Acta Pharmacologica Sinica (2011) 32: 465–477 npg © 2011 CPS and SIMM All rights reserved 1671-4083/11 $32.00 www.nature.com/aps Original Article Effects of diltiazem and propafenone on the inactivation and recovery kinetics of fKv1.4 channel currents expressed in Xenopus oocytes Dong ZHANG1, 2, * , Shi-min WANG1, Hui CHEN2, Xue-jun JIANG1, Sheng-ping CHAO2 1Department of Cardiology, Renmin Hospital, Wuhan University, Wuhan 430073, China; 2Department of Cardiology, Zhongnan Hospi- tal, Wuhan University , Wuhan 430071, China Aim: To investigate the effects of diltiazem, an L-type calcium channel blocker, and propafenone, a sodium channel blocker, on the inactivation and recovery kinetics of fKv1.4, a potassium channel that generates the cardiac transient outward potassium current. Methods: The cRNA for fKv1.4ΔN, an N-terminal deleted mutant of the ferret Kv1.4 potassium channel, was injected into Xenopus oocytes to express the fKv1.4ΔN channel in these cells. Currents were recorded using a two electrode voltage clamp technique. Results: Diltiazem (10 to 1000 μmol/L) inhibited the fKv1.4ΔN channel in a frequency-dependent, voltage-dependent, and concen- tration-dependent manner, suggesting an open channel block. The IC50 was 241.04±23.06 μmol/L for the fKv1.4ΔN channel (at +50 mV), and propafenone (10 to 500 μmol/L) showed a similar effect (IC50=103.68±10.13 μmol/L). After application of diltiazem and propafenone, fKv1.4ΔN inactivation was bi-exponential, with a faster drug-induced inactivation and a slower C-type inactivation. Dilti- azem increased the C-type inactivation rate and slowed recovery in fKv1.4ΔN channels. However, propafenone had no effect on either the slow inactivation time constant or the recovery. Conclusion: Diltiazem and propafenone accelerate the inactivation of the Kv1.4ΔN channel by binding to the open state of the channel. Unlike propafenone, diltiazem slows the recovery of the Kv1.4ΔN channel. Keywords: inactivation; recovery; Kv1.4; potassium channel; diltiazem; propafenone; two electrode voltage clamp technique Acta Pharmacologica Sinica (2011) 32: 465–477; doi: 10.1038/aps.2010.234 Introduction is controlled by the slower C-type mechanism[11], which makes Transient outward potassium currents (Ito) contribute to the it physiologically important. early repolarization phase of the cardiac action potential[1, 2]. The L-type calcium channel blocker diltiazem and the Two types of Ito are known: Ito (fast), which shows fast recov- sodium channel blocker propafenone are widely used in clin- ery kinetics, and Ito (slow), which shows slow recovery kinetics ics for the treatment of cardiovascular diseases of hyperten- that are related to accumulated inactivations[3]. As the major sion, cardiac angina (for diltiazem) and arrhythmias[12–14]. The component of Ito (slow), the Kv1.4 channel plays an important therapeutic effects are generally believed to be related to the role in the repolarization of cardiac myocytes. Kv1.4 chan- L-type calcium channel (for diltiazem) and the sodium chan- nels were inactivated by two well-established processes: N- nel (for propafenone). Recent studies demonstrated that and C-type inactivation. N-type inactivation results from the diltiazem inhibited the hKv1.5 channel, which conducts ultra occlusion of the intracellular side of the pore by a “ball and rapid delayed rectifier currents (Ikur), and Ito, encoded by Kv4.3 chain” mechanism formed by the NH2 terminus of the channel by binding to the open and the inactivated states of the chan- molecule[4–9], while C-type inactivation involves conforma- nels[15–17]. There is evidence that diltiazem decreases Kv1.4 tional changes on the extracellular side of the pore[10]. These channel currents expressed in the oocytes of Xenopus laevis[16], two mechanism are coupled[5]; C-type inactivation is more and propafenone was shown to be an open channel antagonist rapid in the presence of N-type inactivation[11] and can be of Kv1.4 channel currents[18], but their detailed characteristics affected by open channel blockers. Recovery from inactivation have not been studied. The present study, in which we used an N-terminal deletion construct of Kv1.4 (Kv1.4ΔN) that lacks rapid N-type inacti- * To whom correspondence should be addressed. [19] E-mail [email protected] vation but exhibits robust C-type inactivation , was there- Received 2010-06-28 Accepted 2010-12-18 fore designed for the following: (1) to study the properties of npg www.nature.com/aps Zhang D et al 466 diltiazem blockade of the fKv1.4ΔN channel; (2) to study the time was used to allow equilibration of the drug with the effect of diltiazem on Kv1.4 channel C-type inactivation and oocytes. After this wash-on period, a series of 500 ms depolar- recovery, and (3) to compare the electrophysiological effects of izing pulses (from -90 mV to +50 mV at a frequency of 1 Hz diltiazem on fKv1.4ΔN with those of propafenone. for 1 min) was employed to ensure a steady-state block before beginning the experimental protocols[20]. Materials and methods Molecular biology Data analysis The constructs and sequences of the cDNA fKv1.4ΔN used in Data were recorded with a personal computer installed with this study have been previously described[19–21] and were a gift pCLAMP 9.0 (Axon, USA) and analyzed using Clampfit 9.0 from professor Randall L RASMUSSON (University at Buf- (Axon, USA) and Microsoft Excel software (Microsoft, USA). falo, SUNY). The construction of fKv1.4ΔN was performed Unless otherwise stated, raw data traces were not leakage or by removal of 2–146 amino acid residues from the N-terminal capacitance subtracted. Data are shown as means±SEM. Sig- domain of Kv1.4, which results in the loss of the fast compo- nificant differences were determined using Student’s paired nent of inactivation but leaves the C-type inactivation path- t-tests. way intact[19–21]. Transcribed fKv1.4ΔN cRNA was prepared in vitro using an mMessage mMachine kit (T3 kit, Ambion, USA). Results Effects of diltiazem on fKv1.4ΔN currents Isolation of oocytes and incubation Voltage-, concentration-, and frequency-dependent blockade of Oocytes were collected from mature female Xenopus laevis diltiazem on fKv1.4ΔN currents frogs (Chinese Academy of Science, Beijing, China). Frogs Figure 1A shows representative fKv1.4ΔN current traces were anesthetized (immersion in 1.5 g/L tricaine) for 30 min, recorded by applying 5 s pulses from -100 mV to +50 mV in 10 followed by surgical removal of the ovarian lobes through a mV increments in the control, in the presence of 250 μmol/L lateral incision in the lower abdomen. The incision was then diltiazem, and after the drug washout. The fKv1.4ΔN currents sutured, and the frog was allowed to recover in a container were substantially inhibited by the application of 250 μmol/L with a small amount of water. When the frogs did not pro- diltiazem and the effect recovered after washout of the drug duce a high quality of oocytes, they were humanely killed via for 10 min. a high dose of tricaine. All procedures were approved by the Peak-voltage relationships from the control, 250 μmol/L Institutional Animal Care and Use Committee of the Wuhan diltiazem-treated and drug washout oocyte groups were plot- University of China. ted against clamp potential in Figure 1B. In this figure, the The follicular layer was removed enzymatically by placing IDIL/ICON ratio was plotted as a function of the membrane 2+ the ovarian lobes in a collagenase-containing, Ca -free OR2 potential. Diltiazem decreased the peak currents at transmem- solution (mmol/L: 82.5 NaCl, 2 KCl, 1 MgCl2 and 5 Hepes, pH brane potentials positive to the activation threshold (-30 mV). 7.4, with 1–1.5 mg/mL collagenase (Type I, Sigma, USA). The The blockade increased steeply in the voltage range coinciding oocytes were gently shaken for about 1 h and washed several with that of channel activation (between -40 mV and -20 mV) 2+ [13] times with Ca -free OR2 solution as previously described . and remained constant at voltages above this range. The peak Finally, defolliculated (stage IV) oocytes were selected and current was blocked by 52.21%±4.63% when the cell mem- placed in ND96 solution (mmol/L): 96 NaCl, 2 KCl, 1 MgCl2, brane was depolarized to +50 mV in 250 μmol/L diltiazem, 1.8 CaCl2 and 5 Hepes, pH 7.4. Each oocyte was injected with and the effect was reversed by 95% after the drug washout. about 25–50 nL of fKv1.4ΔN cRNA using a microinjector (WPI, There was a voltage dependence to the action of diltiazem, a Sarasota) and incubated in an 18 °C environment in ND96 phenomenon typical of open channel block. solution with 100 IU/mL penicillin for over 16 h. Figure 1C shows the concentration dependence of fKv1.4ΔN current inhibition by diltiazem. Inhibition of the currents in a Electrophysiology concentration-dependent manner was measured at the end of The experiment was carried out using a two electrode voltage a 300 ms pulse of +50 mV. A nonlinear least-squares fit of the clamp technique. Oocytes were clamped using a preampli- Hill equation to the individual data points yielded an appar- fier CA-1B (DAGAN, USA), and the current signals were ent dissociation constant, KD, for an open channel blockade of filtered at 2.5 kHz. Microelectrodes were fabricated from 1.5 241.04±23.06 μmol/L (n=5). mm o.d. borosilicate glass tubing using a two-stage puller To evaluate the longer-term effects of exposing the (NARISHIGE, Japan) to produce electrodes with resistances fKv1.4ΔN channel to diltiazem, we applied a series of 500 ms of 0.5–1.0 MΩ when filled with 3 mmol/L KCl.