//     FLUCHER GROUP

 

Keywords: voltage-gated calcium channels, Cav1.1 structure-function analysis, excitation-contraction (EC) coupling, neuro-muscular junction formation

//     AIM

Our scientific interests in Ca_V1.1 calcium channels range from molecular mechanisms of voltage-sensing and channel gating to its physiological role in skeletal muscle excitation-contraction coupling and the in regulating the development of the neuro-muscular junction. Our research efforts aim at advancing our basic understanding of normal Ca_V channel functions, and to identify mechanisms by which defects of calcium channel structure and function cause neurological diseases.

//     APPROACH

To this end we employ a broad range of biological model systems and experimental approaches: Site-directed mutagenesis is used to alter the protein structure, test molecular interactions, and to examine the functional consequences of disease variants. Wild-type and mutant channel constructs are expressed in cultured muscle cells lacking the endogenous Ca_V1.1, where they incorporate in the native skeletal muscle triad and restore muscle function. Whole-cell patch-clamp analysis and fluorescent calcium indicator techniques are applied to examine functionally important domains in the channel protein as well as effects of disease mutations on channel function and EC coupling. High-resolution immunofluorescence microscopy is applied to study the subcellular distribution, protein-protein interactions and the assembly of functional channel complexes. Protein-structure modeling (e.g. molecular dynamics simulation) is applied to characterize the effects of mutations on the channel structure, to predict functionally relevant structures, and to develop mechanistic models for channel function. Mouse models are applied to study the functions of Ca_V1.1 in skeletal muscle development and the formation of the neuro-muscular synapse.

//     CURRENT RESEARCH

Voltage-gated calcium channels are capable of sensing changes of the cell membrane potential and to respond by opening of the channel pore with specific sensitivities and gating properties. One current project in our Lab aims at understanding the structural correlates of the specific voltage-sensitivity and kinetics of channel gating. Particularly, we aim at understanding the molecular mechanisms that regulate voltage-sensor action and the specific contributions of the four voltage-sensors to channel gating and activation of EC coupling. As Ca_V1.1 serves dual functions as calcium channel and voltage-sensor for EC coupling, and because these two functions are activated at different voltages and with different kinetics, the current hypothesis is that distinct voltage-sensors regulate the different functions. Lately our structure-function analysis has been greatly enhanced by the application of molecular structure modeling which aids in the mechanistic interpretation of our experimental results and generates predictions to be tested experimentally.

The mechanism of skeletal muscle EC coupling has been a major research focus of the Lab over the past three decades. With the recent identification of STAC3 as a critical component of the EC coupling mechanism research in this field has received new momentum. Our latest most exciting result is finding out which one of the four voltage-sensing domains of Ca_V1.1 controls EC coupling.

Voltage-gated calcium channels are involved in numerous diseases, so called calcium channelopathies. Our expertise in studying structure-function relationships in Ca_V channels in muscle and nerve cells enables us to analyze the disease mechanisms of genetically caused Ca_V channelopathies. Lately this line of research led us on an excursion to the class of T-type calcium channels, for one of which we characterized novel disease mutations and linked it to neurological disease.

Before Ca_V1.1 takes up its function in skeletal muscle EC coupling it functions as regular calcium channel with various roles in the regulation of developmental processes. Previously we identified its role in specifying muscle fiber types. Currently we examine its involvement in the formation of the neuro-muscular synapse. Surprisingly, Ca_V1.1-triggered calcium signals are not only important for the organization of acetylcholine receptors in the postsynaptic membrane, but also determine the differentiation and targeting of the presynaptic nerve terminals. Now we search for the signaling mechanisms connecting postsynaptic calcium signals to presynaptic differentiation.

//     LAB MEMBERS

• Group leader: Bernhard E. Flucher
• Doctoral candidates:
  • Martin Heiss
  • Simone Pelizzari

//     ALUMNI

• • Yousra El Ghaleb
  • Mehmet Kaplan
  • Monica Fernández-Quintero
  • Pierre Coste de Bagneaux
  • Nasrin Sultana
  • Vincenzo Mastrolia
  • Bruno Benedetti
  • Ruslan Stanika
  • Solmaz Etemad
  • Benedikt Nimmervoll
  • Prakash Subramanyam
  • Marta Campiglio
  • Petronel Tuluc
  • Valentina Di Biase
  • Nicole Kasielke
  • Gerald Obermair
  • Ulli Gerster
  • Birgit Neuhuber

 

//     ADDRESS

Division of Physiology,
Department Physiology and Medical Physics
Medical University of Innsbruck
Schöpfstrasse 41
A-6020 Innsbruck, Austria

//     PUBLICATIONS

El Ghaleb, Y., and Flucher, B.E. (2023) Ca_V3.3 channelopathies. Handbook Exp Pharmacology, doi: 10.1007/164_2022_631.

Tuinte, W.E., Török, E., Mahlknecht, I., Tuluc, P., Flucher, B.E. and Campiglio, M. (2022) STAC3 determines the slow activation kinetics of Ca_V1.1 currents and inhibits its voltage-dependent inactivation. J. Cellular Physiol. doi.org/10.1002/jcp.30870

El Ghaleb, Y., Ortner, N.J., Posch, W., Fernández-Quintero, M., Tuinte, W.E., Monteleone, S., Draheim, H.J., Liedl, K.R., Wilfingseder, E., Striessnig, J., Tuluc, P., Flucher, B.E. and Campiglio, M., (2022) Calium current modulation by the ₁ subunit depends on alternative splicing of Ca_V1.1. J. Gen. Physiol. 154:1-17. doi: 10.1085/jgp.202113028
Preprint server – bioRxiv: doi: https://doi.org/10.1101/2021.11.10.468074

Kaplan, M.M. and Flucher, B.E. (2022) Counteractive and cooperative actions of muscle β -catenin and CaV1.1 during early neuromuscular synapse formation. iScience, 25:104025. doi: 10.1016/j.isci.2022.104025

El Ghaleb, Y., Fernández-Quintero, M., Monteleone, S., Tuluc, P., Campiglio, M., Liedl, K.R., and Flucher, B.E. (2021) Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate Ca_V1.1 channel gating.  Biophys. J., 120:4429-4441. doi: 10.1016/j.bpj.2021.09.004. PMID: 34506774

El Ghaleb, Y., Schneeberger, P.E., Fernández-Quintero, M.L., Geisler, S.M., Pelizzari, S., Polstra, A.M., van Hagen, J.M., Denecke, J., Campiglio, M., Liedl, K.L., Stevens, C.A., Person, R.E., Rentas, S., Marsh, E.D., Conlin, L.K., Tuluc, P., Kutsche, K., and Flucher, B.E. (2021) CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders. Brain, in press doi: 10.1093/brain/awab101. PMID: 33704440

Fernández-Quintero, M., El Ghaleb, Y., Tuluc, P., Campiglio, M., Liedl, K.R., and Flucher, B.E. (2021) Structural determinants of voltage-gating properties in calcium channels.  eLife, in press doi: 10.7554/eLife.64087. Online ahead of print. PMID: 33783354

Flucher, B.E. (2020) Skeletal muscle Ca_V1.1 channelopathies, Pflügers Archive – European J. Physiology, 472:739-754. doi: 10.1007/s00424-020-02368-3.

Costé de Bagneaux, P., Campiglio, M., von Elsner, L., Bierhals,, T., Campiglio, M., Johannsen, J., Obermair, G.J., Hempel, M., Flucher, B.E., Kutsche, K. (2020) Homozygous CACNB4 variant impairs b_4b channel and non-channel functions and causes a neurodevelopmental disorder. PLOS Genetics, 16(3):e1008625.

Kaplan, M.M. and Flucher, B.E. (2019) Postsynaptic Ca_V1.1-driven calcium signaling coordinates presynaptic differentiation at the developing neuromuscular junction. Sci. Rep. 9:18450.

El Ghaleb, Y., Campiglio, M., Flucher, B.E. (2019) Correcting the R165K substitution in the first voltage-sensor of Ca_V1.1 right-shifts the voltage-dependence of skeletal muscle calcium channel activation. Channels, 13:62-71.

Flucher, B.E. and Campiglio, M. (2018) STAC proteins: The missing link in skeletal muscle EC coupling and new regulators of calcium channel function. Biochim. Biophys. Acta Mol. Cell Res., 1866:1101-1110.

Costé de Bagneaux, P., Campiglio, M., Benedetti, B., Tuluc, P., Flucher, B.E. (2018) Role of putative voltage-sensor counter-charge D4 in regulating gating properties of Ca_V1.2 and Ca_V1.3 calcium channels. Channels, 12:249-261.

Campiglio, M., Kaplan, M.M., and Flucher, B.E. (2018) STAC3 incorporation into skeletal muscle triads occurs independently of the dihydropyridine receptor. J. Cell. Physiol., 233:9045-9051.

Kaplan, M.M., Sultana, N., Benedetti, A., Obermair, G.J., Linde, N.F., Papadopoulos, S., Dayal, A., Grabner, M., and Flucher, B.E. (2018) Calcium influx and release cooperatively regulate AChR patterning and motor axon outgrowth during neuromuscular junction formation. Cell Rep., 23:3891-3904.

Campiglio, M., Costé de Bagneaux, P., Ortner, N., Tuluc, P., Van Petegem, F., and Flucher, B.E. (2018) STAC proteins associate to the IQ domain of Ca_V1.2 and inhibit calcium-dependent inactivation Proc. Natl. Acad. Sci. USA, 115:1376-1381.

Mastrolia, V., Flucher, S.M., Obermair, G.J., Drach, M., Hofer, H., Renström, E., Schwartz, A., Striessnig, J., Flucher, B.E., and Tuluc, P. (2017) Loss of α₂δ-1 calcium channel subunit function increases the susceptibility for diabetes. Diabetes, 66:897-907.

Campiglio, M. and Flucher, B.E. (2017) STAC3 stably interacts through its C1 domain with Ca_V1.1 in skeletal muscle triads. Sci. Rep., 7:41003. doi: 10.1038/srep41003

Flucher, B.E. and Tuluc, P. (2017) How and why are calcium currents curtailed in the skeletal muscle voltage-gated calcium channels? J. Physiol., 595:1451-1463.

Tuluc, P., Benedetti, B., Coste de Bagneaux, P., Grabner, M., and Flucher, B.E. (2016) Two distinct voltage sensing domains control voltage-sensitivity and kinetics of current activation in Ca_V1.1 calcium channels. J. Gen. Physiology, 147:437-449.

Benedetti, B., Benedetti, A., and Flucher, B.E. (2016) Loss of the calcium channel β₄ subunit impairs parallel fiber volley and purkinje cell firing in cerebellum of adult ataxic mice. Eur.  J. Neurosci. 43:1486-1498.

Sultana, N., Dienes, B., Benedetti, A., Tuluc, P., Szentesi, P., Sztretye, M., Rainer, J., Hess, M.W., Schwarzer, C., Obermair, G.J., Csernoch, L., and Flucher, B.E. (2016) Restricting calcium currents is required for correct fiber type specification
in skeletal muscle. Development, 143:1547-1559.

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//     FUNDING

Funding list