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

Abstract

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.

The Molecular Basis of Oligomeric Organization of the Human M3Muscarinic Acetylcholine Receptor

“…One is that dimeric interactions may be able to occur in a number of distinct ways, including via each of the TMD V-TMD V, TMD VI-TMD VII, TMD IV-TMD V, and TMD I-TMD II interactions (McMillin et al, 2011). Observations that dimer/oligomer interactions of muscarinic receptor subtypes may be transient (Hern et al, 2010;Nenasheva et al, 2013) are certainly consistent with this concept if it predominantly reflects a "kiss and run" phenomenon (Milligan, 2000). A second interpretation of the results of both Wess et al (McMillin et al, 2011) and those we report herein is that the contributions of multiple regions of the receptor to structural organization reflects that higher order complexes beyond dimers can form.…”

G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives

“…These conclusions require that the fluorescent ligands bind with similar affinity to both protomers in the dimer (see below for discussion of ligand cooperativity within receptor dimers/oligomers; see Section II.B), and it is possible that the fluorescent ligand might stabilize the receptor in a nonrepresentative conformational state. Despite these concerns, similar studies have reached equivalent conclusions on the state of the muscarinic acetylcholine M 2 receptor in cardiac muscle (Nenasheva et al, 2013). More recently, TIR-FM was used together with SNAP-tag technology to directly label cell-surface GPCRs with organic fluorophores to dynamically monitor individual b 1 -and b 2 -adrenoceptors as well as GABA B receptors on the surface of living, transiently transfected cells (Calebiro et al, 2013).…”

Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

“…Overall, our measurements fall within a rather heterogeneous background of previous measurements of GPCR oligomerization. In particular, for the three receptors that we investigated, previous research showed either a largely monomeric [32], [29], constitutive dimeric [15] or higher order oligomeric state [29,33,34]. In one case, concentration-dependent effects were observed for both β 1 -ARs and β 2 -ARs, with a different equilibrium constant depending on the receptor [12].…”

“…The only reports supporting a higher oligomerization state for β 1 -ARs come from whole-cell BRET measurements [33] and the results may have been influenced by receptor interactions not on the plasma membrane. For M 2 Rs, single molecule imaging data [32] suggest a predominantly monomeric fingerprint, whereas fluorescence lifetime-based FRET measurements have indicated tetramers [34]. Photon counting histogram analysis indicates constitutive dimers [15].…”