Search

1. The influx of Zn2+ through the channels of fetal and adult mouse muscle nicotinic acetylcholine receptors (gamma- and epsilon-AChRs) and its effects on receptor function were studied in transiently transfected human BOSC 23 cells, by combining patch-clamp recordings with digital fluorescence microscopy. 2. ACh-induced whole-cell currents were reversibly reduced by external ZnCl2, with half-maximal inhibitory concentrations of 3 and 1 mM for gamma- and epsilon-AChRs, respectively. 3. Both gamma- and epsilon-AChR channels were permeable to Zn2+, as shown by fluorescence measurements using Zn2+-sensitive dyes. The fractional current carried by Zn2+ (Pf,Zn; 0.5 mM Zn2+ in Ca2+- and Mg2+-free medium) through gamma- and epsilon"-AChR channels was 1.7 and 4 %, respectively. 4. Pf,Zn increased with the concentration of ZnCl2, but was little affected by physiological concentrations of Ca2+ and Mg2+ in the external medium. 5. The conductance of ACh-evoked unitary events, measured by cell-attached or outside-out recordings, decreased when the patched membrane was exposed to ZnCl2 (1 or 3 mM). Simultaneous application of ACh and Zn2+ to the extra-patch membrane lengthened channel open duration (tau op) by 50%. No obvious increment of tau op was observed following exposure of inside-out patches to Zn2+. 6. The possible physiological relevance of zinc-induced modulation of AChR channels is discussed.

Implantation of myoblasts is a strategy used to enhance the regeneration of skeletal muscle tissue in vivo. In mouse models, myogenic cell lines and primary cells have been employed with different yields of adult muscle tissue formed. The present work is a study of some developmental features of expanded primary mouse myoblasts (i28), which have been shown to form muscle tissue. i28 myoblasts were differentiated in vitro and the expression of acetylcholine receptor channels and maturation of the excitation–contraction coupling mechanism were investigated using patch clamp and videoimaging techniques. In all the developing cells the embryonic isoform of the acetylcholine receptors was present. Skeletal muscle-type excitation–contraction coupling (i.e., a mechanical link between voltage-dependent calcium channels and ryanodine receptor channels) was detected in about 75% of differentiating i28 myotubes. Only these cells showed spontaneous changes in cytosolic free calcium concentration associated with twitches. Our findings are the first description of the physiological properties of expanded primary myoblasts which are used for implantation and confirm that they are a heterogeneous cell population. In comparison to permanent cell lines, the Ca2+ signaling is more similar to that described in mature nonexpanded muscle fibers. This suggests that cultured primary cells are, so far, the most suitable cell type for muscle regeneration.

It has long been known that extracellular Ca2+ and Mg2+ modulate synaptic transmission at the neuromuscular junction, acting both pre- and post-synaptically. Relevant questions concerning the modulation of acetylcholine (ACh) receptors (AChRs) are however still open: are the fetal (gamma-AChR) and adult (epsilon-AChR) receptors modulated differently? Does the ACh concentration influence the effect of divalent cations? Is the effect on channel open duration dependent on type and concentration of divalent cation? These questions were addressed by studying the modulation of the single-channel behaviour of gamma- and epsilon-AChRs by Ca2+ and Mg2+ at the endplate of muscle fibres acutely dissociated from 12- to 14-day-old mice. Ca2+ reduced the conductances of the two receptor channels comparably. Mg2+ had a stronger effect than Ca2+ and reduced the conductance of epsilon-AChR significantly more than that of gamma-AChR. With 0.1 microM ACh, Ca2+ and Mg2+ increased the mean open duration of gamma- and epsilon-AChR channels comparably. At 100 microM ACh, gamma- and epsilon-AChR channels opened in bursts of strikingly similar duration, which was unaffected by divalent cations. These findings indicate that Ca2+, and even more so Mg2+, may regulate synaptic transmission by modulating the function of AChRs in addition to the well-established effects on transmitter release.