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1 Department of Physiology, Ruhr-University Bochum, D-4480 Bochum, Germany
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(Received 2 August 2004;
accepted after revision 27 September 2004;
first published online 30 September 2004)
Corresponding author L. Pott: Department of Physiology, Ruhr-University Bochum, D-44780 Bochum, Germany. Email: lutz.pott{at}rub.de
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Introduction |
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subunits released from heterotrimeric G proteins upon agonist stimulation of appropriate 7-helix receptors. It is generally believed that these channels are preferentially – but not exclusively – activated by ß released from pertussis-toxin-sensitive Gi/o (Bender et al. 1998; Leaney et al. 2000; Wellner-Kienitz et al. 2001). Upon rapid application of a suitable receptor agonist, GIRK current in myocytes and neurones but also in heterologous expression systems is activated on a time scale in the order of 1 s or less, depending on receptor species, receptor density and agonist concentration (Wellner-Kienitz et al. 2000; Bender et al. 2002; Hommers et al. 2003). In the presence of the agonist, activation is followed by a decrease in current, termed desensitization that is also dependent on these parameters. A comparable fade of ACh-induced bradycardia has been observed in the isolated sino-atrial node (Boyett & Roberts, 1987). Desensitization, which is a phenomenological rather than a mechanistical term, occurs in virtually all receptor–effector systems and is important for cellular adaptation to external inputs. In the system under study desensitization, as revealed by the decrease in current and agonist sensitivity during or following agonist exposure, has been shown to consist of several components, depending on the species of the receptor, concentration of agonist(s) and duration of agonist exposure (Bünemann et al. 1996a). Whereas slow components of GIRK current desensitization observed after exposure to agonist on a time scale of minutes to hours are likely to occur on the receptor level, as described for many other G protein coupled receptors (GPCR) (Shui et al. 1997; Bünemann et al. 1999), a fast component (‘fast’ or ‘acute’ desensitization) is unique to the receptor–GIRK channel pathway. This component is characterized by a relaxation of current within seconds immediately following exposure to a high concentration of agonist, provided the expression level of the corresponding GPCR is high enough (Wellner-Kienitz et al. 2000; Bösche et al. 2003a). Moreover, it appears to be heterologous between different receptor species (Bünemann et al. 1996b; Wellner-Kienitz et al. 2001). Although it is commonly accepted that this component of desensitization is localized downstream of the receptor, the underlying mechanism is still a matter of debate (Kurachi et al. 1987; Shui et al. 1998; Chuang et al. 1998; Kim, 1993; Saitoh et al. 2001).
In the present study we show that in atrial myocytes changes in GIRK current analogous to acute desensitization can be induced by opening or induction of other types of K+ channels and are dependent on K+ current flow rather than stimulation of a GPCR or activation of GIRK channels. We provide evidence that acute desensitization is caused by a reduction in electrochemical driving force resulting from K+ current flow, regardless of the K+ current pathway. Comparison of acute desensitization of GIRK current and ACh-induced hyperpolarization demonstrates that acute desensitization determines the time course of the hyperpolarization, i.e. the physiological signal. The present data are in line with an accompanying study demonstrating a negative interference between different inwardly rectifying K+ currents in atrial and ventricular myocytes most likely via a subsarcolemmal K+ gradient (Wellner-Kienitz et al. 2004).
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Methods |
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The method of enzymatic isolation of adult rat atrial myocytes and serum-free culture conditions have been described in detail elsewhere (e.g. Bünemann et al. 1996a). Atrial and ventricular myocytes were cultured in 35 mm culture dishes under identical conditions. If not stated otherwise, myocytes were used experimentally from about 24 h until 5 days after isolation. Time in culture did not affect the key properties of the membrane currents investigated.
Solutions and chemicals
For whole-cell measurements of membrane currents an extracellular solution of the following composition was used (mmol l–1): NaCl 120; KCl 20; CaCl2 0.5; MgCl2 1.0; Hepes/NaOH 10.0, pH 7.4. The pipette solution contained (mmol l–1): potassium aspartate 110; KCl 20; NaCl 5.0, MgCl2 7.0; Na2ATP 5.0; EGTA 2.0; GTP 0.025; Hepes/KOH 20.0, pH 7.4. The K+ reversal potential under these conditions was calculated as –48 mV. Standard chemicals were from Merck (Darmstadt, Germany). EGTA, Hepes, MgATP, GTP, acetylcholine-chloride, adenosine, pinacidil, glibenclamide and 2,4-dinitrophenole (DNP) were from Sigma (Deisenhofen, Germany). Rilmakalime was obtained from Dr H. Gögelein (Aventis, Frankfurt, Germany).
Current measurement
Membrane currents were measured at ambient temperature (22–24°C) using whole-cell patch clamp as described in detail previously (Wellner-Kienitz et al. 2000). If not otherwise stated, cells were voltage clamped at a holding potential of –90 mV, i.e. negative to EK, resulting in inward K+ currents. Current–voltage relations were routinely determined by means of voltage ramps between –120 and +60 mV within 500 ms. Rapid exposure to agonist-containing solutions was performed by means of a custom-made solenoid-operated flow system.
Adenovirus constructs and gene transfer
The pAd-Easy-1 and shuttle plasmid (pAd-Track-CMV) were kindly provided by Dr B. Vogelstein (Johns Hopkins University, Baltimore, MD, USA). Production and purification of the recombinant virus were performed as described in detail by He et al. (1998). Briefly, the cDNA of rat Kir2.1 subunit (obtained from Dr A. Karschin, Würzburg, Germany), was subcloned into pAd-Track-CMV, using HindIII and EcoRV, to yield pAdTrack-Kir2.1. Recombinant adenovirus (Ad-Kir2.1) was generated by homologous recombination between pAdTrack-Kir2.1 and pAd-Easy-1 in E. coli. An adenoviral vector containing rat GIRK4 (Kir3.4) cDNA, provided by Dr Y. Kurachi, was constructed correspondingly (Ad-Kir3.4) using KpnI and XbaI.
For infection, cells were incubated for 3 h, starting about 24 h after plating, with 1 ml culture medium containing approximately 104 transducing particles. Electrophysiological recordings were made on days 3 and 4 after exposure to the virus. Infected cells (
80%) were identified by epifluorescence of green fluorescent protein (GFP). Time-matched GFP-positive cells infected with the empty (GFP-encoding) virus served as controls.
Statistical analysis
Wherever possible, data are presented as mean ± S.E.M. and were analysed using Student's unpaired t test. P < 0.05 was considered significant.
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Results |
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A key property of acute desensitization, as illustrated in Fig. 1A, is represented by its quasi-instantaneous reversibility. As soon as deactivation upon washout of agonist is complete, a full-sized current can be activated by a subsequent challenge by agonist. On average when the current was deactivated to < 10% of its peak following washout of ACh, the response to a second challenge by ACh was 95.3 ± 1.7%
(n
= 8) Acute desensitization, distinguished from other components of desensitization by this criterion, in terms of a distinct relaxation of current from a peak to a steady-state requires a saturating concentration of agonist and a high expression level of the activating receptor, which in native atrial myocytes is the case only for the M2AChR (Bösche et al. 2003a). The dependence of acute desensitization on ACh concentration is illustrated in Fig. 1B, which shows responses to three different concentrations of ACh (0.5, 1 and 20 µmol l–1). The increase in peak IK(ACh) at concentrations 0.5 µmol l–1 corresponds to the development of an increasing ‘desensitizing component’. In a series of paired measurements mean desensitization at 0.5 µmol l–1 ACh was 5.1 ± 1.5% as compared with 28.5 ± 2.6% at saturating ACh concentration (20 µmol l–1; n
= 12). Traces of currents evoked by different concentrations of agonist do not crossover but relax to a common quasi-steady-state level as indicated by the dotted line. The representative data presented in Fig. 1 are neither in support of nor incompatible with a distinct mechanism of acute desensitization. Phenomenologically they might suggest that acute desensitization is related to the size of the current. The experiments to be described in the following were performed to study validity of this novel hypothesis.
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45% of peak IK(ACh), devoid of a desensitizing component. The effects of ACh and Ado, applied simultaneously, are subadditive. The total level of current in this cell amounted to about 80% of peak IK(ACh) and approximately corresponded to the steady-state level of IK(ACh) in the presence of 20 µmol l–1 ACh. An analogous behaviour was observed when a low concentration of ACh (e.g. 0.2 µmol l–1) was used for fractional activation of IK(ACh). As shown in Fig. 4B the effects of 20 µmol l–1 ACh applied following pre-stimulation of the system with 2 µmol l–1 ACh yields a subadditive response. Due to slight variations in the concentration–response curves for activation of IK(ACh) between individual cells, the inhibition by low concentrations of ACh of the response to a saturating concentration showed a high degree of variability, as shown in Fig. 4C. In that cell 0.2 µmol l–1 ACh induced a current of only 18% of peak IK(ACh); concomitantly the response to 20 µmol l–1 of the agonist was reduced by less than 10%. Raising [ACh] to 0.5 µmol l–1 resulted in a current of 75% of Imax and no additional activation by 20 µmol l–1 ACh.
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In a recent study we have provided evidence that in atrial and ventricular myocytes the total macroscopic inward current through inwardly rectifying K+ channels (IK1, IK(ATP), IK(ACh)) is limited due to a reduction in driving force caused by subsarcolemmal K+ accumulation and or depletion in an extracellular membrane-adjacent compartment (Wellner-Kienitz et al. 2004). Based on the data of that study and the data presented in Figs 2–4 of the present study, we hypothesized that acute desensitization of IK(ACh) might be related to K+ current flow rather than to a bona fide cellular signal. In that case one would expect activation of a different endogenous inwardly rectifying K+ current to exert an inhibitory effect on GIRK current comparable with the apparent desensitization caused by low level GIRK activation.
Activation of IK(ATP) causes inhibition of IK(ACh)
Atrial and ventricular myocytes express functional ATP-dependent K+ (K(ATP)) channels that can be opened by ATP depletion via metabolic inhibition or by channel opening drugs (e.g. Terzic et al. 1995). In Fig. 5 representative recordings of membrane current from myocytes, in which IK(ATP) was activated by mitochondrial uncoupling using DNP are illustrated. As a rule, exposure to the uncoupling agent resulted in activation of IK(ATP) within seconds. In this series of experiments there was a large range of variability regarding the amplitude of IK(ATP) in individual cells. In the cell represented by Fig. 5A and B stimulation by ACh (20 µmol l–1) caused activation of a current exhibiting a distinct desensitizing component. Exposure to DNP (50 µmol l–1) resulted in activation of a small inward current, reminiscent of the current evoked by Ado or a low concentration of ACh (compare Fig. 4). However, comparison of the current–voltage relations of IK(ACh) and DNP-induced current (Fig. 5B) clearly identifies the latter as IK(ATP) by its weak inward rectification (e.g. Brandts et al. 1998; Wellner-Kienitz et al. 2004). Analogous to an experiment using application of ACh in the presence of Ado (Fig. 4A), IK(ACh) was substantially reduced in amplitude and was devoid of a desensitizing component in the presence of DNP. Analogous results, i.e. a subadditivity of IK(ACh) and IK(ATP) and a reduction in the desensitizing component, were obtained in all 22 myocytes studied using a protocol as in Fig. 5, though amplitudes or densities, respectively, of IK(ATP) showed a large variability. In the cell represented by Fig. 5C, IK(ATP) was activated with a slower time course. Superimposed pulses of ACh reveal a gradual inhibition of IK(ACh) in parallel with activation of IK(ATP) and a reduction of the desensitizing component.
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Taken together, the data presented in Figs 5 and 6 demonstrate a negative interference of inward current carried by different inward-rectifying K+ channels and suggest that acute desensitization might be related to K+ current rather than to a genuine cellular signal. To further challenge this hypothesis we overexpressed Kir2.1 driven by adenoviral gene transfer. Kir2.1 subunits form constitutively active inwardly rectifying K+ channels and are likely to assemble with other Kir2.x subunits to form cardiac IK1 channels (Liu et al. 2001; Zaritsky et al. 2001; Zobel et al. 2003). Representative current recordings from this series of experiments are shown in Fig. 7. Panel A displays a current recording from a GFP-positive myocyte infected with the empty viral vector. Ba2+ (2 mmol l–1) was used to quantify the background K+ current, which on average was about 25% of peak IK(ACh). In Fig. 7B an analogous experiment on a representative time-matched cell infected with pAd-Kir2.1 is illustrated. In line with a recent study, atrial cells overexpressing the Kir2.1 subunit are characterized by a large inward Ba2+-sensitive holding current, which shows strong inwardly rectifying properties but displays an I–V relation which differed from that of IK(ACh) by its negative slope at positive membrane potentials (Wellner-Kienitz et al. 2004). Two exposures to ACh (20 µmol l–1) caused hardly discernable currents. This behaviour is reminiscent of ventricular myocytes, whose background current–voltage relation is governed by IK1, and which show little receptor-activated GIRK current (e.g. Ito et al. 1995). The differences in background inwardly rectifying current probed by 2 mmol l–1 Ba2+, and ACh-induced current between mock-infected and Ad-Kir2.1-infected cells were highly significant, as demonstrated by the summarized data in Fig. 7C.
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In a previous study we have shown that GIRK4 subunits or concatemeric GIRK4 constructs expressed in CHO cells form functional homomeric channels that can be activated by stimulation of co-expressed A1AdoR. Overexpression of monomeric GIRK4 subunits in atrial myocytes resulted in ACh-induced currents that were completely devoid of a desensitizing component (Bender et al. 2001). Based on these data it was proposed that acute desensitization represents a property related to the heterotetrameric GIRK1/GIRK4 channel. This is contradictory to the hypothesis that acute desensitization results from K+ current flow. We therefore re-investigated the properties of ACh-induced current in myocytes overexpressing GIRK4 by adenoviral gene transfer. Figure 8 compares ACh-induced current and Ba2+-sensitive background current in GFP-positive myocytes infected with the empty vector (A) and Ad-Kir3.4, respectively (B). The representative traces and summarized data (C) reveal a significantly larger Ba2+-sensitive constitutive current in the Ad-Kir3.4-infected cell as compared with the control myocyte. The larger constitutive K+ current was paralleled by a significant reduction in amplitude of receptor-stimulated GIRK current. In line with our previous publication, ACh-induced currents in GIRK4 (Kir3.4) overexpressing myocytes are completely devoid of acute desensitization. In accord with our current hypothesis, the lack of desensitization of receptor-activated current carried by homomeric GIRK4 channels probably results from an increased constitutive K+ current carried by GIRK4 homomers.
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If acute desensitization reflects a reduction in driving force, the reduction in current should be paralleled by a corresponding shift in the reversal potential of IK(ACh).
Using the standard ramp protocol, i.e. a linear change in membrane potential from –130 to +60 mV within 500 ms, corresponding to a dV/dt of 360 mV s–1, the reversal potential of the agonist-activated current was constant throughout, regardless of if it was determined near the peak of IK(ACh) or in the desensitized state (not shown). We hypothesized that because of the slow rate of the ramp, a hypothetical submembrane [K+] gradient dissipates, and the system is in equilibrium throughout the ramp.
We therefore generated more rapid voltage ramps and compared the resulting I–V curves at peak IK(ACh) and in the desensitized state. Following a step from –90 mV to –60 mV a ramp was generated to –30 mV within 7.5 ms (4 V s–1). To eliminate contamination by current through voltage-activated Na+ and Ca2+ channels the solution contained TTX (10 µmol l–1) and CdCl (1 mmol l–1). All curves were background subtracted as described above to eliminate an offset caused by capacitive current.
Figure 9A shows a representative current trace of IK(ACh) elicited by 20 µmol l–1 ACh. The current desensitized by > 50% within 5 s. The I–V curve determined by the standard slow ramp intersected the voltage axis at –40.5 mV as illustrated in Fig. 9B. Figure 9C shows I–V curves determined by application of fast ramps at the times labelled a and b in panel A (for further details see legend). In line with our hypothesis, in the desensitized state the reversal potential was consistently more negative than at peak IK(ACh) (10 mV in the experiment shown). Moreover, the I–V curve at peak had a reversal potential that was consistently a few millivolts more negative than the Erev obtained by the slow ramp protocol, which is in line with the hypothesis that desensitization already develops in parallel with activation. The summarized data from 10 myocytes analysed in the same way (Fig. 9C) demonstrate significant differences in reversal potentials between slow (‘equilibrium’) voltage ramps and fast ramps applied at peak IK(ACh) and in the desensitized state.
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The data of the present study were obtained using an unphysiological K+ gradient and holding potential. These conditions were chosen to measure K+ channel currents in the inward direction, which is standard in numerous studies related to GIRK current because of its strong inward rectification. It has been shown previously that outward IK(ACh) in atrial myocytes shows comparable desensitization properties, though with faster kinetics (Brandts et al. 1997). Nevertheless, the question remains, if acute desensitization is of physiological relevance in terms of shaping the ACh-induced hyperpolarization in myocytes or IPSPs in neuronal synapses. Therefore in a series of experiments we applied ACh to the same cell under both voltage-clamp and current-clamp conditions at two different [K+]o. A representative result from a total of eight cells is shown in Fig. 10. The left trace in panel A shows the inward IK(ACh) induced by ACh at 20 mmol l–1 [K+]o and –90 mV holding potential. The unclamped cell had a resting potential of about –35 mV (panel B). The corresponding hyperpolarization (panel B) was about 5 mV in amplitude. When [K+]o was reduced to 5 mmol l–1, resting potential stabilized at –42 mV. Rapid exposure to 20 µmol l–1 ACh resulted in a hyperpolarization of 24 mV. The corresponding outward current, recorded at –40 mV holding potential, was small, as compared with inward IK(ACh) due to the strong inwardly rectifying properties of the charge-carrying channel species, but showed desensitization, which was similar but consistently faster and stronger than for inward IK(ACh) (Brandts et al. 1997). In both conditions also the ACh-induced hyperpolarization desensitized with comparable time courses. Although this might be coincidental, it might suggest that the rapid fade (Boyett & Roberts, 1987) of the bradycardic action of ACh in the sinoatrial node at least partially reflects current-dependent desensitization.
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Discussion |
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Rapid desensitization of atrial GIRK current was first demonstrated and discussed by Kurachi et al. (1987) as possibly related to the coupling G protein. Due to its apparent heterologous nature it is generally believed to be localized downstream of the receptor(s). A kinase phosphatase mechanism was suggested by Kim (1993). In that study, however, desensitization had a half-time of approximately 20 s as opposed to 
5 s in the present and previous studies and was not instantaneously reversible, and thus, conceivably reflected or was contaminated by slower receptor desensitization. A different mechanism, related to the G protein cycle, had been proposed by Chuang et al. (1998). These authors studied responses of GIRK channels in inside-out macro patches to rapid application of guanosine nucleotides (GDP/GTP). The key observations were: (i) that priming the channels with GDP followed by exposure to GTP resulted in a current of slower activation kinetics, smaller amplitude and less desensitization than upon activation with GTP from a GDP-unbound state; and (ii) that desensitization was enhanced by RGS4. Using a simple model, desensitization could be accounted for by the nucleotide exchange and hydrolysis cycle. The experimental data as such are not contradictory to the present hypothesis, since a distinct ‘desensitizing component’ requires fast activation of IK(ACh). As discussed in that publication, however, the model cannot satisfactorily explain desensitization in an intact cell, where an empty (guanosine nucleotide-free) state of the G
subunit is unlikely to exist. Moreover, RGS4 has been shown to increase the rate of activation of heterologously expressed GIRK channels (Doupnik et al. 1997; Saitoh et al. 1997), which, in line with the present study, would also result in an augmentation of the ‘desensitizing component’. More recently a similar hypothesis was put forward for fast desensitization of receptor-activated GIRK currents in neurones (Sickmann & Alzheimer, 2003) and for heterologously expressed GIRK channels (Leaney et al. 2004). These authors found that intracellular loading with GDP or GDP-ß-S reduced fast desensitization. These experimental manoeuvers without doubt interfere with the G protein cycle. This, however, affects the activation rate and also, as shown in the present study, the amplitude of the receptor-evoked current, and therefore those data are not contradictory to the hypothesis of the present study. Thus, the kinetics of the G protein cycle is likely to have an effect on the ‘desensitizing component’ of the current by virtue of its slowing effect on the activation kinetics and, more importantly, the reduction in current amplitude. Our data using Ba2+ to partially block IK(ACh), clearly show that reducing only the amplitude of the current per se causes a significant reduction in desensitization.
Kobrinsky et al. (2000) proposed that fast desensitization of IK(ACh) is brought about by depletion of PIP2 due to co-stimulation of muscarinic M3 receptors linked to phospholipase C (PLC). This hypothesis, however, is not in line with the observation that in atrial myocytes stimulation of any receptor, endogenous or heterologously expressed, causes acute desensitization (Wellner-Kienitz et al. 2001; Bösche et al. 2003a). Moreover, inhibition of GIRK current upon activation of receptors that cause depletion of PIP2 is too slow to account for this phenomenon (Meyer et al. 2001; Bender et al. 2002; Cho et al. 2002).
In a recent study acute desensitization has been discussed in relation to a novel G protein-independent inhibition of atrial GIRK current (Bösche et al. 2003b) that is particularly pronounced when GIRK current is activated via overexpressed A1AdoR (see also Leaney et al. 2004). The present data suggest that both phenomena are unlikely to share a common mechanism.
The results of the present study clearly demonstrate that acute desensitization is due to flux of K+ ions. This in turn suggests that the decrease in current reflects a reduction in driving force caused by current-dependent changes in subsarcolemmal K+ concentration and/or extracellular depletion. Both alternatives are difficult to distinguish experimentally, particularly in the case of a current pathway that conducts very little outward current. A similar hypothesis has been suggested previously to account for an inhibitory interference between delayed rectifier K+ current and IK(ATP) in smooth muscle cells (McHugh & Beech, 1995).
Integrating IK(ACh) from the start of activation to peak, and relating this charge to the volume of a sphere estimated from the approximated surface (capacitance) measurement yields a global rise in [K+]i in the order of magnitude of 1–5 mmol l–1, a change that would result in a negligible change in Nernst potential. However, the assumption that a significant gradient of [K+]i from the membrane to the centre of the cell during K+ current flow is realistic and supported by experimental data. In a recent study it has been demonstrated that the subsarcolemmal rise in [Na+]i caused by Na+ inward current via voltage-gated Na+ channels can exceed the increment in bulk [Na+]i by a factor of 60 (Weber et al. 2003). The resulting concentration gradient has been shown to dissipate with a time constant of 15 ms, i.e. slower than one would expect assuming a normal diffusion coefficient. Using fast ramp protocols we could detect a shift in the reversal potential of ACh-activated current related to desensitization, which can only be accounted for by a shift in driving force unless one assumes a change in ion selectivity of GIRK channels associated with desensitization. As stated above, current-related changes in driving force could also be accounted for by changes in K+ concentration in an extracellular membrane-adjacent compartment. Accumulation of K+ in such a structurally not yet defined compartment has been suggested to affect delayed (outward) rectifier current by virtue of the driving force in vascular smooth muscle cells (Smirnov & Aaronson, 1994).
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