14Human ether-Ă -go-go-related gene (Kv11.1, or hERG) is a potassium channel that 15 conducts the delayed rectifier potassium current (I Kr ) during the repolarization phase of cardiac 16 action potentials. hERG channels have a larger pore than other K + channels and can trap many 17 unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine, a cyclin-dependent 18 kinase (CDK) inhibitor that also inhibits L-type calcium channels, inhibits open hERG channels 19 but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus 20 oocytes expressing wild-type (WT) or mutant (T623A, S624A, Y652A, F656A) hERG channels 21 demonstrated that, compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 ÂľM R-22 roscovitine was ~ 48 %, 29 %, and 73 % weaker, respectively. In contrast, S624A hERG was 23 inhibited more potently than WT hERG, with an ~ 34 % stronger inhibition. These findings were 24 further supported by the IC 50 values, which were increased for T623A, Y652A and F656A (by 25~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data 26 suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 27 may not be. This relatively unique feature, coupled with R-roscovitine's tolerance in clinical 28 trials, could guide future drug screens. We discuss our findings and how they lend support for the 29 recent Comprehensive In Vitro Proarrhythmia Assay (CiPA) guidelines on the re-evaluation of 30 potentially useful drugs that had failed testing due to unintended interactions with hERG. 31 32 Introduction 33 Human ether-Ă -go-go-related gene, or hERG [Kv11.1], is a voltage-gated potassium 34 channel critical for nerve and cardiac function [1,2]. In the heart, hERG channels initially open 3 35 during the depolarization phase of the cardiac action potential (cAP) but immediately inactivate. 36 Upon cAP repolarization, hERG channels de-inactivate and reopen, which allows the ensuing 37 large K + efflux to speed cAP repolarization [1], limit cardiac excitability, and maintain normal 38 QT intervals [3]. Consequently, mutations in hERG are one of the leading causes of congenital 39 long QT syndrome (cLQTS), with a neonatal incidence rate of up to 1 in 2,500 [4]; abnormal 40 cardiac phenotypes are usually triggered during exercise, arousal, or rest [5].41 hERG channels are tetrameric proteins, with each monomer consisting of six 42 transmembrane alpha helices (S1-S6) and cytoplasmic amino and carboxy termini [6]. Similar to 43 other voltage-gated channels, S1-S4 is considered the primary voltage-sensing region, with S4 44 containing positively-charged residues that move slowly outward to induce the characteristically 45 slow activation kinetics of hERG [7,8]. The four S5 and S6 helices and their intervening 46 sections, including the P-loops and P-helices, form the pore and selectivity filter of the channel 47 [9,10]. The pore is thought to have a region between the pore helix and the S6 segments that may 48 provide p...