Readthrough of long-QT syndrome type 1 nonsense mutations rescues function but alters the biophysical properties of the channel

Abstract

<jats:p>The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause LQT1 (long-QT syndrome type 1) and result in a complete loss of IKs channel function. In the present study we attempted to rescue the function of these mutants, in HEK (human embryonic kidney)-293 cells, by promoting readthrough of their PTCs (premature termination codons) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418 acted to promote full-length channel protein expression from R518X at 100 μM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P&lt;0.05) increase current density and peak-tail current density, at +80 mV for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced from R518X, while similar, were not identical with wild-type as the voltage-dependence of activation was significantly (P&lt;0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible, it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.</jats:p>

Readthrough-Promoting Drugs Gentamicin and PTC124 Fail to Rescue Na v 1.5 Function of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes Carrying Nonsense Mutations in the Sodium Channel Gene SCN5A

“…Increased sustained I Na can cause Long QT syndrome, which is characterized by life-threatening arrhythmia and sudden cardiac death. Indeed, previous studies on readthrough of nonsense mutations in the gene KCNQ1 (encoding the channel responsible for the slow delayed rectifier potassium current) showed changes in the kinetics of the corresponding channel in response to readthrough, 10 highlighting this potential risk. In conclusion, in this study, the readthrough-promoting agents PTC124 and gentamycin failed to suppress translational termination by premature stop codons in hiPSC-CMs carrying the mutations R1638X and W156X in SCN5A.…”

“…have been shown to restore protein expression significantly, [8][9][10][11] although, to date, this has only been evaluated in heterologous expression systems, in which expression of the SCN5A gene was realized by expressing the cDNA in a noncardiac cell. However, heterologous systems lack the complete architecture and components of native cardiomyocytes.…”

Stop-codon and C-terminal nonsense mutations are associated with a lower risk of cardiac events in patients with long QT syndrome type 1

“…The limited experimental data that currently exists for functional effects of stop-codon mutations. First, previously published data show that when expressed without the WT subunit, Q530X and R518X do not produce any functional channels 18, 24 and consistently are associated with deafness in patients 25 . Second, in contrast to our data, when expressed together with wild-type subunits, these mutant channels do not show significant difference from haploinsufficient expression of the channel.…”

“…Second, in contrast to our data, when expressed together with wild-type subunits, these mutant channels do not show significant difference from haploinsufficient expression of the channel. These studies did not compare currents of the WT and the haploinsufficient channel 18 . In our hands, low levels of DNA transfection are necessary to probe differences between WT and haploinsufficient channels in heterologous expression systems.…”

“…Recent studies have focused in repair as possible treatment for inherited channelopathies by read-through of nonsense mutations 13–17 . In particular, with read-through of nonsense mutations also being a target of interest in LQT1 18 , it became important to further investigate the clinical significance of different subtypes of non-missense mutations in LQT1.…”