Nanoscale organization of CaV1.3 L-type Ca2+ channel clusters in electrically excitable cells
Nanoscale cluster organization of L-type calcium channels
by Niko Schwenzer
Date of Examination:2024-04-15
Date of issue:2024-06-18
Advisor:Dr. Christian Vogl
Referee:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Silvio O. Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-10518
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Abstract
English
A key player of excitable cells in the heart and brain is the L-type calcium channel CaV1.3, which translates electrical signals into a cellular response. In the heart, CaV1.3 holds a central role in pacemaking, signal conduction, local calcium handling, and atrial excitation-contraction coupling, and therefore presents an attractive therapeutic target. In neurons, CaV1.3 is a crucial regulator controlling postsynaptic signal transmission. Despite the longstanding investigation of its electrophysiology and regulatory interactions, the molecular mechanisms behind CaV1.3 channel clustering in the plasma membrane have remained elusive. However, the nanoscale arrangement and dynamics of clustered channels likely contribute to the functional modulation of channel activity and subcellular calcium signaling. To comprehensively characterize CaV1.3 channel clustering, novel strategies for nanoscale imaging of CaV1.3 channels and clusters across spatiotemporal scales were developed in this thesis. For this purpose, live-cell Stimulated Emission Depletion (STED) nanoscopy of fluorescently Halo-tagged CaV1.3 channels was established in HEK293 cells and atrial cardiomyocytes, derived from human induced pluripotent stem cells. This unique approach enabled the quantification of CaV1.3 cluster size and molecular channel density in living cells for the first time, revealing a significantly lower channel density than previously expected when assuming densely packed channel clusters. This imaging strategy further evidenced the localization of CaV1.3 clusters in cell-surface endosomal pools and was readily transferable to a cell-derived system of pore-spanning plasma membrane bilayers, thereby opening new experimental avenues. In atrial cardiomyocytes, CaV1.3 channel clusters were immobilized at defined membrane sites, which were identified as calcium release units containing Junctophilin-2 and Ryanodine receptor type 2. Further, a novel experimental approach to image CaV1.3 channel clusters was demonstrated: DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) achieved molecular resolution fluorescence imaging of a voltage-gated calcium channel for the first time and evidenced a relatively large and irregular spacing of individual CaV1.3 channels within clusters. Importantly, cluster dimensions and channel numbers reflected those measured by live-cell STED nanoscopy. Lastly, single particle tracking of individual channels confirmed the dynamic nature of channels in the clustered state, coherent with an emerging nanodomain model, which may facilitate functional channel regulation by various interaction partners. Taken together, these findings provide a foundation for future studies correlating the nanoscale structure of LTCC clusters with distinct functional states that meet cell type-specific signaling demands. Importantly, the novel imaging strategies that were established are suitable for a wide range of ion channels and cell types.
Keywords: CaV1.3; calcium channel clusters; cluster size; atrial cardiomyocytes; hipsc-cm; super-resolution; STED; LTCC clustering; DNA-PAINT; single particle tracking; thesis
