Caveolae Localize Protein Kinase A Signaling to Arterial ATP-Sensitive Potassium Channels

Abstract

Abstract-Arterial ATP-sensitive K ϩ (K ATP ) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of K ATP channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that K ATP channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the K ATP pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of K ATP channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-␤-cyclodextrin. This reduces the cAMP-dependent protein kinase A-sensitive component of whole-cell K ATP current, indicating that the integrity of caveolae is important for adenylyl cyclase-mediated channel modulation. These results suggest that to be susceptible to protein kinase A-dependent activation, arterial K ATP channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate K ATP channel activity.

Protein Kinase C-ε Induces Caveolin-Dependent Internalization of Vascular Adenosine 5′-Triphosphate–Sensitive K + Channels

“…15,37 However, the mechanism by which PKC inhibits K ATP channels is unknown. Both PKC-and Kir6.1 are localized in the caveolae of arterial smooth muscles, 16,17 which are specialized microdomains in the plasma membrane and serve to compartmentalize and integrate numerous signaling events. 38 Caveolin-1 is a major integral membrane component of caveolar membranes in vascular smooth muscles.…”

“…16,17 We evaluated the effect of methyl-␤-cyclodextrin (M␤CD) on PMA-induced inhibition of K ATP currents in HEK293 cells transfected with Kir6.1/SUR2B. M␤CD is a drug that disrupts caveolae and lipid rafts by depleting the cell membrane of cholesterol.…”

“…14,15 Recent studies have revealed that Kir6.1 is mainly localized in the caveolae of aortic smooth muscle cells. 16 Angiotensin II, a potent physiological vasoconstrictor, was reported to recruit a novel PKC isoform, PKC-, to arterial smooth muscle caveolae, indicating a potential role for caveolae in the regulation of K ATP channel function. 17 However, the molecular mechanism by which PKC inhibits vascular K ATP channels remains unknown.…”

Caveolae as Organizers of Pharmacologically Relevant Signal Transduction Molecules

“…MβCD has similar functional effects in cardiac myocytes, but in these cells Kv1.5 is found in cholesterol-rich fractions that are not caveolae, implying cell-specific localization of such channels (74). In smooth muscle cells, a subunit of ATP-sensitive potassium channels (K ATP ), which are important for the regulation of vascular tone, forms a complex with adenylyl cyclase (AC) in caveolae, thereby allowing for sustained activation by protein kinase A (75). Although some channels (e.g., TRPC1) directly interact with caveolins, others, such as Kv1.5, lack caveolin-binding motifs, which suggests that other proteins mediate their localization to caveolae (33).…”

Cyclic AMP directs IP3-evoked Ca2+ signalling to different intracellular Ca2+ stores

“…After loading with a Ca 2+ indicator (and, where appropriate, ciIP 3 ), cells were depleted of intracellular cholesterol by incubation with bMCD (2% w/v, ,15 mM) for either 10 minutes at 37˚C or for up to 2 hours at 20˚C (Rodal et al, 1999;Sampson et al, 2004). After washing, cells were used for experiments.…”