Microdomains of muscarinic acetylcholine and Ins(1,4,5) P ₃ receptors create ‘Ins(1,4,5) P ₃ junctions’ and sites of Ca ²⁺ wave initiation in smooth muscle

Summary

Increases in cytosolic Ca ²⁺ concentration ([Ca ²⁺] _c) mediated by inositol (1,4,5)-trisphosphate [Ins(1,4,5) P ₃, hereafter InsP ₃] regulate activities that include division, contraction and cell death. InsP ₃-evoked Ca ²⁺ release often begins at a single site, then regeneratively propagates through the cell as a Ca ²⁺ wave. The Ca ²⁺ wave consistently begins at the same site on successive activations. Here, we address the mechanisms that determine the Ca ²⁺ wave initiation site in intestinal smooth muscle cells. Neither an increased sensitivity of InsP ₃ receptors (InsP ₃R) to InsP ₃ nor regional clustering of muscarinic receptors (mAChR3) or InsP ₃R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP ₃R1 localisation, by centre of mass analysis, revealed that there was a small percentage (∼10%) of sites that showed colocalisation. Indeed, the extent of colocalisation was greatest at the Ca ²⁺ wave initiation site. The initiation site might arise from a selective delivery of InsP ₃ from mAChR3 activity to particular InsP ₃Rs to generate faster local [Ca ²⁺] _c increases at sites of colocalisation. In support of this hypothesis, a localised subthreshold ‘priming’ InsP ₃ concentration applied rapidly, but at regions distant from the initiation site, shifted the wave to the site of the priming. Conversely, when the Ca ²⁺ rise at the initiation site was rapidly and selectively attenuated, the Ca ²⁺ wave again shifted and initiated at a new site. These results indicate that Ca ²⁺ waves initiate where there is a structural and functional coupling of mAChR3 and InsP ₃R1, which generates junctions in which InsP ₃ acts as a highly localised signal by being rapidly and selectively delivered to InsP ₃R1.