Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node

Abstract

The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization.

Fig. 1

Activation of AVN I_KACh and sensitivity to M2 receptor inhibition and tertiapin-Q (A) Left panel shows voltage protocol used: from a holding potential of −40 mV, a step to +20 mV for 50 ms preceded a descending voltage-ramp to −120 mV over 250 ms. The start-to-start interval between successive applications of the protocol was 2 s. Right panel shows representative currents elicited in control (normal Tyrode’s) solution (black trace) and immediately following rapid superfusion of 1 μM ACh (grey trace). (B) Mean current–voltage (I–V) relation for current activated by 1 μM ACh (n = 6; obtained by digital subtraction of current in control from that in ACh and normalised to cell membrane capacitance). The black trace denotes the mean current values at each voltage, with grey traces indicating ±SEM values. (C) Mean current–density (pA/pF) of the maximal I_KACh at −120 mV (1 μM ACh sensitive current) in the presence of ACh alone (black bar; n = 6), with 1 μM of the M2 muscarinic receptor inhibitor AFDX-116 (grey bar; n = 5), and in the presence of 300 nM of the GIRK channel inhibitor tertiapin-Q (white bar; n = 6). Asterisks denote statistically significant differences (^∗∗∗p < 0.001).

Fig. 2

Time-course of fade of the ACh response. (A and B) show representative continuous time-plots of current at +20 mV (A) and −120 mV (B) before and during exposure to 1 μM ACh. Time of ACh application is denoted by the horizontal bar in panel A. (C and D) show mean data-plots (n = 6) in which for each cell the net current amplitude was normalised to the maximum response in the presence of ACh at +20 mV (C) and −120 mV (D). Data were fitted by a two-phase decay equation. At +20 mV 56.1 ± 8.8% of the declining current was characterised by a fast rate of decline (K_fast) of 0.67 ± 0.42 s⁻¹ (equivalent to a τ_f of 1.5 s) whilst the remainder of current decline was characterised by a slow rate of decline (K_slow) of 0.029 ± 0.012 s⁻¹ (equivalent to a τ_s of 34.5 s). At −120 mV 62.2 ± 6.7% of current decline was characterised by a K_fast of 0.50 ± 0.20 s⁻¹ (p > 0.7 versus +20 mV; equivalent to a τ_f of 2 s), whilst the remainder of current decline was characterised by a K_slow of 0.013 ± 0.011 s⁻¹ (p > 0.3; equivalent to a τ_s of 76.9 s).

Fig. 3

Fade of ET-1 activated K⁺ current. (A) shows representative continuous time-plots of current at +20 mV (upper panel) and −120 mV (lower panel) before and during exposure to 10 nM ET-1. Time of ET-1 application is denoted by the horizontal bar in upper panel. Voltage protocol same as Fig. 1A. (B) shows mean current density–voltage relation for ET-1 activated current (n = 6). (C, D) show mean data indicating time-course of decline of response to ET-1 at +20 mV (C) and −120 mV (D). Data were normalised as for ACh in Fig. 2. The fade in response exhibited monophasic decline with rate constants of 0.018 ± 0.001 s⁻¹ and 0.019 ± 0.001 s⁻¹ for +20 and −120 mV, respectively (p > 0.4; equivalent to τ values of 55.5 and 52.6 s).

Fig. 4

Relationship between K⁺ current response magnitude and decline rate, and effect of combined ACh and ET-1 application. (A and B) Plots of time constants of current fade (A: τ_fast (τ_f); B τ_slow (τ_s)) against magnitude of initial response to 1 μM ACh (n = 6; filled circles). Open circles show data for 0.1 μM ACh. For this concentration of ACh 3 cells exhibited biphasic decay and 3 cells monophasic decay; for the latter, the single τ values are plotted in B. (C) Plot of time constant of current fade against magnitude of initial response to 10 nM ET-1 (n = 6; filled circles). (A)–(C) time-course measured for responses at −120 mV. (D) Plot of E_rev for I_KACh activated by 1 μM ACh at the initial peak of the response (black bar), at 6 s following the maximal response (grey bar) and at 2 min following maximal response (open bar). There was no significant difference in E_rev values at the different time-points (n = 6; p > 0.1). (E and F) Effects of 10 nM ET-1 following prior exposure to 1 μM ACh. (E) shows representative currents (plotted as current density against voltage) for responses in the same cell to 1 μM ACh (I_KACh) measured as ACh-activated current at maximal response (black trace) and 2 min after the maximal response (light grey trace). The dark grey trace shows maximal current in 10 nM ET-1 (plotted as ET-1 activated current compared to control) following 2 min exposure to ACh. In the concomitant presence of ACh, ET-1 elicited little additional current. (F) Maximal current densities for current at −120 mV for ACh-activated current (black bar; n = 6), ET-1 (grey bar; n = 6,) and the ET-1 difference current (ET-1 minus ACh) when ET-1 was applied following fade of the ACh response. Asterisks denote statistical significance (^∗p < 0.05, ^∗∗p < 0.01 and ^∗∗∗p < 0.001).