Worm Breeder's Gazette 14(4): 72 (October 1, 1996)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

exp-2 encodes a potassium channel

M. Wayne Davis, Joe Dent, Leon Avery

Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas TX 75235-9038

exp-2(sa26sd) was isolated based on a severe defect in the expulsion step of the defecation cycle (Thomas, Genetics 124: 855-872, 1990; Reiner et. al., Genetics 141:961-976, 1995). This allele is semi-dominant and is homozygous lethal. sa26/+ worms also have egg laying defects and very brief pharyngeal contractions. Electropharyngeograms show that these worms have very short pharyngeal muscle action potentials, indicating that the pharyngeal defects are due to an electrical defect in the muscle cells. We isolated a loss-of-function allele, exp- 2(sa26 ad1201), in a reversion screen. ad1201 mutants have long pharyngeal muscle contractions and long pharyngeal action potentials with no clear currents associated with the repolarization phase of the action potential (fig. 1).

We cloned exp-2 using a candidate gene approach. Since the pharyngeal electrical defect was in the relaxation phase of the action potential, we hypothesized that exp-2 might encode a potassium channel. Using a BLAST search, we found a potassium channel in the center of the cosmid F12F3, which is within the bounds of the exp-2 position based on physically mapped deficiencies. This gene has the highly conserved potassium channel pore signature sequence MTTVGYGD, is a member of the six transmembrane family of potassium channels, and is most similar to DRK1, a delayed rectifier K+ channel from rat. We showed that F12F3 restores the repolarization phase current in exp-2(ad1201) (fig.1), and that the transgene in a wild-type background gives a slight exp-2 gain-of- function phenotype. Furthermore, sequencing the K+ channel gene from mutants shows a cys to tyr mutation in the sixth transmembrane segment (S6) in the gain-of-function allele exp-2(sa26). The revertant allele exp-2(sa26ad1201) has both this mutation and a gly to arg mutation in the pore domain. 

                  pore                             S6
           ----------------------       ---------------------
....DEEGTPFTSIPAAYWWCIVTMTTVGYGDAVPATTMGKIIASAAIMCGVLVLALPITIIVDNFIKVAQDEQQAE....
                              ^G-R(ad1201)      ^C-Y (sa26)

DRK1 channels with the second gly of the pore region GYG mutated to cys do not express functional channels in Xenopus oocytes, while substitutions in the S6 region of DRK1 sometimes lead to altered inactivation kinetics (R. Joho, pers. comm.). These results are consistent with the gain and loss of function phenotypes we observe in exp-2. Together, these results indicate that exp-2 encodes a potassium channel that is responsible, at least in part, for the correct repolarization timing of the pharynx, as well as other muscles in the worm. Figure 1

figure 1 EPGs from wild-type, ad1201 and ad1201+F12F3 rescued worms. Asterisks indicate relaxation transients in a pump.

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