STRUCTURAL DETERMINANTS OF CHANNEL CONDUCTANCE IN FETAL AND ADULT-RATMUSCLE ACETYLCHOLINE-RECEPTORS

1 The structural basis of the developmentally regulated increase in en dplate channel conductance in rat, where the gamma-subunit of the feta l muscle acetylcholine receptor (gamma-AChR) is replaced by the epsilo n-subunit in the adult muscle receptor (epsilon-AChR), was investigate d by analysing the structure of gamma- and epsilon-subunit genes and b y expressing recombinant AChR channels of different molecular composit ion in Xenopus oocytes and measuring their single-channel conductance. 2. The gamma- and epsilon-subunit genes each have twelve exons. In bo th subunits, the four homologous segments, designated M1, M2, M3 and M 4, which are thought to contribute to the formation of the pore, are e ncoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilo n(gamma)- or gamma(epsilon)-subunits were constructed from sequences d erived from the parental epsilon- and gamma-subunits, respectively. Ex changing the four hydrophobic segments (M1-M4) of the gamma-subunit fo r those of the s-subunit and vice versa completely reversed the differ ence in conductance between gamma-AChR and epsilon-AChR channels. 4. E ffects of single- and multiple-point mutations in M1-M4 segments of ga mma- and epsilon-subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels ar e located in the M2 segment. The key differences are the exchange of a lanine/threonine (gamma-subunit) for serine/isoleucine (epsilon-subuni t) in M2, and the lysine (gamma-subunit) for glutamine (epsilon-subuni t) exchanges in the regions flanking the M2 segment.