Super-Resolution Imaging of Cav1.2 Channel Clusters

Abstract

L-type Cav1.2 channels regulate multiple processes in neurons, muscle and endocrine cells, including action potential duration, excitation-contraction coupling, gene expression, neurotransmitter and insulin release. The amplitude and functional impact of local Ca2+ signals (“Cav1.2 sparklets”) depends on the spatial distribution of Cav1.2 channels in the surface membrane. Here, we used TIRF microscopy in combination with STORM and GSD super-resolution imaging to determine the organization of Cav1.2 channels in the surface membrane of arterial myocytes and tsA-201 cells. The spatial resolution of our super-resolution maps was ∼30 nm. The localization of Cav1.2 was determined based on immunofluorescence or EGFP fluorescence. As shown in the super-resolution map in Fig. 1, we found that in arterial myocytes, Cav1.2 channels were expressed in clusters broadly throughout the cell membrane. However, the size and geometry of these clusters varied, suggesting that number of Cav1.2 channels within clusters was dissimilar. The average size of the clusters was ∼3 μm2. Up to 12 channels could occupy clusters of this size assuming a monomer occupies ∼240 nm2. Similar findings were obtained from tsA-201 cells expressing Cav1.2-EGFP or immuno-labeled Cav1.2 channels. Data will be presented on the functional implications of these findings.View Large Image | View Hi-Res Image | Download PowerPoint Slide