Supplementary Information to
MOD-1 is a serotonin-gated chloride channel that modulates C. elegans
locomotory behaviour
Rajesh Ranganathan*, Stephen C. Cannon#, and H. Robert Horvitz*
*Howard
Hughes Medical Institute, Department of Biology, Room 68-425
Massachusetts Institute of Technology, 77 Massachusetts Avenue
Cambridge, MA 02139
#Department
of Neurobiology, Harvard Medical School, Boston, MA 02115
and Department of Neurology, Massachusetts General Hospital, Boston, MA
02114
A281V may block channel pore
When the alpha helix that forms the M2 domain of MOD-1 is viewed in a helicalwheel configuration1, Ala-281 is on the side of the alpha helix analogous to the
pore-facing side of the nAChR2Moreover, Ala-281 is in a position analogous to
that of Ser-252 in nAChR and Thr-265 in GABA-A 1, both of which have been
predicted to face the lumen of the channel pore as a result of their accessibility
to modification by polar sulfhydryl reagents2. We suggest that the addition of the
two methyl side groups caused by the A281V substitution predicted in
mod-1(n3034) mutants alters the pore of the channel and prevents ion flow.
mod-1 deletion mutants
The ok103 deletion removes 4135 bp, starting four bp after the first exon. The
nr2043 deletion removes 1209 bp, starting 146 bp 5' of exon 3, and adds the
sequence ATAAGTTTTTT. If the end of exon 2 splices onto the next available
splice acceptor site at the start of exon 6, an additional 15 unrelated amino acids
would be present before the protein ends at a premature stop codon. The cDNA
predicted by the C. elegans Genome Sequencing Project3 omits the first and
seventh exons and does not include the first 54 bp of the second exon.
Methods
Mapping and cloning of mod-1
mod-1 was mapped to LG V based upon its two-factor linkage to dpy-11 unc-76
V. The following three-factor data were obtained: dpy-11 (14/36) mod-1 (22/36)
unc-76, dpy-11 (27/40) mod-1 (13/40) unc-42, mod-1 (0/32) unc-42 (32/32) sma1, dpy-11 (38/74) stP23 (25/74) mod-1 (11/74) unc-42, dpy-11 (38/74) pkP5056
(25/74) mod-1 (11/74) unc-42, dpy-11 (36/45) mod-1 (6/45) pkP520 (3/45)
2
unc-42, let-434 (19/33) mod-1 (14/33) unc-42. stP23, pkP5056, and pkP520 are
Tc1-transposon polymorphisms present in strains RW7000 and KR1787,
respectively3. All mapping experiments were performed by mating each
homozygous recombinant with wild-type (N2) males and scoring the F1 cross
progeny for semi-dominant 5-HT resistance at 20 min. The presence of the Tc1
polymorphisms was assayed by performing PCRs with a Tc1-specific primer and
a primer from genomic sequence adjacent to the Tc1 insert site on crude wholeworm lysates prepared from each homozygous recombinant strain.
References
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Flinta, C., von Heijne, G. & Johansson, J. Helical sidedness and the
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Karlin, A. & Akabas, M. H. Toward a structural basis for the function of
nicotinic acetylcholine receptors and their cousins. Neuron 15, 1231-44
(1995).
3.
Genome sequence of the nematode C. elegans: a platform for
investigating biology. The C. elegans Sequencing Consortium. Science
282, 2012-8 (1998).
3