Inhibitory actions of the phosphatidylinositol 3‐kinase inhibitor LY294002 on the human Kv1.5 channel

Abstract

*These authors made equal contribution to this studyBackground and purpose: Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (IKur), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K). Experimental approach: hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method. Key results: LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC50 of 7.9 mmol·L -1 ). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k+1) and unbinding (k-1) rate constants were calculated to be 1.6 mmol·L -1-1 ·s -1 and 5.7 s -1 respectively. Inhibition by LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A. Conclusions and implications: LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for LY294002.

Interaction of propofol with voltage-gated human Kv1.5 channel through specific amino acids within the pore region

“…As summarized in Figs. 2D and 2E, 100 M propofol significantly shifted V h to a negative potential but had little effect on k, which has also been shown for the blocking action of mibefradil (Perchenet and Clément-Chomienne, 2000), papaverine (Choe et al, 2003) and LY294002 (Wu et al, 2009) on the hKv1.5 current.…”

“…Electrophysiological recordings and data analysis 5 Whole-cell membrane currents (Hamill et al, 1981) were recorded from green fluorescent protein (GFP)-positive cells 48 h after transfection, using an EPC-8 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany). The recording technique used in this study was essentially the same as that described previously (Ding et al, 2014;Wu et al, 2009). The firepolished patch electrodes were fabricated from 1.5-mm capillary glass (Narishige Scientific Instrument Lab., Tokyo, Japan) using a P-97 horizontal puller (Sutter Instrument, Novato, CA, USA).…”

“…The hKv1.5 mutants (H463C, T480A, I502A, I508A, L510A and V516A) were constructed using polymerase chain reaction (PCR)-based site-directed mutagenesis with QuikChange Kit (Stratagene, La Jolla, CA, USA), as previously described (Ding et al, 2014;Wu et al, 2009). All PCR products were fully sequenced (ABI3100x/, Applied Biosystems, Foster City, CA, USA) to confirm the presence of appropriate mutations.…”

“…Furthermore, alanine-scanning mutagenesis studies of hKv1.5 channels have detected several amino acids that reside within the pore region as putative binding sites for the open channel blockers (Decher et al, 2006(Decher et al, , 2004Eldstrom et al, 2007;Franqueza et al, 1997;Herrera et al, 2005;Wu et al, 2009;Yeola et al, 1996). Thus, hKv1.5 channel has been utilized as 4 useful tool to investigate and identify the putative binding sites of various drugs within the channel protein.…”

Open channel block of Kv1.5 currents by citalopram

“…4) The actions of citalopram in inhibiting Kv1.5 were usedependent, with effects enhanced at higher rates of channel activation. One of the features of open channel blockers is a use-dependent inhibition because the blockers would have a higher chance to bind to channel pores as the channels open more frequent ly [21,25,26,28] . This is consistent with the actions of citalopram on the open state of Kv1.5.…”