Yoanne M. Clovis, Ph.D.

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Background

Humans have over 70 potassium channel genes, but only some of these have been linked to disease. In this respect, the KCNQ family of potassium channels is exceptional: mutations in four out of five KCNQ genes underlie diseases including cardiac arrhythmias, deafness, and epilepsy. For example, mutations in KCNQ1 are associated with heart arrhythmias such as long-QT syndrome (LQTS), in which the cardiac action potential is prolonged. Mutations in KCNQ2 and KCNQ3 are associated with benign neonatal epilepsy (1, 2).

Homologs of KCNQ genes are found in a wide range of model organisms, where they often have functions analogous to those in humans (see Appendix). Mice bearing mutations in the mouse gene Kcnq1 exhibit epileptic seizures, coupled with aberrant cardiac events such as long pauses (3). In the Drosophila heart, a null mutation in the fly gene KCNQ, which is homologous to the human gene KCNQ1, mimics both LQTS and the effect of aging on the heart, resulting in longer cardiac contractions, increased inter-pump duration, and lower heart rate (4).

 

Purpose

In this technical note, we investigated the effect of mutations of orthologous KCNQ-like genes in the nematode C. elegans on electrical excitability. kqt-1 is orthologous to the subfamily of human KCNQ genes that comprise KCNQ2 to KCNQ5, and is mainly expressed in the muscles of the pharynx, a rhythmic muscular pump involved in feeding (5). kqt-2, which is  only expressed in intestinal cells, does not possess a human orthologue (5). kqt-3 is orthologous to the human gene KCNQ1 and although it is not expressed in the pharynx, kqt-3 is present in mechanosensory and chemosensory neurons, which can regulate feeding behavior (5). We hypothesized that mutations in kqt-1 and kqt-3 induce abnormalities in pharyngeal pumping that are reminiscent of cardiac arrhythmias. Taking advantage of the NemaMetrix ScreenChip system, we tested this hypothesis by measuring the duration of pharyngeal action potentials and related parameters of pumping behavior.

 

Methods

Strains and cultivation
The null mutant strains kqt-1(aw3), kqt-3a(aw1) and kqt-3b(aw4) were kindly donated by Dr. Aguan Wei (6). kqt-1(aw3) mutants have a 620 bp deletion of the 2.4 Kb long kqt-1 gene. kqt-3a(aw1) and kqt-3b(aw4) mutants have deletions of 1674 bp and 596 bp of the 3.1 Kb long kqt-3 gene, respectively. Synchronized worms were cultivated at 20 oC until the first day of adulthood on plates containing nematode growth medium (NGM) seeded with E. coli (OP50).

Recording electropharyngeograms (EPGs) and data analysis
Worms were pre-incubated for 20-30 min in M9 buffer containing 10 mM serotonin to induce pumping. EPGs were recorded using the NemaMetrix ScreenChip System, following methods described in the ScreenChip User Guide (7). Each recording was 2 to 3 min in duration. Recordings were analyzed using NemAnalysis software which automatically identifies individual pumps and extracts mean pump duration, mean inter-pump interval (IPI) duration and the mean pump frequency for each worm.

 

Results

In kqt-1(aw3) mutants, the absence of functional kqt-1 in pharyngeal muscles was associated with a significant increase in the duration of pharyngeal action potentials (35%; panel A) and length of inter-pump intervals (41%; panel B), together with a reduction in pump frequency (23%; panel C). Also, these mutants occasionally exhibited periods in which pumping appeared to cease altogether for up to three seconds. The absence of functional kqt-3, in the alleles kqt-3a(aw1) and kqt-3b(aw4), was also associated with prolonged action potential duration (Panel A), providing evidence for indirect effects on pharyngeal activity.

Figure 1: Effect of mutations in kqt-1 and kqt-3 on pharyngeal pumping. The EPGs of 1-day adult N2 (dark blue bars) and mutant (light blue bars) worms were recorded for an average of 130 seconds per worm. (A) Mean duration of pharyngeal pumps. (B) Mean duration of inter-pump interval (IPI). Large standard error in the case of kqt-1(aw3) mutants is likely attributable to occasional periods in which pumping ceased. (C) Mean frequency of pharyngeal pumping. Data were plotted as mean ± S.E.M. Statistics: One-way analysis of variance followed by Dunnett’s post-hoc test to compare mutants to N2 worms, n > 17 worms for each mean. Symbols: n.s., not significant; *, p < 0.05; **, p < 0.01.

Figure 1: Effect of mutations in kqt-1 and kqt-3 on pharyngeal pumping. The EPGs of 1-day adult N2 (dark blue bars) and mutant (light blue bars) worms were recorded for an average of 130 seconds per worm.
(A) Mean duration of pharyngeal pumps.
(B) Mean duration of inter-pump interval (IPI). Large standard error in the case of kqt-1(aw3) mutants is likely attributable to occasional periods in which pumping ceased.
(C) Mean frequency of pharyngeal pumping. Data were plotted as mean ± S.E.M. Statistics: One-way analysis of variance followed by Dunnett’s post-hoc test to compare mutants to N2 worms, n > 17 worms for each mean. Symbols: n.s., not significant; *, p < 0.05; **, p < 0.01.

Discussion

We present evidence that two C. elegans genes, kqt-1 and kqt-3 are necessary for normal pharyngeal pumping, consistent with the well-known role of KCNQ potassium channel mutations in generating cardiac arrhythmias in humans and model organisms (see Appendix). Both genes are required for normal pump frequency as elicited by serotonin, whereas kqt-1 is also required for normal inter-pump intervals. We propose that the strains tested here, and the recording methodology used, could be the basis of future screens to identify pharmacological agents to mitigate certain arrhythmias.

Taken together, these data demonstrate the feasibility of using C. elegans to identify candidate genes for heart disease and to assess the effects of new therapeutic agents in high-volume, whole-animal screens in an unbiased manner.

 

Future Directions

The present study focused on using C. elegans as a model for cardiac arrhythmias (10), but C. elegans can also be used as a model for other KCNQ-related phenotypes, including epilepsy (8,9). C. elegans epilepsy models exhibit localized or whole-body contractions which can silence pharyngeal pumping (8,9,11). The ScreenChip system makes pharyngeal pumping easier to quantify than body contraction, providing a novel means of screening for compounds that might prevent epileptic convulsions.

 

References

  1. Singh et. al, Nat. Genet. (1998).
  2. Singh et al., Brain 126, 2726–37 (2003).
  3. Goldman, A. M. et al. , Sci. Transl. Med. 1, 2ra6 (2009).
  4. Ocorr, K. et al., Proc. Natl. Acad. Sci. U. S. A. 104, 3943–8 (2007).
  5. Wei et al., J. Biol. Chem. 280, 21337–21345 (2005).
  6. Wei et al. Nucleic Acids Res. 30, e110 (2002).
  7. NemaMetrix ScreenChip System User Guide – nemametrix.com
  8. Williams et al., Hum. Mol. Genet. 13, 2043–59 (2004).
  9. Petersen et al., PNAS (2004).
  10. Schüler et al., Sci. Rep. (2015).
  11. Pandey et al., Seizure (2010).

 

Appendix

Recapitulation of the physiological effects of KCNQ mutations in various models.

Recapitulation of the physiological effects of KCNQ mutations in various models.