Role of M2 domain residues in conductance and gating of acetylcholine receptors in developing Xenopus muscle.

1. The contributions of specific residues in gamma- and epsilon-subunits to the developmental changes in conductance and open time of Xenopus muscle acetylcholine receptors (AChRs) were investigated. This study was directed primarily at residues in the M2 domains of gamma- and epsilon-subunits; however, the results of additional mutations in the extracellular region flanking M2 and in the amphipathic region between M3 and M4 are also described. 2. The M2 domains of gamma- and epsilon-subunits differ at only three amino acid residues, two of which are adjacent to each other and located near the narrowest part of the pore. These two residues (NI in gamma, SV in epsilon) were found to be major determinants of the difference in conductance and open time of AChRs bearing gamma- or epsilon-subunits. 3. Mutation of N to S in the gamma-subunit converted the long open time of receptors bearing the gamma-subunit (gamma-AChRs) to the brief open time characteristic of receptors bearing an epsilon-subunit (epsilon-AChRs). Conversely, epsilon-AChRs with SV mutated to NI in the epsilon-subunit exhibited a long open time characteristic of gamma-AChRs. 4. Mutation of N to S in the gamma-subunit increased the conductance of gamma-AChRs but did not confer the full conductance of wild-type epsilon-AChRs. Conversely, mutation of SV to NI in the epsilon-subunit reduced the conductance of epsilon-AChRs, but not completely to the level of wild-type gamma-AChRs.

Sequential expression of acetylcholine receptor isoforms in mesodermalized Xenopus animal caps.

Exposure of Xenopus animal pole explants to transforming growth factor beta 2 (TGF-beta 2) induced the sequential expression of muscle nicotinic acetylcholine receptor (AChRs) isoforms and their corresponding mRNAs in cells which normally give rise to ectoderm. Single channel recordings revealed two functional classes of receptors with properties similar to those expressed during normal development of skeletal muscle in vivo. The predominant class of receptors in all patches corresponded to those of embryonic myotomal muscle. Additional receptors resembling those of mature myotomal muscle were observed in older explants. Levels of transcripts encoding the embryonic and adult AChR subunit isoforms varied accordingly. TGF-beta 2 appears to initiate a developmental program of AChR gene expression which is similar to that found in normally developing muscle.

Structure and expression of the nicotinic acetylcholine receptor beta subunit of Xenopus laevis.

A cDNA encoding the beta subunit of the Xenopus muscle nicotinic acetylcholine receptor (AChR) was cloned from an embryonic Xenopus cDNA library. The predicted mature polypeptide has 469 amino acids and four membrane spanning regions corresponding to the M1-M4 regions identified in other AChR subunit clones. The polypeptide bears greater homology to beta subunits of Torpedo and mouse than to alpha, gamma or delta subunits of Xenopus. The earliest beta subunit transcripts were detected by RNase protection assays at the neural plate stage of development (stage 14) and the level of transcripts, as a fraction of total RNA, continued to increase through the age of hatching (stages 34-36). Co-injection of Xenopus alpha, beta, gamma and delta cRNAs into Xenopus oocytes led to expression of functional AChRs. Micromolar concentrations of ACh activated depolarizing AChR currents which reversed at -5 mV and were blocked by alpha bungarotoxin. Injection of alpha, gamma and delta subunits alone did not yield detectable ACh responses. With the cloning of the Xenopus beta subunit, structure/function relations of AChRs can now be studied using receptors composed entirely of Xenopus subunits.

Calcitonin gene-related peptide lengthens acetylcholine receptor channel open time in developing muscle.

The effect of calcitonin gene-related peptide (CGRP) on nicotinic acetylcholine receptor (AChR) function was examined in developing Xenopus myotomal muscle. Short term exposure to CGRP was found to prolong the open time of AChR channels in intermediate stage muscle. Of the two predominant conductance classes of AChRs expressed in Xenopus muscle, only the low conductance channels exhibited a lengthened open time in response to CGRP. High conductance channels were not affected, nor were low conductance channels at an earlier developmental stage. The lengthening of open time was reflected by an increase in the duration of synaptic currents at intermediate stages in response to CGRP. Application of 8 bromo-cAMP also prolonged synaptic currents, suggesting that the CGRP effects were mediated by cAMP. CGRP is known to be present in motor nerve terminals of Xenopus, and its release at developing synapses could potentiate the effects of ACh on muscle membrane.