Functional Clustering of L-Type CaV1.3 Channels

Abstract

CaV1.3 channels play a critical role in pace-making of sinoatrial node cells and in the repetitive firing of neurons. We investigated the function and spatial organization of CaV1.3 channels in tsA-201 cells and in human iPSC-derived cardiomyocytes (hiPSC-CM) using patch-clamp, TIRF and super resolution GSD microscopy. The activation of CaV1.3 channels in these cells produced local Ca2+ signals called “CaV1.3 sparklets”. Sparklet activity varied regionally with some regions of the surface membrane showing higher activity than others. The amplitude of the elementary CaV1.3 sparklet was about 40 nM. Sparklets with discrete amplitudes of 80, 120 and 160 nM were frequently observed, suggesting the simultaneous opening of 1-4 CaV1.3 channels. We used GSD super resolution imaging, with a spatial resolution of approximately 30 nm, to determine the organization of CaV1.3 channels. As shown in the super resolution map in Fig. 1, we found that CaV1.3 channels were expressed in clusters, of variable sizes and geometry, distributed at irregular intervals throughout the cell membrane. Our data suggest that CaV1.3 channels organize in clusters and that the simultaneous activation of channels within these clusters can produce high-amplitude Ca2+ signals. (NIH Grants HL085870, HL085686, NS077863)View Large Image | View Hi-Res Image | Download PowerPoint Slide