Abstract
The steady state of the acetylcholine receptor (AChR) density at the neuromuscular junction (NMJ) is critical for efficient and reliable synaptic transmission. However, little is known about signaling molecules involved in regulating the equilibrium between the removal and insertion of AChRs that establishes a stable postsynaptic receptor density over time. In this work, we tested the effect of activities of two serine/threonine kinases, PKC and PKA, on the removal rate of AChRs from and the re-insertion rate of internalized recycled AChRs into synaptic sites of innervated and denervated NMJs of living mice. Using an in vivo time-lapse imaging approach and various pharmacological agents, we showed that PKC and PKA activities have antagonistic effects on the removal and recycling of AChRs. Inhibition of PKC activity or activation of PKA largely prevents the removal of pre-existing AChRs and promotes the recycling of internalized AChRs into the postsynaptic membrane. In contrast, stimulation of PKC or inactivation of PKA significantly accelerates the removal of postsynaptic AChRs and depresses AChR recycling. These results indicate that a balance between PKA and PKC activities may be critical for the maintenance of the postsynaptic receptor density.
Highlights
The maintenance of a high density of nicotinic acetylcholine receptors (AChRs) at the postsynaptic membrane of a neuromuscular junction (NMJ) is essential for the effectiveness of synaptic impulse transmission
In muscles treated with calphostin C, fluorescence intensity of pre-existing AChRs decreased by only 4% (96 ± 6% of original fluorescence; n = 33 NMJs, 3 mice), compared to untreated muscles (p < 0.001), where the fluorescence intensity decreased by 12% (88 ± 5% of original fluorescence; n = 19 NMJs, 3 mice) (Figure 1 B, C)
In muscles treated with calphostin C, the loss of fluorescence intensity of pre-existing AChRs was only 8% of the original fluorescence (92 ± 7%, n = 18 NMJs, 3 mice, p < 0.001) compared to 29% of receptor loss in nontreated denervated synapses (71 ± 9%; n = 22 NMJs, 3 mice, p < 0.001)
Summary
The maintenance of a high density of nicotinic acetylcholine receptors (AChRs) at the postsynaptic membrane of a neuromuscular junction (NMJ) is essential for the effectiveness of synaptic impulse transmission. This high concentration of AChRs is established by rates of removal, re-insertion of recycled, insertion of newly synthesized and lateral diffusion of AChRs [1,2,3]. Several studies have reported that serine/threonine kinases PKC and PKA activities are implicated in the clustering and stability of AChRs in cultured muscle [10,11,12,13,14,15]. The skeletal muscle abundantly expresses cAMP-dependent PKA, whose Rαisoform is enriched in the NMJ region [21]
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