Nanoscale architecture and dynamics of CaV1.3 channel clusters in cardiac myocytes revealed by single channel nanoscopy

The clustering of L-type calcium channels in cardiac myocytes presents an important mechanism for functional regulation of calcium signaling. Here we applied targeted super-resolution imaging techniques for the study of atrial-specific Ca _V 1.3 channel clusters in human iPSC-derived atrial cardiomyocytes (hiPSC-aCM). We thereby clarified cluster localization, dimensions, architecture, and dynamics, which were largely unexplored previously. Live-cell STimulated Emission Depletion (STED) imaging identified that cell surface-localized clusters contained 9 channel molecules within 120 nm diameter on average. DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) optimized for molecular mapping revealed an irregular arrangement of channels with significant spacing. Single Particle Tracking (SPT) further evidenced that clustered channels do not associate into rigidly packed structures (oligomers or lattices), but rather co-diffuse in confined and stationary membrane nanodomains. Immunofluorescence showed consistent cell-surface colocalization with Ryanodine Receptor type 2 and Junctophilin-2 forming stable calcium release units, similar to dyadic junctions containing Ca _V 1.2 in ventricular cardiomyocytes. Lastly, novel genetic constructs for live-cell imaging showed that the cytosolic C-terminal tail of Ca _V 1.3 by itself is sufficient for cluster formation. In conclusion, a novel strategy for LTCC clustering studies in atrial cells was established, suitable for a wide range of super-resolution imaging techniques. Based on live-cell STED, DNA-PAINT and SPT data, we propose that Ca _V 1.3 channel clusters consist of mobile individual channels inside defined membrane nanodomains.