAC3 variants may cause myopathy.
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http://dx.doi.org/10.1016/j.str.2020.05.005DOI Listing
August 2020

A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions.

PLoS Genet 2020 03 16;16(3):e1008625. Epub 2020 Mar 16.

Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b.
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http://dx.doi.org/10.1371/journal.pgen.1008625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176149PMC
March 2020

Presynaptic αδ-2 Calcium Channel Subunits Regulate Postsynaptic GABA Receptor Abundance and Axonal Wiring.

J Neurosci 2019 04 25;39(14):2581-2605. Epub 2019 Jan 25.

Division of Physiology, Medical University Innsbruck, 6020 Innsbruck, Austria, and

Presynaptic αδ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual αδ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of αδ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABARs. Strikingly, presynaptic αδ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of αδ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with αδ-2 can modulate postsynaptic GABAR abundance. Finally, exclusion of the alternatively spliced exon 23 of αδ-2 is essential for the trans-synaptic mechanism. The novel function of αδ-2 identified here may explain how abnormal αδ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders. Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. αδ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual αδ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single αδ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.
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http://dx.doi.org/10.1523/JNEUROSCI.2234-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445987PMC
April 2019

Correcting the R165K substitution in the first voltage-sensor of Ca1.1 right-shifts the voltage-dependence of skeletal muscle calcium channel activation.

Channels (Austin) 2019 12;13(1):62-71

a Department of Physiology and Medical Physics , Medical University Innsbruck , Innsbruck , Austria.

The voltage-gated calcium channel Ca1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant Ca1.1e, which lacks exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a Ca1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both Ca1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of Ca1.1 channel gating.
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http://dx.doi.org/10.1080/19336950.2019.1568825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380215PMC
December 2019

STAC proteins: The missing link in skeletal muscle EC coupling and new regulators of calcium channel function.

Biochim Biophys Acta Mol Cell Res 2019 07 10;1866(7):1101-1110. Epub 2018 Dec 10.

Department of Physiology and Medical Physics, Medical University Innsbruck, Schöpfstraße 41, A6020 Innsbruck, Austria.

Excitation-contraction coupling is the signaling process by which action potentials control calcium release and consequently the force of muscle contraction. Until recently, three triad proteins were known to be essential for skeletal muscle EC coupling: the voltage-gated calcium channel Ca1.1 acting as voltage sensor, the SR calcium release channel RyR1 representing the only relevant calcium source, and the auxiliary Ca β subunit. Whether Ca1.1 and RyR1 are directly coupled or whether their interaction is mediated by another triad protein is still unknown. The recent identification of the adaptor protein STAC3 as fourth essential component of skeletal muscle EC coupling prompted vigorous research to reveal its role in this signaling process. Accumulating evidence supports its possible involvement in linking Ca1.1 and RyR1 in skeletal muscle EC coupling, but also indicates a second, much broader role of STAC proteins in the regulation of calcium/calmodulin-dependent feedback regulation of L-type calcium channels.
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http://dx.doi.org/10.1016/j.bbamcr.2018.12.004DOI Listing
July 2019

STAC3 incorporation into skeletal muscle triads occurs independent of the dihydropyridine receptor.

J Cell Physiol 2018 12 2;233(12):9045-9051. Epub 2018 Aug 2.

Department of Physiology, Medical University, Innsbruck, Innsbruck, Austria.

Excitation-contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine-rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.
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http://dx.doi.org/10.1002/jcp.26767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334165PMC
December 2018

Role of putative voltage-sensor countercharge D4 in regulating gating properties of Ca1.2 and Ca1.3 calcium channels.

Channels (Austin) 2018 ;12(1):249-261

a Department of Physiology and Medical Physics , Medical University of Innsbruck , Innsbruck , Austria.

Voltage-dependent calcium channels (Ca) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of Ca1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of Ca1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of Ca1.3 and in VSDs II and IV of Ca1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of Ca1.3 and in two splice variants of Ca1.2. In both channels the D4N (VSD IV) mutation resulted in a  ̴5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in Ca1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined Ca1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle Ca1.1 channel.
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http://dx.doi.org/10.1080/19336950.2018.1482183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161609PMC
August 2019

STAC proteins associate to the IQ domain of Ca1.2 and inhibit calcium-dependent inactivation.

Proc Natl Acad Sci U S A 2018 02 23;115(6):1376-1381. Epub 2018 Jan 23.

Division of Physiology, Medical University Innsbruck, 6020 Innsbruck, Austria;

The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (Ca) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with Ca1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in Ca1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal Ca1 channels remained unknown. Using Ca1.2/2.1 chimeras expressed in dysgenic (Ca1.1) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of Ca1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of Ca1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with Ca1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the Ca1.2 IQ domain with that of Ca2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the Ca1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of Ca1.2 currents.
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http://dx.doi.org/10.1073/pnas.1715997115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819422PMC
February 2018

Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca1.2 calcium channels in hippocampal neurons.

J Biol Chem 2018 01 27;293(3):1040-1053. Epub 2017 Nov 27.

From the Institute of Biophysics, Medical University of Graz, 8010 Graz, Austria,

L-type voltage-gated Ca1.2 calcium channels (Ca1.2) are key regulators of neuronal excitability, synaptic plasticity, and excitation-transcription coupling. Surface-exposed Ca1.2 distributes in clusters along the dendrites of hippocampal neurons. A permanent exchange between stably clustered and laterally diffusive extra-clustered channels maintains steady-state levels of Ca1.2 at dendritic signaling domains. A dynamic equilibrium between anchored and diffusive receptors is a common feature among ion channels and is crucial to modulate signaling transduction. Despite the importance of this fine regulatory system, the molecular mechanisms underlying the surface dynamics of Ca1.2 are completely unexplored. Here, we examined the dynamic states of Ca1.2 depending on phosphorylation on Ser-1700 and Ser-1928 at the channel C terminus. Phosphorylation at these sites is strongly involved in Ca1.2-mediated nuclear factor of activated T cells (NFAT) signaling, long-term potentiation, and responsiveness to adrenergic stimulation. We engineered Ca1.2 constructs mimicking phosphorylation at Ser-1700 and Ser-1928 and analyzed their behavior at the membrane by immunolabeling protocols, fluorescence recovery after photobleaching, and single particle tracking. We found that the phosphomimetic S1928E variant increases the mobility of Ca1.2 without altering the steady-state maintenance of cluster in young neurons and favors channel stabilization later in differentiation. Instead, mimicking phosphorylation at Ser-1700 promoted the diffusive state of Ca1.2 irrespective of the differentiation stage. Together, these results reveal that phosphorylation could contribute to the establishment of channel anchoring mechanisms depending on the neuronal differentiation state. Finally, our findings suggest a novel mechanism by which phosphorylation at the C terminus regulates calcium signaling by tuning the content of Ca1.2 at signaling complexes.
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http://dx.doi.org/10.1074/jbc.M117.799585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777246PMC
January 2018

Structural insights into binding of STAC proteins to voltage-gated calcium channels.

Proc Natl Acad Sci U S A 2017 11 23;114(45):E9520-E9528. Epub 2017 Oct 23.

Department of Biochemistry and Molecular Biology, University of British Columbia, V6T 1Z3 Vancouver, BC, Canada;

Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (Ca1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different Ca isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of Ca1.2.
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http://dx.doi.org/10.1073/pnas.1708852114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5692558PMC
November 2017

Stapled Voltage-Gated Calcium Channel (Ca) α-Interaction Domain (AID) Peptides Act As Selective Protein-Protein Interaction Inhibitors of Ca Function.

ACS Chem Neurosci 2017 06 17;8(6):1313-1326. Epub 2017 Mar 17.

Molecular Biophysics & Integrated Imaging Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein-protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein-protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop meta-xylyl (m-xylyl) stapled peptides that target a prototypic VGIC high affinity protein-protein interaction, the interaction between the voltage-gated calcium channel (Ca) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Caβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Caβ interactions and reduce the entropic penalty associated with AID binding to Caβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Caα:Caβ interaction that modulate Ca function in an Caβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein-protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca modulator design.
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http://dx.doi.org/10.1021/acschemneuro.6b00454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481814PMC
June 2017

STAC3 stably interacts through its C1 domain with Ca1.1 in skeletal muscle triads.

Sci Rep 2017 01 23;7:41003. Epub 2017 Jan 23.

Department of Physiology and Medical Physics, Medical University of Innsbruck, 6020 Innsbruck, Austria.

The adaptor protein STAC3 is essential for skeletal muscle excitation-contraction (EC) coupling and a mutation in the STAC3 gene has been linked to a severe muscle disease, Native American myopathy (NAM). However the function of STAC3, its interaction partner, and the mode of interaction within the EC-coupling complex remained elusive. Here we demonstrate that STAC3 forms a stable interaction with the voltage-sensor of EC-coupling, Ca1.1, and that this interaction depends on a hitherto unidentified protein-protein binding pocket in the C1 domain of STAC3. While the NAM mutation does not affect the stability of the STAC3-Ca1.1 interaction, mutation of two crucial residues in the C1 binding pocket increases the turnover of STAC3 in skeletal muscle triads. Thus, the C1 domain of STAC3 is responsible for its stable incorporation into the Ca1.1 complex, whereas the SH3 domain containing the NAM mutation site may be involved in low-affinity functional interactions in EC-coupling.
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http://dx.doi.org/10.1038/srep41003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5253670PMC
January 2017

Splice variants of the Ca1.3 L-type calcium channel regulate dendritic spine morphology.

Sci Rep 2016 10 6;6:34528. Epub 2016 Oct 6.

Division of Physiology, Medical University Innsbruck, 6020 Innsbruck, Austria.

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 Ca1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length Ca1.3 and two C-terminally truncated splice variants (Ca1.3 and Ca1.3) 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 Ca1.3 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 Ca1.3 induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with Ca1.3 and correlated with increased Ca1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of Ca1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of Ca1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.
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http://dx.doi.org/10.1038/srep34528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052568PMC
October 2016

A Ca3.2/Stac1 molecular complex controls T-type channel expression at the plasma membrane.

Channels (Austin) 2016 Sep 5;10(5):346-354. Epub 2016 May 5.

a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic , Prague , Czech Republic.

Low-voltage-activated T-type calcium channels are essential contributors to neuronal physiology where they play complex yet fundamentally important roles in shaping intrinsic excitability of nerve cells and neurotransmission. Aberrant neuronal excitability caused by alteration of T-type channel expression has been linked to a number of neuronal disorders including epilepsy, sleep disturbance, autism, and painful chronic neuropathy. Hence, there is increased interest in identifying the cellular mechanisms and actors that underlie the trafficking of T-type channels in normal and pathological conditions. In the present study, we assessed the ability of Stac adaptor proteins to associate with and modulate surface expression of T-type channels. We report the existence of a Ca3.2/Stac1 molecular complex that relies on the binding of Stac1 to the amino-terminal region of the channel. This interaction potently modulates expression of the channel protein at the cell surface resulting in an increased T-type conductance. Altogether, our data establish Stac1 as an important modulator of T-type channel expression and provide new insights into the molecular mechanisms underlying the trafficking of T-type channels to the plasma membrane.
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http://dx.doi.org/10.1080/19336950.2016.1186318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988463PMC
September 2016

The role of auxiliary subunits for the functional diversity of voltage-gated calcium channels.

J Cell Physiol 2015 Sep;230(9):2019-31

Division of Physiology, Department of Physiology and Medical Physics, Medical University Innsbruck, Innsbruck, Austria.

Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α(1) subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein-protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein-protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity.
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http://dx.doi.org/10.1002/jcp.24998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672716PMC
September 2015

The juvenile myoclonic epilepsy mutant of the calcium channel β(4) subunit displays normal nuclear targeting in nerve and muscle cells.

Channels (Austin) 2014 ;8(4):334-43

Voltage-gated calcium channels regulate gene expression by controlling calcium entry through the plasma membrane and by direct interactions of channel fragments and auxiliary β subunits with promoters and the epigenetic machinery in the nucleus. Mutations of the calcium channel β(4) subunit gene (CACNB4) cause juvenile myoclonic epilepsy in humans and ataxia and epileptic seizures in mice. Recently a model has been proposed according to which failed nuclear translocation of the truncated β(4) subunit R482X mutation resulted in altered transcriptional regulation and consequently in neurological disease. Here we examined the nuclear targeting properties of the truncated β(4b(1–481)) subunit in tsA-201 cells, skeletal myotubes, and in hippocampal neurons. Contrary to expectation, nuclear targeting of β(4b(1–481)) was not reduced compared with full-length β(4b) in any one of the three cell systems. These findings oppose an essential role of the β(4) distal C-terminus in nuclear targeting and challenge the idea that the nuclear function of calcium channel β(4) subunits is critically involved in the etiology of epilepsy and ataxia in patients and mouse models with mutations in the CACNB4 gene.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203735PMC
http://dx.doi.org/10.4161/chan.29322DOI Listing
June 2015

Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex.

J Cell Sci 2013 May 27;126(Pt 9):2092-101. Epub 2013 Feb 27.

Department of Physiology and Medical Physics, Medical University Innsbruck, A-6020 Innsbruck, Austria.

Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary β subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether β subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and β subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of β relative to the α1 subunit, altered the specific dynamic properties of the β subunits. In contrast, a single residue substitution in the α interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the β subunit isoforms.
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http://dx.doi.org/10.1242/jcs.jcs124537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148589PMC
May 2013

Surface traffic of dendritic CaV1.2 calcium channels in hippocampal neurons.

J Neurosci 2011 Sep;31(38):13682-94

Department of Physiology and Medical Physics, Medical University of Innsbruck, 6020 Innsbruck, Austria and Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany.

In neurons L-type calcium currents function in gene regulation and synaptic plasticity, while excessive calcium influx leads to excitotoxicity and neurodegeneration. The major neuronal Ca(V)1.2 L-type channels are localized in clusters in dendritic shafts and spines. Whereas Ca(V)1.2 clusters remain stable during NMDA-induced synaptic depression, L-type calcium currents are rapidly downregulated during strong excitatory stimulation. Here we used fluorescence recovery after photobleaching (FRAP), live cell-labeling protocols, and single particle tracking (SPT) to analyze the turnover and surface traffic of Ca(V)1.2 in dendrites of mature cultured mouse and rat hippocampal neurons, respectively. FRAP analysis of channels extracellularly tagged with superecliptic pHluorin (Ca(V)1.2-SEP) demonstrated ∼20% recovery within 2 min without reappearance of clusters. Pulse-chase labeling showed that membrane-expressed Ca(V)1.2-HA is not internalized within1 h, while blocking dynamin-dependent endocytosis resulted in increased cluster density after 30 min. Together, these results suggest a turnover rate of clustered Ca(V)1.2s on the hour time scale. Direct recording of the lateral movement in the membrane using SPT demonstrated that dendritic Ca(V)1.2s show highly confined mobility with diffusion coefficients of ∼0.005 μm² s⁻¹. Consistent with the mobile Ca(V)1.2 fraction observed in FRAP, a ∼30% subpopulation of channels reversibly exchanged between confined and diffusive states. Remarkably, high potassium depolarization did not alter the recovery rates in FRAP or the diffusion coefficients in SPT analyses. Thus, an equilibrium of clustered and dynamic Ca(V)1.2s maintains stable calcium channel complexes involved in activity-dependent cell signaling, whereas the minor mobile channel pool in mature neurons allows limited capacity for short-term adaptations.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325119PMC
http://dx.doi.org/10.1523/JNEUROSCI.2300-11.2011DOI Listing
September 2011