Fig. 5. twister mutation is a point mutation in the M2 domain of chrna1. (A-C) Molecular and genetic mapping of twister locus to LG6. (A) PCR amplification using SSLP marker z6601 on DNA obtained from the two grandparents (G₀) and from pools of F₂ homozygous mutants, heterozygous mutants and wild-type siblings, respectively. The shorter PCR fragment from the G₀ Tüebingen heterozygote segregates with the homozygous mutant pool, from which the longer, WIK-specific allele is absent. (B) PCR amplification of DNA obtained from individual mutant embryos with marker z6601. Recombinant embryos (R) were detected by the presence of a larger WIK-specific PCR fragment. (C) The twister locus maps between markers z6601 and z9739. (D) Sequence analysis of chrna1 in twister revealed a single nucleotide change, TC, giving rise to a leucine to proline amino acid substitution at codon 258. (E) The L258P mutation is located in the second transmembrane (M2) domain, which contributes to the cation-selective channel pore. (F) Cross-species sequence alignment of the CHRNA1 M2 domain. The leucine 258 residue affected in twister mutants is 100% conserved across species. GenBank Accession numbers are in parentheses. (G) Whole-cell recordings of spontaneous synaptic currents from nic-1^b107 homozygous fish expressing receptors containing either wild-type -subunits or the L258P mutant subunits. The holding potential was –90 mV and the vertical calibration corresponds to 20 pA for wild-type and 60 pA for L258P mutant. (H) The distribution of mean values for exponentially fitted current decays from 8 muscles expressing wild-type and 9 mutant twister -subunit receptors. Between 6 and 276 synaptic events were used to compute each mean value (overall mean and standard errors are indicated).