Thouta S et al. (2018), Investigating the state dependence of drug bind...

XB-ART-56355

Sci Rep 2018 Mar 21;81:4962. doi: 10.1038/s41598-018-23346-x.

Show Gene links Show Anatomy links

Investigating the state dependence of drug binding in hERG channels using a trapped-open channel phenotype.

Thouta S , Lo G , Grajauskas L , Claydon T .

???displayArticle.abstract???
The hERG channel is a key player in repolarization of the cardiac action potential. Pharmacological blockade of hERG channels depletes the cardiac repolarization reserve, increasing the risk of cardiac arrhythmias. The promiscuous nature of drug interactions with hERG presents a therapeutic challenge for drug design and development. Despite considerable effort, the mechanisms of drug binding remain incompletely understood. One proposed mechanism is that high-affinity drug binding preferentially occurs when channels are in the inactivated state. However, this has been difficult to test, since inactivation is rapid in hERG and access to the drug binding site is limited by slower opening of the activation gate. Here, we have directly assessed the role of inactivation in cisparide and terfenadine drug binding in mutant (I663P) hERG channels where the activation gate is trapped-open. We firstly demonstrate the utility of this approach by showing that inactivation, ion selectivity and high affinity drug binding are preserved in I663P mutant channels. We then assess the role of inactivation by applying cisapride and terfenadine at different membrane voltages, which induce varying degrees of inactivation. We show that the extent of block does not correlate with the extent of inactivation. These data suggest that inactivation is not a major determinant of cisapride or terfenadine binding in hERG channels.

???displayArticle.pubmedLink??? 29563525
???displayArticle.pmcLink??? PMC5862968
???displayArticle.link??? Sci Rep

Species referenced: Xenopus laevis
Genes referenced: kcnh2

???attribute.lit??? ???displayArticles.show???

	Figure 1. hERG I663P trapped open channels exhibit inactivation that is similar to that in WT channels. (A–C) Typical hERG WT, I663P and I663P/S620T current traces evoked during 4 s repolarizing voltage steps from +40 to −120 mV applied following a 500 ms step to +60 mV to activate channels (holding potential was −80 mV for WT and −30 mV for I663P and I663P/S620T). (D) Fully activated hERG WT, I663P and I663P/S620T I-V relations constructed from peak tail currents. Current amplitudes were normalized in order to compare rectification in the two constructs. (E) Voltage-dependence of inactivation of hERG WT and I663P channels. Rectification factor was calculated (see Materials and Methods) from the fully activated tail current data in (D). Data were fitted with a Boltzmann function. V1/2 and k values were −62 ± 3.1 and 17 ± 0.4 mV for WT (n = 6), and −58.6 ± 0.5 and 5.4 ± 0.5 mV for I663P (n = 6), respectively.
	Figure 2. WT-like ion selectivity is preserved in I663P trapped open channels. (A and B) Plot of the permeability ratio (PX/PK) for each ion (see Materials and Methods section) in hERG WT (A) and I663P (B) channels. PX/PK ratios for WT were: PLi/PK = 0.02 ± 0.002; PNa/PK = 0.06 ± 0.01; PRb/PK = 1.2 ± 0.01; and PCs/PK = 0.42 ± 0.01 (n = 5). PX/PK ratios for I663P were: PLi/PK = 0.03 ± 0.004; PNa/PK = 0.06 ± 0.01; PRb/PK = 0.97 ± 0.03; and PCs/PK = 0.47 ± 0.09 (n = 3). n numbers are shown in parenthesis. The error bars are displayed behind the data points and some are so small that they are not visible.
	Figure 3. High affinity block by terfenadine and cisapride is preserved in I663P trapped open channels. (A–D) Typical WT and I663P currents recorded with 30 mM external K+ evoked during repetitive application of the voltage protocol shown in the insets and in the presence of the indicated concentration of terfenadine (A and B) or cisapride (C and D). The pulse frequency was 0.14 Hz, such that channels were held at the holding potential for 1 s between successive sweeps. In both WT and I663P channels, peak tail current was inhibited in a concentration dependent manner. Traces shown represent steady-state conditions at each concentration. (E and F) Concentration-effect relationship for block of WT and I663P channels by terfenadine (E) and cisapride (F), plotted from peak tail current amplitudes recorded in experiments such as in (A–D). Peak tail current amplitudes in the presence of drug were normalized to peak tail currents in the absence of drug. Fits of WT data to the Hill equation (see Material and Methods) yielded values for IC50 and n of: 4.4 ± 3.0 µM and 0.7 ± 0.04 for terfenadine block (n = 5); 1.1 ± 2.0 µM and 1.3 ± 0.09 for cisapride block (n = 5). Fits of I663P data yielded equivalent values of: 4.0 ± 4.0 µM and 0.4 ± 0.03 for terfenadine block (n = 4); 0.85 ± 0.2 µM and 0.7 ± 0.05 for cisapride block (n = 4).
	Figure 4. State-dependence of drug binding in trapped open hERG I663P channels. (A and B) Plots of percentage block by terfenadine (A) or cisapride (B) versus membrane voltage for I663P channels collected using the protocols shown. Pulse frequency was 0.5 Hz, such that channels were held at the holding potential indicated for 1.5 s between sweeps. n numbers are shown in parenthesis. Dashed lines in (A and B) represent the rectification factor calculated from hERG I663P fully activated currents (Fig. 1E).

References [+] :

Barrows, Extracellular potassium dependency of block of HERG by quinidine and cisapride is primarily determined by the permeant ion and not by inactivation. 2009, Pubmed, Xenbase