DOI: 10.1113/jp291474 ISSN: 0022-3751

Ca V 1.2 dynamics in native male arterial myocytes

Miguel Martin‐Aragon Baudel, Hannah Voorhees, Arsalan U. Syed, Maryann K. Ferrara, Mary Horne, Johannes W. Hell, Luis F. Santana, Rose E. Dixon, Madeline Nieves‐Cintron, Manuel F. Navedo
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Abstract

L‐type Ca V 1.2 channels are key regulators of cellular function in diverse cell types, controlling processes that range from gene expression to muscle contraction. In arterial myocytes, Ca V 1.2 channels traffic to the plasma membrane and assemble into clusters that control Ca 2+ influx. Changes in the size and number of these Ca V 1.2 clusters have been linked to altered channel and vascular function in diabetic hyperglycaemia, angiotensin II (Ang II) signalling and hypertension. Yet, the dynamics governing the distribution of vascular Ca V 1.2 at the plasma membrane remain poorly understood. Here, we found that microtubule‐mediated transport actively shapes Ca V 1.2 distribution under hyperglycaemia and Ang II signalling. Employing high‐resolution imaging, we tracked live Ca V 1.2 trafficking in male arterial myocytes using a dual approach: unpassaged cells expressing vascular Ca V 1.2‐ and freshly isolated cells from a Ca V 1.2–mClover2 knock‐in mouse, which enabled endogenous channel visualization in native tissue. By recreating diabetic hyperglycaemia (20 mM

d
‐glucose) and enhancing Ang II signalling in vitro , we uncovered distinct Ca V 1.2 trafficking patterns in both cultured and freshly isolated arterial myocytes that were dependent on an intact microtubular network. Importantly, microtubule disruption abolished Ca V 1.2 dynamics and prevented Ang II‐induced calcium elevations in both isolated myocytes and intact arteries. These findings identify microtubule‐dependent Ca V 1.2 trafficking as a key mechanism by which hyperglycaemia and Ang II signalling may drive channel reorganization and calcium dysregulation. The results have implications for vascular dysfunction in diabetes and hypertension, and open new potential therapeutic avenues targeting the cellular transport machinery. Moreover, the Ca V 1.2–mClover2 mouse provides a useful tool for investigating native channel behaviour in living tissues across physiological and pathological contexts. image

Key points

Live‐cell total internal reflection fluorescence and spinning‐disk confocal imaging revealed that vascular Ca V 1.2 channels exhibit distinct perimembrane behaviours in male arterial myocytes, including kiss‐and‐stay, kiss‐and‐run, and homotypic fusion and fission events.

Microtubules are essential for vascular Ca V 1.2 structural dynamics, with nocodazole‐induced microtubule disruption significantly reducing channel mobility, speed and displacement.

Hyperglycaemic or angiotensin II‐stimulated environments rapidly increased vascular Ca V 1.2 clustering at the plasma membrane, and microtubule disruption abolished these pathological elevations in clustering and intracellular Ca 2+ in both isolated myocytes and intact arteries.

A CRISPR/Cas9‐generated Ca V 1.2–mClover2 knock‐in mouse enabled endogenous channel visualization in native tissue, providing a transformative tool for investigating Ca V 1.2 behaviour in physiological and pathological contexts.

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