Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors

Abstract

CALCIUM release from intracellular stores is a point of convergence for a variety of receptors involved in cell signalling¹. Consequently, the mechanism(s) by which cells differentiate between individual receptor signals is central to transmembrane communication^2–5. There are significant differences in timing and magnitude of Ca²⁺ release stimulated by the m2 and m3 muscarinic acetyl-choline receptors^6–11. The m2 receptors couple to a pertussis toxin-sensitive G protein to activate phosphatidyl inositol hydrolysis weakly and to stimulate small, delayed and oscillatory chloride currents. In contrast, m3 receptors potently activate phosphatidyl inositol hydrolysis and stimulate large, rapid and transient chloride currents by a pertussis toxin-insensitive G protein pathway. Using confocal microscopy, we now show that the m2- and m3-coupled Ca²⁺ release pathways can also be spatially distinguished. At submaximal acetylcholine concentrations, both receptors stimulated pulses of Ca²⁺ release from discrete foci in random, periodic and frequently bursting patterns of activity. But maximal stimulation of m2 receptors increased the number of focal release sites, whereas m3 receptors invariably evoked a Ca²⁺ wave propagating rapidly just beneath the plasma membrane surface. Analysis of pertussis toxin sensitivity and hybrid m2–m3 muscarinic acetyl-choline receptors confirmed that these Ca²⁺ release patterns represent distinct cell signalling pathways.