NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Abstract

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (-34 ± 4 mV) is similar to wild-type (WT) (-37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (-131 ± 4 mV for K525C and -120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.

NS1643 enhances ionic currents in a G604S‐WT hERG co‐expression system associated with long QT syndrome 2

“…Combining biophysical, computational, and electrophysiological evidence, they proposed that NS1643 is coordinated in a putative binding site in proximity to L529 and K525 in WT or mutant hERG channels. 31,32 In our present study, we did not observe that NS1643 showed any effect on activation and deactivation of hERG channels in cells co-expressing G604S-and WT-hERG.…”

Molecular Basis of Cardiac Delayed Rectifier Potassium Channel Function and Pharmacology

“…In addition to ginsenoside Rg3 75 , mallotoxin 83 and KB130015 84 also shift the voltage dependence of hERG1 activation to more negative potentials; however, the binding site for these agents has not yet been defined. NS1643 shifts the voltage dependence of activation to more negative potentials and of inactivation to more positive potentials, but effects vary depending upon the heterologous expression system employed 85–87 . The binding site for NS1643 has eluded definitive identification, but based on detailed molecular modeling and analysis of L529I hERG1 mutant channels, it is likely that NS1643 interacts indirectly with the VSD to facilitate opening of hERG1 channels 88 .…”

Computational membrane biophysics: From ion channel interactions with drugs to cellular function

“…In the case of the NS1643 activating effect, the model shows that the drug should bind to the open state and at least two early closed states; and modify the voltage dependence of the transition rates among the states to reproduce the experimental behavior. Interestingly, these mechanistic findings together with mutagenesis experiments and MD structural analysis suggested a binding site around the voltage sensor domain of hERG imbedded in lipid membrane as a mechanism to alter the voltage-gated properties of hERG[138]. …”