Abundance, distribution, mobility and oligomeric state of M₂ muscarinic acetylcholine receptors in live cardiac muscle

M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~ 1 μm− 2, increasing at birth (to ~ 3 μm− 2) and decreasing back to ~ 1 μm− 2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening.
Graphical abstract Individual M2 muscarinic receptors were visualized and tracked in live cardiac tissue slices to establish their abundance, distribution, mobility and oligomeric state in situ. Highlights ► The first observation of single molecules moving on the surface of living tissue ► Only ca. 10% of murine cardiomyocytes expressed M2 receptors at appreciable levels. ► M2 density at birth was 3-fold higher than that in embryonic and adult myocytes. ► M2 receptors were primarily monomeric but formed reversible dimers. ► M2 receptors diffused 4-times faster in heart slices than in cultured myocytes.