Apamin does not inhibit human cardiac Na+ current, L-type Ca2+ current or other major K+ currents

Abstract

Background: Apamin is commonly used as a small-conductance Ca2+-activated K+ (SK) current inhibitor. However, the specificity of apamin in cardiac tissues remains unclear.

Objective: To test the hypothesis that apamin does not inhibit any major cardiac ion currents.

Methods: We studied human embryonic kidney (HEK) 293 cells that expressed human voltage-gated Na+, K+ and Ca2+ currents and isolated rabbit ventricular myocytes. Whole-cell patch clamp techniques were used to determine ionic current densities before and after apamin administration.

Results: Ca2+ currents (CACNA1c+CACNB2b) were not affected by apamin (500 nM) (data are presented as median [25th percentile;75th percentile] (from -16 [-20;-10] to -17 [-19;-13] pA/pF, P = NS), but were reduced by nifedipine to -1.6 [-3.2;-1.3] pA/pF (p = 0.008). Na+ currents (SCN5A) were not affected by apamin (from -261 [-282;-145] to -268 [-379;-132] pA/pF, P = NS), but were reduced by flecainide to -57 [-70;-47] pA/pF (p = 0.018). None of the major K+ currents (IKs, IKr, IK1 and Ito) were inhibited by 500 nM of apamin (KCNQ1+KCNE1, from 28 [20]; [37] to 23 [18]; [32] pA/pF; KCNH2+KCNE2, from 28 [24]; [30] to 27 [24]; [29] pA/pF; KCNJ2, from -46 [-48;-40] to -46 [-51;-35] pA/pF; KCND3, from 608 [505;748] to 606 [454;684]). Apamin did not inhibit the INa or ICaL in isolated rabbit ventricular myocytes (INa, from -67 [-75;-59] to -68 [-71;-59] pA/pF; ICaL, from -16 [-17;-14] to -14 [-15;-13] pA/pF, P = NS for both).

Conclusions: Apamin does not inhibit human cardiac Na+ currents, L-type Ca2+ currents or other major K+ currents. These findings indicate that apamin is a specific SK current inhibitor in hearts as well as in other organs.

Figure 1. The endogenous K⁺ currents of HEK 293 cells.

(A) The representative tracing obtained with ramp protocol shown in the inset at time point indicated by arrow a in (B). The pipette and bath solutions are the same as the ones used in measuring I _SK2. (B) The time course of I _SK2 measured at 0 mV. (C) The representative tracings obtained with the pulse protocol shown in the inset with the pipette and bath solution used in measuring I _Ks. (D) The representative tracings obtained with the pulse protocol shown in the inset with the pipette and bath solutions used in measuring I _Kr, I _K1 and I _to.

Figure 2. Effect of apamin on I _SK2 in transfected HEK 293 cells.

(A) The representative I _SK2 tracings obtained by the descending voltage ramp protocol shown in the inset before (a) and after (b) apamin at time points indicated by arrows a and b in (B). (B) The time course of I _SK2 at 0 mV. (C) The summary of current density before and after apamin.

Figure 3. Effects of apamin on I _Na and I _Ba in transfected HEK 293 cells.

(A) The representative I _Na tracings obtained by the pulse protocol shown in the inset before apamin (a), after apamin (b) and after flecainide (c) at time points indicated by arrows a through c, respectively, in (B). (B) The time course of peak I _Na measured at –10 mV. (C) The summary of drug effects normalized to baseline. (D) The representative I _Ba tracings at 0 mV obtained by the pulse protocol shown in the inset before apamin (a), after apamin (b) and after nifedipine (c) at time points indicated by arrows a through c, respectively, in (E). (E) The time course of peak I _Ba measured at 0 mV. (F) The summary of drug effects normalized to baseline.

Figure 4. Effects of different concentrations of apamin on the rundown course of I _Ks in transfected HEK 293 cells.

(A) An observation experiment without apamin treatment showing time-dependent rundown, obtained with the pulse protocol shown in the inset with chromanol 293B at the end. (B) The representative time course of I _Ks treated with different concentrations of apamin. (C) The time constant (τ) of the rundown curve with (n = 10) and without (n = 3) apamin.

Figure 5. Effects of apamin on I _Ks and I _Kr in transfected HEK 293 cells.

(A) The representative tracings of I _Ks obtained by pulse protocol shown in the inset before apamin (a), after apamin (b) and after chromanol (c) at time points indicated by arrows a through c, respectively, in (B). (B) The time course of peak I _Ks at 40 mV. (C) The summary of drug effects normalized to baseline. (D) The representative tracings of I _Kr obtained by a pulse protocol shown in the inset before apamin (a), after apamin (b) and after E4031 (c) at time points indicated by arrows a through c in (E). (E) The time course of peak I _Kr at 20 mV. (F) The summary of drug effects normalized to baseline.

Figure 6. Effects of apamin on I _K1 and I _to in transfected HEK 293 cells.

(A) The representative tracings of I _K1 by ascending voltage ramp protocol shown in the inset before apamin (a), after apamin (b) and after CsCl (c) at time points indicated by arrows a through c, respectively, in (B). (B) The time course of I _K1 at –100 mV. (C) The summary of drug effects normalized to baseline. (D) The representative tracings of I _to obtained by a pulse protocol shown in the inset before apamin (a), after apamin (b) and after 4-AP (c) at time points indicated by arrows a through c, respectively, in (E). (E) The time course of peak I _to at 20 mV. (F) The summary of drug effects normalized to baseline.

Figure 7. Effects of apamin on I _Ca and I _Na in rabbit cardiomyocytes.

(A) The representative I _Ca tracings obtained by a pulse protocol shown in the inset before apamin (a), after apamin (b) and after nifedipine (c) at time points indicated by arrows a through c, respectively, in (B). (B) The time course of peak I _Ca measured at 0 mV. (C) The summary of drug effects normalized to baseline. (D) The representative tracings of I _Na obtained by a pulse protocol shown in the inset before apamin (a), after apamin (b) at time points indicated by arrows a and b, respectively, in (E). (E) The time course of peak I _Na at –40 mV. (F) The summary of current densities before and after apamin.