Caveolae are 50–100 nm large membrane invaginations found in many cell types. A considerable body of information has accumulated pointing towards an essential functional role of these structures in smooth muscle from experimental animals, but comparative data in humans is scarce. Here we aimed to assess whether M3 and Kir6.1 are associated with human detrusor caveolae and to probe the functional relevance of this organization.

Smooth muscle strips were dissected from human detrusors and used in ultrastructural, biochemical and mechanical studies. Caveolae were disrupted by cholesterol desorption using methyl-b-cyclodextrin (mbcd). Quantitative RT-PCR (RT-qPCR) was used to assess subunit composition of human detrusor KATP channels. The subcellular distribution of M3 muscarinic receptors and Kir6.1 was examined using sucrose density fractionation and immunoelectron microscopy. Desorption of cholesterol right-shifted the concentration-response curve for the muscarinic receptor agonist carbachol ~3-fold. This effect was inhibited by glibenclamide, PNU-37883 and chelerythrine, and mimicked by levcromakalim, suggesting involvement of KATP channels. RT-qPCR demonstrated expression of Kir6.1, Kir6.2, and SUR2A. Sucrose density fractionation and Western blotting revealed co-fractionation of detrusor M3 receptors and Kir6.1, in partial overlap with caveolin-1. Immunoelectron microscopy showed M3 and Kir6.1 to be enriched 6-fold in caveolae. Thus, ultrastructural imaging, unique in its kind using the human urinary bladder, demonstrates that M3 receptors co-localize with Kir6.1 in caveolae. Functional assays moreover reveal that this organization facilitates cholinergic detrusor activation. Changes in caveolae-associated signaling may thus underlie functional bladder disturbances in man.