Microdomains of muscarinic acetylcholine and Ins(1,4,5)P₃ receptors create 'Ins(1,4,5)P₃ junctions' and sites of Ca²+ wave initiation in smooth muscle

• Published in: Journal of cell science Vol. 125; no. Pt 22; pp. 5315 - 5328
• Main Authors: Olson, Marnie L, Sandison, Mairi E, Chalmers, Susan, McCarron, John G
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists 15-11-2012
• Subjects: Animals; Calcium - metabolism; Calcium Signaling - drug effects; Carbachol - pharmacology; Colon - drug effects; Colon - metabolism; Guinea Pigs; Inositol 1,4,5-Trisphosphate Receptors - metabolism; Male; Membrane Microdomains - drug effects; Membrane Microdomains - metabolism; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - metabolism; Photolysis - drug effects; Protein Isoforms - metabolism; Protein Transport - drug effects; Receptor, Muscarinic M3 - metabolism
• DOI: 10.1242/jcs.105163
• EISSN: 1477-9137
• ISSN: 0021-9533

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(3)R) to InsP(3) nor regional clustering of muscarinic receptors (mAChR3) or InsP(3)R1 explained the selection of an initiation site. However, examination of the overlap of mAChR3 and InsP(3)R1 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(3)Rs 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(3)R1, which generates junctions in which InsP(3) acts as a highly localised signal by being rapidly and selectively delivered to InsP(3)R1.