Microdomains of muscarinic acetylcholine and InsP3 receptors create InsP3 junctions and sites of Ca2+ wave initiation in smooth muscle

Inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c) regulate activities which include division, contraction and cell death. InsP3-evoked Ca2+ release often begins in a single site then regeneratively propagates through the cell as a Ca2+ wave. The Ca2+ wave consistently begins at the same site on successive activations. We addressed the mechanisms that determine the Ca2+ wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP3 receptors (InsP3R) to InsP3 nor regional clustering of muscarinic receptors (mAChR3) or InsP3R1 explained the initiation site. However, examination of the overlap of mAChR3 and InsP3R1 by centre of mass analysis revealed a small percentage (~10%) of sites which showed colocalisation. Indeed, the extent of colocalisation was greatest at Ca2+ wave initiation site. The initiation site may arise from a selective delivery of InsP3 from mAChR3 activity to particular InsP3R to generate faster local [Ca2+]c increases at sites of co-localization. In support, a localized subthreshold ‘priming’ InsP3 concentration applied rapidly but at regions distant from the initiation site shifted the wave to the site of priming InsP3 release. Conversely, when the Ca2+ rise at the initiation site was rapidly and selectively attenuated the Ca2+ wave again shifted and initiated at a new site. These results indicate that Ca2+ waves initiate where there is a structural and functional coupling of mAChR3 and InsP3R1 which generates junctions in which InsP3 acts as a highly localized signal by being rapidly and selectively delivered to InsP3R