The open duration of fetal ACh receptor‐channel changes during mouse muscle development

Abstract

We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the γ subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5. We studied unitary ACh‐evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6). At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24‐26 °C. On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the γs subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period.

5-Hydroxytryptamine blocks the fetal more potently than the adult mouse muscle acetylcholine receptor

“…There is no obvious difference in the effect of 5-HT on γ-AChRs in transfected cells and E18 or P14 fibres in spite of the different mean channel open times, suggesting that whatever the cause of the difference in τ op , this does not influence the 5-HT-induced block. However, this paper confirms that the mean open duration of γ-AChR channel becomes shorter during postnatal mouse development [10], and extends this observation to γ-AChRs expressed in the end-plate region. In addition, it demonstrates that, as in the rat [20], both γand ε-AChR are present at the endplate of P14 mice.…”

“…Cells were used for recordings 36-48 h after transfection. Flexor digitorum brevis (FDB) muscles were dissected from the hindlimbs of fetal (as described in [10]) or 14-day-old Balb/C mice, using procedures F. Grassi that comply with national guidelines for animal experiments. The muscles obtained from 14-day-old mice, killed by cervical dislocation, were incubated with Type I collagenase (3 mg/ml, Sigma) for 30 min at 37°C in Minimum Essential Medium (MEM, Gibco).…”

Functional maturation of nicotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system

“…These differences may reflect the occurrence of two types of embryonic AChRs, as previously reported [14,17,24,35].…”

“…Thus, in the motor unit model we developed, the e subunit of AChR appears to be progressively expressed, in addition to the g subunit, as occurs during muscle maturation and development in vivo [31]. Although several muscle cell lines have been described by some [28,34,36], but not all labs [17,24], to express in a minor fashion the adult AChR form in the absence of neuronal cells, physiological expression of the e subunit in vivo is under the control of nerve signals [32]. Therefore, our co-culture system, in which the expression of the adult form of AChR is induced following innervation by spinal cord explants, may well be a reliable physiological model for studying the differentiation of muscle fibers and the development of neuromuscular junctions.…”

“…The g S AChR variant appears to be the only form of g-subunit found in newborn muscle. Channel openings are longer for the g S AChR when compared to gAChRs in transfected BOSC 23 cells [14], C2 myotubes [35] and developing mouse myotubes in culture [17].…”