Vascular smooth muscle cells (VSMCs) regulate blood flow through dynamic control of vessel diameter. This is achieved through a balance of vasoconstrictive and vasodilatory signalling pathways, where the latter is focussed upon in this research. Central to vasodilation is the sarcoplasmic reticulum (SR)–plasma membrane (PM) nanodomain, a specialised junction in which Ca²⁺ released from clusters of type-2 ryanodine receptors (RyR2) activates large-conductance Ca²⁺-activated K⁺ (BK) channels at the PM, driving membrane hyperpolarisation and promoting vasodilation. Previous modelling has estimated that SR-PM distances must be <20 nm for local [Ca²⁺] released from RyRs, known as a Ca²⁺ spark, to reach concentrations sufficient for BK channel activation, highlighting the critical importance of nanodomain geometry for functional coupling. We have recently demonstrated that BK channel dysfunction is associated with impaired cerebral blood flow in both Alzheimer’s disease and hypertension (Taylor et al., 2022; 2023), yet the nanoscale structural organisation of the SR-PM nanodomain in VSMCs remains poorly characterised, with the field largely relying on assumptions drawn from cardiac and other non-vascular cell types.
To address this, we employed volume electron microscopy (vEM) with 3D reconstruction, and the super-resolution microscopy technique of DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT), to characterise the SR-PM nanodomain ultrastructure in VSMCs, using both isolated cells and fixed cerebral arteries. vEM enabled direct visualisation of SR-PM proximity and precise junctional geometry within cerebral arteries from C57BL/6J mice, while DNA-PAINT utilised a GFP-tagged RyR mouse model (RyR2D4365-GFP), provided RyR2 cluster architecture and spatial organisation, all at nanometre resolution.
vEM analysis reveals that SR-PM junctions in VSMCs have a modal junctional distance of approximately 10 nm, well within the suggested distance required for efficient Ca²⁺ spark–BK channel coupling. The SR appears to adopt a linear arrangement running parallel to the cell, as well as isolated contact sites, suggesting two spatially orientated nanodomains that were previously unknown. DNA-PAINT further reveals that RyR2 organise into small discrete clusters of 1–8 RyRs in VSMCs, in stark contrast to the larger clusters and ‘super-clusters’ of up to and exceeding 100 RyRs identified within cardiac muscle, indicating a VSMC-specific organisation. These findings provide the first direct nanoscale characterisation of the SR-PM nanodomain in cerebral artery SMCs, establishing a structural framework for the vasodilatory signalling axis and offering new insight into how disruption of nanodomain architecture may contribute to cerebrovascular dysfunction in disease.