Abstract
Objective- This study examined whether caveolae position CaV3.2 (T-type Ca2+ channel encoded by the α-3.2 subunit) sufficiently close to RyR (ryanodine receptors) for extracellular Ca2+ influx to trigger Ca2+ sparks and large-conductance Ca2+-activated K+ channel feedback. Approach and Results- Using smooth muscle cells from mouse mesenteric arteries, the proximity ligation assay confirmed that CaV3.2 reside within 40 nm of caveolin 1, a key caveolae protein. Methyl-β-cyclodextrin, a cholesterol depleting agent that disrupts caveolae, suppressed CaV3.2 activity along with large-conductance Ca2+-activated K+-mediated spontaneous transient outward currents in cells from C57BL/6 but not CaV3.2-/- mice. Genetic deletion of caveolin 1, a perturbation that prevents caveolae formation, also impaired spontaneous transient outward current production but did so without impairing Ca2+ channel activity, including CaV3.2. These observations indicate a mistargeting of CaV3.2 in caveolin 1-/- mice, a view supported by a loss of Ni2+-sensitive Ca2+ spark generation and colocalization signal (CaV3.2-RyR) from the proximity ligation assay. Vasomotor and membrane potential measurements confirmed that cellular disruption of the CaV3.2-RyR axis functionally impaired the ability of large-conductance Ca2+-activated K+ to set tone in pressurized caveolin 1-/- arteries. Conclusions- Caveolae play a critical role in protein targeting and preserving the close structural relationship between CaV3.2 and RyR needed to drive negative feedback control in resistance arteries.
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