Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

Microtubules position ion channels close to each other to ensure autoregulation of vascular tone in cerebral arteries. Supporting ion channel coupling in smooth muscle Cerebral resistance arteries must autoregulate their vascular tone to maintain a constant supply of blood flow to the brain regardless of fluctuations in blood pressure. Vasoconstriction of arterial smooth muscle is initiated by Ca2+ influx through plasma membrane channels, which also triggers type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum to generate Ca2+ sparks. This process balances vasoconstriction by causing vasodilation, which results from the activation of BK channels in the plasma membrane by Ca2+ sparks. Pritchard et al. found that arching microtubule structures maintained the close opposition of the sarcoplasmic reticulum and the plasma membrane at regions called peripheral coupling sites. In rodent cerebral arteries, pharmacological disruption of microtubule networks caused the sarcoplasmic reticulum and plasma membrane at these sites to move farther apart, spread out the Ca2+ sparks over a greater area, decreased BK channel activity, and impaired the ability of cerebral arteries to autoregulate vascular tone. These effects were not seen upon disruption of the actin cytoskeleton. Thus, microtubules play a critical structural role in smooth muscle in ensuring the functional coupling of the activities of RyR2s in the sarcoplasmic reticulum and BK channels in the plasma membrane. Junctional membrane complexes facilitate excitation-contraction coupling in skeletal and cardiac muscle cells by forming subcellular invaginations that maintain close (≤20 nm) proximity of ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) with voltage-dependent Ca2+ channels in the plasma membrane. In fully differentiated smooth muscle cells, junctional membrane complexes occur as distributed sites of peripheral coupling. We investigated the role of the cytoskeleton in maintaining peripheral coupling and associated Ca2+ signaling networks within native smooth muscle cells of mouse and rat cerebral arteries. Using live-cell confocal and superresolution microscopy, we found that the tight interactions between the SR and the plasma membrane in these cells relied on arching microtubule structures present at the periphery of smooth muscle cells and were independent of the actin cytoskeleton. Loss of peripheral coupling associated with microtubule depolymerization altered the spatiotemporal properties of localized Ca2+ sparks generated by the release of Ca2+ through type 2 RyRs (RyR2s) on the SR and decreased the number of sites of colocalization between RyR2s and large-conductance Ca2+-activated K+ (BK) channels. The reduced BK channel activity associated with the loss of SR–plasma membrane interactions was accompanied by increased pressure–induced constriction of cerebral resistance arteries. We conclude that microtubule structures maintain peripheral coupling in contractile smooth muscle cells, which is crucial for the regulation of contractility and cerebral vascular tone.