Ca_V1.3 channel clusters characterized by live-cell and isolated plasma membrane nanoscopy

A key player of excitable cells in the heart and brain is the L-type calcium channel Ca_V1.3. In the heart, it is required for voltage-dependent Ca²⁺-signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of Ca_V1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify Ca_V1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify Ca_V1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. Ca_V1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism.