Experiments for April NemaMetrix Workshop
- Goal: To test the role of kqt-1 and kqt-3 potassium channel subunits for pharyngeal pumping.
- Medical relevance: cardiac arrhythmia, epilepsy
- Rational and background:
Potassium channels serve diverse functions such as shaping the dynamic electrical activity of neurons (Connor, 1980), modulating the immune response (Cahalan and Chandy, 1997) and controlling intestinal function (Koh et al., 1995). Interestingly, many of the 70 potassium channels in C. elegans have close mammalian orthologues. The human genome contains five KCNQ genes that encode a family of K channel subunits. Functional channels are assembled from four subunits, and may be either homo- or heterotetramers, depending on cell type. These channels serve a wide range of physiological roles. In the heart, KCNQ1 is co-assembled with the product of the KCNE1 gene to form the cardiac IKs delayed rectifier-like K current (see figure below).
Mutations in either KCNQ1 or KCNE1, two potassium channel alpha subunits cause inherited long QT syndrome (LQT1 or LQT5, respectively), a condition leading to arrhythmia (Romano-Ward syndrome) and characterized by a delay in ventricular repolarization.
In C. elegans pharynx, mps-4 mimics alQTS: suppressed hyperpolarization, increased number of pumping pauses due to extended muscle excitation caused by delay in repolarization, action potentials of various length and delayed after-depolarization in the absence of mps-4 (Park et al., 2007).
Kqt-1 = KCNQ2-5 homologue (mutants: kqt-1(aw3); kqt-1(aw1))
- The only kqt expressed in C.elegans pharyngeal muscles (Wei et al, 2005)
- Strongly blocked through M1 muscarinic Rc (90%)
- Strongly inhibited by OAG and PMA through PKC activation
- Blocked by linopirdine, drug used to block human KCNQ1 (tested on AD patients, no success)
- muscarinic agonists suppress KCNQ2/KCNQ3 currents via the M1 receptors (Shapiro et al., 2000)
Kqt-3 = KCNQ1 homologue (kqt-3(aw4))
- Expressed mechano-sensory, chemosensory neurons, other head neurons and intestinal cells, not in pharynx
- Mildly blocked through M1 muscarinic Rc (16%)
- Mildly inhibited by OAG and PMA (1000-fold less than kqt-1)
- Blocked by linopirdine , drug used to block human KCNQ1
Kqt-1 and -3 have not yet been tested for pharyngeal phenotype.
KCNE1 (mps-4) only associates with KCNQ1 (kqt-3), not KCNQ2 to 5 (kqt-1). However, several mutations in either of KCNQ2 to 5 genes cause a form of neonatal epilepsy in humans (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998). In C. elegans, epilepsy is characterized by a lack of pharyngeal pumping (Shelly et al., 2004) (due to disrupted GABA transmission). Worms under tonic seizure (rigid, unresponsive to touch but with pharyngeal pumping) are still viable. In addition, kqt-3 is expressed in mechanosensory neurons (Wei et al., 2005), and mechanosensory inputs influence C. elegans pharyngeal activity via Ivermectin Sensitivity Genes (avr- 15, also involved in spitting behavior under light exposure (Bhatla et al., 2015)) (Keane et al., 2003).
- Mutation of kqt-1, which is expressed in pharyngeal muscles, will destabilize formation of K channels, leading to pharyngeal pumping phenotype similar to mps-4 mutants.
- Mutation of kqt-3, expressed in mechanosensory neurons may be indirectly involved in pharyngeal pumping arrest (epilepsy-like behavior) or spitting behavior in response to light exposure, which can be linked to aortic regurgitation syndrome (use avr-15 as a positive control).
- Mutantcontrol:mps-4KO(D74NmutationcorrespondingtohumanD76NmutationofKCNE1) (Park et al, 2007)
kqt-3(aw4), kqt-1(aw3), kqt-1(aw1) were published in Wei et al., 2002, table 1.
mps-4 KO was published in Park et al., 2007: The D74N MPS-4 mutation was produced in wild-type MPS- 4 in pCI-neo by plaque-forming unit- based mutagenesis (Stratagene). The construct was confirmed by automated DNA sequencing and quantified with spectroscopy.
Potentially interesting notes:
- KCNQ2 is not expressed in human heart (http://www.proteinatlas.org/ENSG00000075043- KCNQ2/tissue/heart+muscle).
- KCNQ3 is expressed in 20% of human cardiomyocytes (http://www.proteinatlas.org/ENSG00000184156- KCNQ3/tissue/heart+muscle) but is cited in publications as not expressed in heart at all.
- KCNQ2 and KCNQ3 are expressed in numerous brain regions and sympathetic ganglia (Biervert et al., 1998; Wang et al., 1998; Yang et al., 1998).