All animal experimental procedures were performed in accordance with guidelines established by the Committee for Animal Experiments at Kangwon National University. Male New Zealand White rabbits (1.7–2.2 kg) were anesthetized by administration of a pentobarbitone (75 mg/kg) and heparin sodium (120 U/kg) mixture through the ear vein. The thoracic cavity was dissected and the heart was rapidly removed and then transferred to normal Tyrode's solution. The second branches of coronary arteries were carefully isolated from the heart, and cleaned of surrounding tissues under a stereoscope. The endothelium was eliminated by flowing an air bubble into the lumen of each coronary arteries. Single coronary artery smooth muscle cells were isolated using a two-step enzymatic procedure. The first enzymatic treatment was performed by submerging coronary arteries in 1 mL Ca²⁺-free normal Tyrode's solution containing papain (1.3 mg/mL), bovine serum albumin (BSA, 1.6 mg/mL), and dithiothreitol (DTT, 2.0 mg/mL) for 25 min at 36.8℃. After washing in Ca²⁺-free normal Tyrode's solution, the arteries were transferred to Ca²⁺-free normal Tyrode's solution containing collagenase (2.6 mg/mL), BSA (1.6 mg/mL), and DTT (2.0 mg/mL) for 18 min at 36.8℃. Enzyme-treated arteries were carefully agitated using a fire-polished Pasture pipette to obtain single smooth muscle cells. The isolated single cells were stored at 4℃ in Kraft-Brühe (KB) solution and used within 10 h.

The normal Tyrode's solution contained the following (in mM): 133 NaCl, 5.2 KCl, 0.36 NaH₂PO₄, 15.6 glucose, 5.5 HEPES, 0.7 MgCl₂, 1.8 CaCl₂, adjusted to pH 7.35 with NaOH. The KB solution contained the following (in mM): 10 HEPES, 24 L-glutamate, 17 taurine, 68 KH₂PO₄, 16 glucose, 0.6 EGTA, 2.5 MgCl₂, 52 KCl, 50 KOH, adjusted to pH 7.30 with KOH. The pipette-filling solution contained the following (in mM): 113 K-aspartate, 3.6 Mg-ATP, 8 EGTA, 10 HEPES, 2.5 MgCl₂, 4.6 NaCl, 27 KCl, adjusted to pH 7.25 with KOH. Propafenone was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Diphenyl phosphine oxide-1 (DPO-1), guangxitoxin, and linopirdine were purchased from Tocris Cookson (Ellisville, MO, USA) and dissolved in dimethyl sulfoxide (DMSO).

Whole-cell currents were measured from single coronary artery smooth muscle cells using a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA, USA). PatchPro software was used for pulse generationand data acquisition. Patch pipettes were prepared from borosilicate tubes using a PP-830 vertical puller (Narishige Scientific Instrument Laboratory, Tokyo, Japan). The patch pipettes exhibited a resistance of 3–4 MΩ when filled with the pipette-filling solution.

Data analysis was conducted using Origin 7.5 software (Microcal Software, Inc., Northampton, MA, USA). A first-order blocking model was applied to describe the channel-drug interaction kinetics [1516]. The IC₅₀ value and Hill coefficient (n) were calculated using concentration-dependent data, and the values were obtained using the Hill equation:

f=1/1+IC50/Dn

where f indicates the fractional suppression of current (f=1−I_drug/I_control) at the test potential, and [D] indicates the propafenone concentration.

Tail currents, which were induced by a returning potential of −40 mV after short (20–50 ms ms) depolarizing steps from −80 to +60 mV in 10 mV increments, were used to obtain the steady-state activation curves. The recorded tail currents for each potential were normalized to the maximal tail current. Activation curves were yielded using the Boltzmann equation:

y=1/{1+exp(–(V–V1/2)/k)}

where V is the test potential, V_1/2 is the potential for the half-activation point, and k is the slope value.

A two-step voltage protocol was applied to obtain the steady-state inactivation curves. Currents were recorded with a 600-ms test potential of +40 mV after 7-s pre-conditioning steps from −80 mV to +30 mV (10 mV increments) in the absence and presence of propafenone. Inactivation curves were yielded using another Boltzmann equation:

y=1/1+expV–V1/2/k

where V is the test potential, V_1/2 is the potential for the half-inactivation point, and k is the slope value.

All data are presented as the mean±standard error of the mean. Student's t-test was used to determine statistical significance, and p values <0.05 were considered statistically significant.

Kv currents, which were expressed in coronary arterial smooth muscle cells, were recorded with the whole-cell patch clamp technique. The Kv currents were elicited by applying 600-ms step depolarizing pulses from −80 to +60 mV at a holding potential of −80 mV. To record only the Kv current, other K⁺ channels were suppressed by including EGTA (to block BK_Ca channels) and ATP (to block K_ATP channels) in the intracellular solution. Furthermore, the involvement of Kir channels was excluded by using the second branches of coronary arteries, as Kir channels have not been found in these branches. Fig. 1A shows the control Kv currents, which rapidly rose to a peak, followed by slow and partial inactivation. Application of 10 µM propafenone induced inhibition of Kv currents (Fig. 1B). This inhibition was reached within 2 min and partially (~60%) washed out. Fig. 1C shows the steady-state current-voltage (I–V) relationships in the absence and presence of 10 µM propafenone. Application of 10 µM propafenone decreased the Kv currents by 51%, as measured at +60 mV.

To determine whether the inhibition of Kv currents by propafenone was concentration-dependent, several concentrations (0, 0.1, 1, 3, 10, 30, and 100 µM) of propafenone were applied to the external solution. The Kv currents were recorded by applying a repetitive one-step depolarizing pulse to +60 mV from a holding potential of −80 mV. The inhibition of Kv current increased progressively with the concentration of propafenone (Fig. 2A). The concentration dependence of Kv channel inhibition by propafenone was analyzed using the Hill equation, as described in the Materials and Methods section. From the fit of the Hill equation, we obtained an IC₅₀ value of 5.04±1.05 µM and a slope value (Hill coefficient) of 0.78±0.06 (Fig. 2B).

To evaluate whether the inhibitory effect of propafenone affects the gating properties of the Kv channels, we tested the effect of propafenone on the steady-state activation and inactivation kinetics of Kv currents. As described in the Materials and Methods section, the steady-state activation curve was obtained from the tail currents and the data were fitted with the Boltzmann equation. Application of 10 µM propafenone did not affect the steady state activation curve (Fig. 3A). The potentials for the half-activation point (V_1/2) and slope value (k) were −10.03±1.36 mV and 16.44±1.01, respectively, under control conditions, and −8.97±0.95 mV and 15.72±0.98, respectively, in the presence of 10 µM propafenone.

We also tested the influence of propafenone on the steady-state inactivation curve. The steady-state curve was acquired using a two-step voltage protocol as described in the Materials and Methods section. Similar to the results for the activation curve, propafenone did not change the steady-state inactivation curve (Fig. 3B). The potentials for the half-inactivation point (V_1/2) and slope value (k) were −27.67±0.47 mV and 2.52±0.42, respectively, under control conditions, and −28.82±0.63 mV and 3.01±1.46, respectively, in the presence of 10 µM propafenone. These data suggest that propafenone does not affect the gating properties of Kv channels.

To determine whether the inhibitory effect of propafenone on Kv channels was use-dependent, we repetitively elicited Kv currents with +60 mV pulses 20 times at frequencies of 1 and 2 Hz. The amplitudes of the Kv currents measured at the 20^th pulse were slightly (10% and 17%) lower at 1 and 2 Hz, respectively, than those induced by the first pulse (Figs. 4A and B). In the presence of 10 µM propafenone, the inhibition of Kv currents was increased to 16% and 25% at 1 and 2 Hz, respectively. The overlapping raw traces of Kv currents elicited with trains of voltage steps together with the each Kv current trace elicited by the 1^st, 2^nd, and 3^rd voltage steps are shown in the upper panels of Figs. 4A and B. The first depolarizing voltage step evoked most of the steady inhibition of the Kv current, indicating that the propafenone-induced inhibition of Kv current is largely through state-independent way. However, some small but sizable portion of the use-dependent inhibition was also evident. From these results, we concluded that propafenone inhibited the Kv current primarily through state-independent manner together with some portion of use-dependency.

To further evaluate the use-dependent inhibition of Kv currents by propafenone, we examined recovery times from the inactivation of Kv currents in the absence and presence of propafenone. During the 150 ms-depolarizing voltage-steps, small but sizable portion of Kv channels are inactivated, and the gradual decline of the Kv currents by the trains of voltage steps shown in Fig. 4 reflects the accumulation of this small portion of inactivation. Therefore, the experiment in Fig. 5 was designed with the same duration of voltage steps to examine recovery time courses of the Kv currents after the 150 ms-voltage steps in the absence and presence of propafenone. The recovery kinetics of the Kv currents under the control conditions and in the presence of 10 µM propafenone were measured using a double-pulse protocol as described in Fig. 5 inset. The interval time was increased from 30 to 7,000 ms. As shown in Fig. 5, the recovery time course was slowed in the presence of propafenone. Data were adapted to a single exponential function, yielding recovery time constants of 1,391.17±267.91 ms under control conditions and 1,741.45±181.74 ms in the presence of 10 µM propafenone. The recovery time constants were statistically significant. This state-dependent effect of propafenone (i.e., retardation of recovery from inactivation after 150 ms-depolarizing pulse) may contribute to the use-dependency shown in Fig. 4.

To determine the major Kv subtypes involved in propafenone-induced inhibition of Kv channels, we pre-treated with Kv1.5 inhibitor (DPO-1, 1 µM), Kv2.1 inhibitor (guangxitoxin, 30 nM), or Kv7 inhibitor (linopirdine, 10 µM) before applying propafenone. The application of DPO-1 reduced the Kv current by ~40% (Fig. 6A). However, the additional application of propafenone did not change the degree of inhibition (Fig. 6B, 56.2% inhibition). Similar to the results for DPO-1, the application of guangxitoxin and linopirdine immediately decreased the Kv current by 22% and 14%, respectively (Figs. 6C and E). The additional application of propafenone induced further inhibition; however, the degree of further inhibition did not differ from that of propafenone alone (Fig. 6D, 49.9% inhibition and Fig. 6F, 43.1% inhibition vs. propafenone alone, 49.4% inhibition). These results suggest that the major targets of inhibitory action by propafenone are not of the Kv1.5, Kv2.1, or Kv7 subtype.