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

## TL;DR

This study uses advanced imaging to show that CaV1.3 calcium channels in heart cells form clusters with mobile channels in small membrane regions, challenging previous assumptions about their dense packing.

## Contribution

The study introduces a new model of L-type calcium channel clustering using super-resolution imaging in human stem cell-derived heart cells.

## Key findings

- CaV1.3 channels are mobile within confined membrane nanodomains, not densely packed.
- The C-terminal tail of CaV1.3 is sufficient for cluster formation.
- Molecular-scale imaging revealed irregular spacing between channels in clusters.

## Abstract

The clustering of L-type calcium channels (LTCC) 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.

Unlabelled Image

•New model of L-type channel clustering: mobile channels in membrane nanodomains.•Super-resolution CaV1.3 cluster imaging in stem cell-derived atrial cardiomyocytes•Live-STED cluster detection and molecule counting by brightness calibration•Single particle tracking of channel diffusion in confined and stationary nanodomains•Molecular scale resolution showed irregular channel distribution within clusters

New model of L-type channel clustering: mobile channels in membrane nanodomains.

Super-resolution CaV1.3 cluster imaging in stem cell-derived atrial cardiomyocytes

Live-STED cluster detection and molecule counting by brightness calibration

Single particle tracking of channel diffusion in confined and stationary nanodomains

Molecular scale resolution showed irregular channel distribution within clusters