Human ether-a-go-go related gene (HERG) encodes a K channel involved in repolarization of the cardiac action potential. Mutations in HERG result in long QT syndrome, a condition that can lead to life-threatening arrhythmias.
unc-103 is the C. elegans homologue of HERG, sharing 70% amino acid identity in the highly conserved transmembrane and pore domains. A gain-of-function mutation (gf), A334T, occurs in an amino acid in transmembrane domain S6 that is conserved in all species variants of ERG isolated to date.
unc-103 (gf) displays a profound mutant phenotype characterized by pharyngeal pumping arrhythmias, egg-laying and locomotion defects, consistent with a hyperpolarizing effect of an inappropriately activated K channel. Quantitation of pharyngeal pauses in (gf) mutant worms reveal a 10 fold increase in pause length, from 0.3 0.1 to 13.0 2.7 seconds in Wild-type and mutant worms respectively (p < 0.05). This pumping defect is partially rescued by the HERG-specific blocker d-sotalol, with 100 mM d-sotalol treatment decreasing pause length by nearly 50% from 14.9 5.1 to 7.6 1.7 seconds in control and drug-treated worms respectively. This result suggests that the drug-binding pocket of ERG may be conserved between worms and humans. Locomotion assays reveal that both the (gf) mutation and a loss-of-function (lf) mutation (
n1213) in
unc-103 result in impaired movement as determined in liquid thrashing assays. Wild-type worms thrashed at a rate of 45.4 0.8 thrashes/sec while the (gf) worm thrashing rate was slowed over ten-fold to 3.1 0.4 thrashes/sec and the (lf) rate was 36.9 1.9 thrashes/sec (p < 0.05 for (gf) and (lf) when compared to Wild-type). Whereas (gf) worms are unable to lay eggs,
unc-103 (lf) worms are impaired in their ability to retain eggs, consistent with a depolarizing effect that may result from the absence of this K channel. These data suggest
unc-103 plays an essential role in feeding, locomotion and egg-laying. In an effort to understand the molecular nature of the mutant phenotype, we have engineered the analogous
unc-103 (gf) mutation into HERG (A653T) and expressed this clone in Xenopus oocytes to study under voltage-clamp conditions. HERG A653T displays a 30 mV hyperpolarizing shift in the I(V) when compared to Wild-type HERG. This profound change in gating kinetics would result in a channel passing considerable outward current at negative membrane potentials. This hyperpolarizing shift may be responsible for setting resting membrane potentials too hyperpolarized in the tissues where it is expressed. Indeed, we find that expression of this mutant channel significantly hyperpolarizes the Xenopus ooctye Vm by 17 mV when compared to Wild-type HERG-expressing oocytes (-80.6 0.8 and -63.6 0.6 for A653T and Wild-type HERG respectively, p < 0.05). We propose that
unc-103 plays a critical role is establishing resting membrane potentials in various tissues and that mutations in this gene disrupt normal resting potentials resulting in the mutant phenotypes observed. We are currently looking at d-sotalol effects on the mutant channel expressed in Xenopus oocytes and plan to use the
unc-103 (gf) worms as a background for a repressor screen to identify interacting proteins.