Acute desensitization of GIRK current in rat atrial myocytes is related to K+ current flow

Abstract

We have investigated the acute desensitization of acetylcholine-activated GIRK current (I(K(ACh))) in cultured adult rat atrial myocytes. Acute desensitization of I(K(ACh)) is observed as a partial relaxation of current with a half-time of < 5 s when muscarinic M2 receptors are stimulated by a high concentration (> 2 micromol l(-1)) of ACh. Under this condition experimental manoeuvres that cause a decrease in the amplitude of I(K(ACh)), such as partial block of M2 receptors by atropine, intracellular loading with GDP-beta-S, or exposure to Ba2+, caused a reduction in desensitization. Acute desensitization was also identified as a decrease in current amplitude and a blunting of the response to saturating [ACh] (20 micromol l(-1)) when the current had been partially activated by a low concentration of ACh or by stimulation of adenosine A1 receptors. A reduction in current analogous to acute desensitization was observed when ATP-dependent K+ current (I(K(ATP))) was activated either by mitochondrial uncoupling using 2,4-dinitrophenole (DNP) or by the channel opener rilmakalim. Adenovirus-driven overexpression of Kir2.1, a subunit of constitutively active inwardly rectifying K+ channels, resulted in a large Ba2+-sensitive background K+ current and a dramatic reduction of ACh-activated current. Adenovirus-driven overexpression of GIRK4 (Kir3.4) subunits resulted in an increased agonist-independent GIRK current paralleled by a reduction in I(K(ACh)) and removal of the desensitizing component. These data indicate that acute desensitization depends on K+ current flow, independent of the K+ channel species, suggesting that it reflects a reduction in electrochemical driving force rather than a bona fide signalling mechanism. This is supported by the observation that desensitization is paralleled by a significant negative shift in reversal potential of I(K(ACh)). Since the ACh-induced hyperpolarization shows comparable desensitization properties as I(K(ACh)), this novel current-dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia.

Figure 1. Properties of acute desensitization of atrial GIRK current activated by Ach

A, instantaneous reversibility of acute desensitization. Superfusion with ACh-containing (20 μmol l⁻¹) solution was performed as indicated. The response marked by the arrow indicates a full-sized response at 94% deactivation. The dotted line was drawn to indicate baseline current. The rapid deflections in this and subsequent figures represent changes in membrane current caused by voltage ramps from −120 to +60 mV (500 ms) that were applied once every 10 s to determine current–voltage relations. B, inward currents activated by rapid superfusion of a cell with solutions containing 0.5, 1.0 and 20 μmol l⁻¹ ACh. The dotted line in this panel indicates the quasi-steady-state current level. Zero current level in this and subsequent figures is indicated by a bar on the right labelled ‘0’.

Figure 2. Effects of the muscarinic receptor antagonist atropine (A) and the G protein antagonist GDP-β-S (B) on I_K(ACh)

ACh and atropine were applied as indicated in the left panels. GDP-β-S (500 μmol l⁻¹) was included in the pipette-filling solution. Right panels show expanded current recordings from sections labelled in the slow recordings (left).

Figure 3. Inhibition of I_K(ATP) and reduction in desensitization by Ba²⁺ (5 μmol l⁻¹)

A, recordings of ACh-induced inward currents with and without Ba²⁺. B, superimposed and expanded traces from A, vertically scaled to match the peaks. C, summarized data from a total of 8 analogous experiments. Bars represent percentage desensitization determined 5 s after switching to ACh-containing solution (P < 0.002).

Figure 4. Desensitization of I_K(ACh) by low-level activation

A, nonadditivity of current evoked by Ado (10 μmol l⁻¹) plus ACh (20 μmol l⁻¹) and removal of desensitizing component. B and C, two examples of nonadditivity and removal of the desensitizing component of I_K(ACh) evoked by 20 μmol l⁻¹ ACh by low-level activation of current using 0.2 μmol l⁻¹ (B) or 0.2 and 0.5 μmol l⁻¹ ACh (B). Agonists were applied as indicated by horizontal bars.

Figure 5. Activation of I_K(ATP) by DNP negatively interferes with I_K(ACh)

A, representative recording of membrane current from a myocyte with small DNP-activated current. ACh and DNP were applied as indicated. B, background-subtracted current–voltage relations of ACh- and DNP-activated current, normalized to current at −120 mV. C, sample recording of membrane current from a cell with large DNP-activated current and inhibition of I_K(ACh) in parallel to activation of I_K(ATP). The arrows indicate peak I_K(ACh) and the current level 3.5 s after the peak.

Figure 6. Activation of I_K(ATP) and inhibition of I_K(ACh) by rilmakalim

A, representative current recording. B, difference I–V curves of ACh- and rilmakalim-activated current.

Figure 7. Reduction of I_K(ACh) in myocytes overexpressing Kir2.1

A, recording of I_K(ACh) and Ba²⁺-sensitive background current from a GFP-positive myocyte infected with the empty virus (left) and background-subtracted I–V relation of I_K(ACh) (right). B, recording of I_K(ACh) and Ba²⁺-sensitive background current from a GFP-positive myocyte infected with Ad-Kir2.1 (left) and I–V relation of Ba²⁺-sensitive background current (right). C, summarized data from 10 time-matched cells in each group comparing fractional Ba²⁺-sensitive background current (I_Ba) and ACh-activated current (n = 8 for each group).

Figure 8. Increased background current and reduced ACh-activated current in myocytes overexpressing GIRK4 (Kir3.4)

A, representative current recordings from two myocytes infected with the empty virus (A) and Ad-Kir3.4 (B). C, summarized data showing I_K(ACh) and Ba²⁺-sensitive background current (I_Ba) as fraction of total current (n = 8 for each group).

Figure 9. Desensitization affects the reversal potential of I_K(ACh)

A, inward current induced by exposure to ACh (20 μmol l⁻¹). B, current–voltage relation obtained by slow voltage-ramp protocol as outlined in the scheme. The arrow indicates the reversal potential (−41.2 mV). C, current–voltage relations obtained by fast voltage ramps, as outlined in the scheme, at peak I_K(ACh) and 5 s later. These curves were obtained during a second exposure to ACh, with the slow ramp protocol turned off, which resulted in an inward current with identical properties as shown in A. The corresponding times have been labelled a and b in panel A. The arrow corresponds to the reversal potential obtained by the slow ramp (B). D, summarized data from 10 myocytes. The column labelled ‘slow’ represents the mean reversal potential obtained by a slow voltage ramp. Peak and 5 s have the same meaning as in C.

Figure 10. Comparison of desensitization of I_K(ACh) and ACh-induced hyperpolarization in a representative myocyte

A, I_K(ACh) recorded at 20 mmol l⁻¹ (left) and 5 mmol l⁻¹ [K⁺]_o (right). Holding potentials were −90 mV (20 mmol l⁻¹) and −40 mV (5 mmol l⁻¹). B, ACh-induced hyperpolarizations recorded in current-clamp mode.