Abstract
Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.
Highlights
The human ether-á-go-go-related gene channels, known as Kv11.1 and KCNH2, are voltage-gated potassium channels that generate rapidly activating, delayed rectifier K+ currents (IKr) in the heart [1, 2]
In addition to the heart, human ether-á-go-go-related gene (hERG) channels are expressed in the brain where they regulate neuronal excitability, and changes in hERG channel currents contribute to increased risk of schizophrenia [6,7,8,9]. hERG channels are frequently overexpressed in cancer, and inhibition of hERG currents has been shown to decrease cancer progression [10,11,12]
We found that the double mutation F860G/L862G in the intrinsic ligand and long QT syndrome (LQTS)-causing mutations R20G and E58D located at the interdomain interface had no effect on the PAS/cyclic nucleotide-binding homology (CNBH) domain binding affinity, while N33T mutation, located at the interdomain interface, and high ionic strength abolished PAS/CNBH domain interactions
Summary
To investigate PAS and CNBH interdomain interactions in hERG channels, we purified PAS and CNBH domains and used. Independent of the PAS/CNBH domain interactions, and have no statistically significant effect on the channel voltage dependence and deactivation kinetics [39] Consistent with these functional observations, our SPR-based direct binding results indicate that R20G (Fig. 2B) and E58D (Fig. 2D) mutations have no statistically significant effect on the binding affinity of the isolated PAS and CNBH domains, as summarized in Table 1 (p > 0.3 for the unpaired Student t test). To test the contribution of electrostatic interactions to the affinity of the PAS and CNBH domain binding, we immobilized CNBH domain on the CM5 sensor chip and recorded SPR response for the free PAS domain applied at 3 μM concentration in solutions containing 150, 300, or 600 mM KCl. Increasing ionic strength of the solution gradually decreased the SPR response, with no binding detected in 600 mM KCl (Fig. 5A). These results indicate that electrostatic interactions play a major role in the PAS/CNBH and PAS/PAS domain binding
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
