A Novel Labeling Approach Identifies Three Stability Levels of Acetylcholine Receptors in the Mouse Neuromuscular Junction In Vivo

Abstract

BackgroundThe turnover of acetylcholine receptors at the neuromuscular junction is regulated in an activity-dependent manner. Upon denervation and under various other pathological conditions, receptor half-life is decreased.Methodology/Principal FindingsWe demonstrate a novel approach to follow the kinetics of acetylcholine receptor lifetimes upon pulse labeling of mouse muscles with 125I-α-bungarotoxin in vivo. In contrast to previous assays where residual activity was measured ex vivo, in our setup the same animals are used throughout the whole measurement period, thereby permitting a dramatic reduction of animal numbers at increased data quality. We identified three stability levels of acetylcholine receptors depending on the presence or absence of innervation: one pool of receptors with a long half-life of ∼13 days, a second with an intermediate half-life of ∼8 days, and a third with a short half-life of ∼1 day. Data were highly reproducible from animal to animal and followed simple exponential terms. The principal outcomes of these measurements were reproduced by an optical pulse-labeling assay introduced recently.Conclusions/SignificanceA novel assay to determine kinetics of acetylcholine receptor turnover with small animal numbers is presented. Our data show that nerve activity acts on muscle acetylcholine receptor stability by at least two different means, one shifting receptor lifetime from short to intermediate and another, which further increases receptor stability to a long lifetime. We hypothesize on possible molecular mechanisms.

Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease

“…Sciatic Denervation, Stimulation of Lumbar Sympathetic Ganglionic Chain, and Pharmacological Treatments. Sciatic denervation was as described (41). For details regarding the stimulation of the sympathetic ganglionic chain, see Fig.…”

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

“…10, [21][22][23] However, it is unclear if this effect is due to a generalized atrophic signaling or specific to the lack of nervederived input. To address this point, we used a recently established in vivo-labeling approach that measures the metabolic stability of CHRN.…”

“…To address this point, we used a recently established in vivo-labeling approach that measures the metabolic stability of CHRN. 23 This assay is based on the irreversible binding to CHRN of 125 I-labeled BGT ( 125 I-BGT), an 8 kDa, non-cell-penetrating snake venom (for scheme, see Fig. 1).…”

“…As previously described, in denervated muscles an increase in size of a short-lived pool of CHRN (half-life of about 1 d) and a reduction in half-life of a long-lived receptor pool from 13 to 8 d had been observed. 23 Now, we applied the approach to fasted mice. Animals were sorted into 4 different groups.…”

“…23 In brief, 0.7 pmol of 125 I-BGT with high specific activity from Perkin Elmer (NEX126H050UC) was injected intramuscularly to the tibialis anterior muscle at day 0 and subsequent 125 I-emission measurements from entire hind legs were performed under mild isoflurane anesthesia using liquid nitrogen-cooled GX-3018 (Canberra GmbH) and electrically cooled GEM-FX5825P4-S (AMETEK GmbH, ORTEC Division) Germanium semiconductor detectors.…”