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 CHO(M2) 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 ~1μm(-2), increasing at birth (to ~3μm(-2)) and decreasing back to ~1μm(-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.
Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. dreminat@mail.ru
Tatiana A
TA
Nenasheva
Marianne
M
Neary
Gregory I
GI
Mashanov
Nigel J M
NJ
Birdsall
Ross A
RA
Breckenridge
Justin E
JE
Molloy
D000818:Animals; D016466:CHO Cells; D002232:Carbocyanines / Q000737:chemistry; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000737:chemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D009928:Organ Specificity; D061251:Primary Cell Culture; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D013194:Staining and Labeling
D002232:Carbocyanines; D005456:Fluorescent Dyes; D043585:Receptor, Muscarinic M2; C089187:cyanine dye 3