An ion channel of the degenerin/epithelial sodium channel superfamily controls the defecation rhythm in Caenorhabditis elegans

Abstract

Ultradian rhythms are widespread phenomena found in various biological organisms. A typical example is the defecation behavior of the nematode Caenorhabditis elegans, which repeats at about 45-sec intervals. To elucidate the mechanism, we studied flr-1 mutants, which show very short defecation cycle periods. The mutations also affect some food-related functions, including growth rate, the expulsion step of defecation behavior, and the regulation of the dauer larva (a nonfeeding, special third-stage larva) formation in the unc-3 (Olf-1/EBF homolog) background. The flr-1 gene encodes a novel ion channel belonging to the DEG/ENaC (C. elegans degenerin and mammalian epithelial sodium channel) superfamily. A flr-1::GFP (green fluorescent protein) fusion gene that can rescue the flr-1 mutant phenotypes is expressed only in the intestine from embryos to adults. These results suggest that FLR-1 may be a component of an intestinal regulatory system that controls the defecation rhythm as well as other functions.

Figure 1

Defecation interval length and the percentage of defecation behaviors having the Exp step. The graphs show the relationship between the length of each defecation interval (abscissa) and that of the next interval (ordinate). The bars on the right of the graphs represent the percentage of defecation behaviors that show the Exp step (Exp/pBoc). We used plates containing 400 μg/ml of NaF in the experiments shown in the middle two graphs and plates without NaF in other experiments. The numbers above the graphs show the average defecation cycle period ± SD (number of defecation intervals measured) and Exp/pBoc (number of pBoc observed). See Materials and Methods for the meaning of the ∗. Data not shown are the following: flr-1 alleles other than ut11, 34.0 ± 15.4 sec (106)* and 31% (121) for flr-1(ut4); 26.6 ± 8.5 sec (116) and 6% (126) for flr-1(ut6); 24.7 ± 6.5 sec (107)* and 64% (120) for flr-1(sa96). Another class 2 flr mutation flr-2(ut5): 55.4 ± 7.7 sec (101) and 96% (111) for flr-2(ut5), and 37.6 ± 12.1 sec (111) and 60% (121) for flr-2(ut5); flr-1(ut11). Unlike L1 larvae, young adults survived on the plates containing 400 μg/ml of NaF for at least 3 days.

Figure 2

Cloning of flr-1 gene and cDNA. (A) Genetic and physical map around flr-1 gene. A genomic DNA fragment flanking the transposon Tc1 that caused the flr-1(ut11) mutation, hybridized to the yeast artificial chromosome Y50B10 and the cosmid C15A2. An overlapping cosmid F02D10 and its subclone pMT21–3 rescued the flr-1 phenotypes by microinjection. The arrow shows the direction of transcription of the only complete ORF contained in pMT21–3. Sl, SalI site; Sc, SacI site. (B, Top) The structure of the 8.5-kb genomic clone (pMT21–3) that rescued the flr-1 phenotypes. The 17 exons are indicated by boxes. The trans-splice leader SL1 and the poly(A) tail also are shown. (Middle) The structure of pMTG25–1, which codes for a GFP(F64L S65T)-tagged FLR-1 protein. (Bottom) The structure of pMTG24–5, a flr-1∷GFP(S65C) fusion gene containing a nuclear localization signal. (C) Rescue of various phenotypes of flr-1(ut11) by pMTG25–1.

Figure 3

Sequence alignment of FLR-1 and other ion channels of the DEG/ENaC superfamily. Shown is a comparison of the predicted amino acid sequences between FLR-1, the C. elegans mechanosensory channel MEC-4 (24), the human amiloride-sensitive epithelial sodium channel beta subunit (ENaCbe) (28), and the snail amiloride-sensitive FMRFamide peptide-gated channel (FaNaCh) (30). The residues similar in three or four proteins are boxed. The numbers in parentheses show those of amino acids omitted. Two putative membrane-spanning domains (MSDI and MSDII) and the extracellular Cys-rich domains (CRDII and CRDIII) are shown by lines with arrowheads. A broken line with arrowheads shows the similarity region including MSDI. N and + indicate five potential N-glycosylation sites in the extracellular region and potential phosphorylation sites in the C-terminal intracellular domain of FLR-1, respectively. The latter consist of the consensus sequences for cyclic AMP-dependent protein kinase (R R X S/T), cyclic GMP-dependent protein kinase (R/K R/K X S/T), and Ca²⁺/calmodulin-dependent protein kinase II (R X X S/T) (32). The mutation sites of (ut1, ut4, ut6), ut2, and sa96 are indicated by ∗ together with the allele names and the resultant amino acid substitution. The Tc1-insertion site in ut11 is indicated by an arrowhead.

Figure 4

Expression of GFP-tagged FLR-1 protein. (A–E) Nomarski images and (F–J) epifluorescence images of the corresponding animals. (A and F) L1 larva of N2 (control). Autofluorescence of gut granules can be seen only faintly in F. (B and G) L1 larva of N2 carrying pMTG24–5. All of the intestinal nuclei show strong GFP fluorescence, whereas weak fluorescent dots in the cytoplasm in G may be gut granules. (C and H) Comma stage embryo of N2 carrying pMTG25–1. (D and I) Three-fold stage embryo of N2 carrying pMTG25–1. (E and J): L1 larva of N2 carrying pMTG25–1. (Scale bar represents 50 μm.)