Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the γ-subunit of the fetal muscle acetylcholine receptor (γ-AChR) is replaced by the ε-subunit in the adult muscle receptor (ε-AChR), was investigated by analysing the structure of γ- and ε-subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single-channel conductance. 2. The γ- and ε-subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric ε(γ)- or γ(ε)-subunits were constructed from sequences derived from the parental ε- and γ-subunits, respectively. Exchanging the four hydrophobic segments (M1-M4) of the γ-subunit for those of the ε-subunit and vice versa completely reversed the difference in conductance between γ-AChR and ε-AChR channels. 4. Effects of single- and multiple-point mutations in M1-M4 segments of γ- and ε-subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (γ-subunit) for serine/isoleucine (ε-subunit) in M2, and the lysine (γ-subunit) for glutamine (ε-subunit) exchanges in the regions flanking the M2 segment.