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

The clustering of L-type calcium channels for functional regulation of intracellular calcium signaling remains poorly understood. Here we applied super-resolution imaging to study CaV1.3 channel clusters in human iPSC-derived atrial cardiomyocytes (hiPSC-aCM) to analyze subcellular localization, dimensions, architecture, and dynamics, which were largely unexplored previously. STimulated Emission Depletion (STED) imaging characterized the localization and structure of CaV1.3 channel clusters in living cardiomyocytes. DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) achieved true molecular resolution, revealing an irregular channel distribution with substantial spacing. Single Particle Tracking (SPT) showed that channels co-diffuse in confined and stationary membrane nanodomains. The cytosolic C-terminal tail of CaV1.3 by itself was found sufficient for cluster formation. In conclusion, our LTCC clustering studies demonstrate that CaV1.3 channel clusters consist of mobile individual channels inside defined membrane nanodomains, in contrast to previous models of dense channel packing.