An M₂-like muscarinic receptor enhances a delayed rectifier K⁺ current in rat sympathetic neurones

Abstract

Background and purpose:

Resting superior cervical ganglion (SCG) neurones are phasic cells that switch to a tonic mode of firing upon muscarinic receptor stimulation. This effect is partially due to the muscarinic inhibition of the M-current. Because delayed rectifier K⁺ channels are essential to sustain tonic firing in central neurones, we asked whether the delayed rectifier current I_KV in SCG neurones was modulated by the muscarinic receptors expressed in these cells.

Experimental approach:

Whole-cell patch-clamp records of M-current and I_KV were done in cultured or acutely dissociated rat SCG neurones. To characterize the receptor that regulates I_KV, cells were bathed with muscarinic agonists and antagonists, relatively specific for receptor subtypes.

Key results:

The muscarinic agonist oxotremorine-M (Oxo-M) enhanced I_KV by ∼46% relative to its basal value. This effect remained unaltered when M-current was suppressed by linopirdine or Ba²⁺. Enhancement of I_KV was insensitive to the M₁-antagonist pirenzepine, whereas it was inhibited (∼60%) by the M_2/4-antagonist himbacine. Further, the relatively specific M₂-agonist bethanechol was as potent as Oxo-M in enhancing I_KV. The modulation of I_KV was insensitive to pertussis toxin (PTX), but was severely attenuated when internal ATP was replaced by its non-hydrolysable analogue AMP-PNP.

Conclusions and Implications:

These results suggest that an M₂-like muscarinic receptor couples to a PTX-insensitive G-protein and to an ATP-dependent pathway to enhance I_KV. Modulation of I_KV must be taken into consideration in order to understand more precisely how muscarinic receptors acting on different ion channels regulate sympathetic excitability.

Introduction

In the nervous system, the slow synaptic actions of acetylcholine (ACh) are mediated by muscarinic ACh receptors (mAChRs) and their coupled G proteins. Stimulation of postsynaptic mAChRs very often elicits a slow membrane depolarization that increases firing frequency or switches firing pattern from a burst to a tonic mode (McCormick, 1993; Marrion, 1997). Most of the knowledge in relation to the signalling pathways activated by mAChRs that regulate cell excitability come from studies carried out in rat superior cervical ganglion (SCG) neurones. According to their similarity in amino-acid sequence and pharmacological profile, mAChRs are grouped into the M₁-like (M₁, M₃ and M₅) and M₂-like (M₂ and M₄) subtypes. Of these, SCG neurones mostly express the M₁, M₂ and M₄ receptors (Brown et al., 1995).

SCG neurones are considered phasic nerve cells because during a maintained depolarizing current stimulus, they respond with few action potentials that show adaptation, that is, their firing rate slows down (Wang and McKinnon, 1995). This electrical behaviour is partially determined by the Kv7.2 and Kv7.3 (KCNQ) K⁺ channel subunits that generate the voltage-activated and non-inactivating M-current (Wang et al., 1998). As M-current dominates the membrane conductance around the cell firing threshold, its slow activation strongly damps the depolarizing effects by inward Na⁺ or Ca²⁺ currents, thereby contributing to spike frequency adaptation (Wang et al., 1998; Yue and Yaari, 2004). Conversely, inhibition of M-current by muscarinic agonists produces a slow depolarization that promotes high-frequency tonic firing (Brown and Constanti, 1980; Adams et al., 1982; McCormick, 1993; Marrion, 1997). In SCG cells, the inhibition of M-current is disrupted by the relatively selective M₁-antagonist pirenzepine and its modulation disappears upon deletion of the m₁ gene (Hamilton et al., 1997). This pharmacological and genetic evidence indicates that the M₁ subtype underlies the muscarinic modulation of the M-current (Marrion et al., 1989; Bernheim et al., 1992; Hamilton et al., 1997). The signalling pathway downstream from the M₁ receptor has been elucidated and involves a Gα_q-activated phospholipase that depletes membrane PIP₂ (phosphatidylinositol-4,5-bisphosphate) as the ultimate signal for M-current inhibition (Haley et al., 1998; Suh and Hille, 2002; Zhang et al., 2003; Winks et al., 2005). A second ionic current modulated by mAChRs is the N-type Ca²⁺ current (I_CaN). Its muscarinic inhibition develops with an initial fast (τ∼1 s) and voltage-dependent component, followed by a slow (τ∼9 s) and voltage-independent component (Zhou et al., 1997). The slower component is blocked by pirenzepine, whereas the voltage-dependent inhibition is disrupted by the relatively selective M_2/4-antagonist himbacine (Bernheim et al., 1992). Genetic deletion strategies indicate that the M₁ subtype mediates the slow component, whereas the M₂ subtype is responsible for the voltage-dependent inhibition of I_CaN (Shapiro et al., 1999).

Despite the well-documented muscarinic inhibition of both M-current and I_CaN, it is not known whether other SCG-endogenous K⁺ currents are modulated by mAChRs. One potential target for muscarinic action might be the delayed rectifier K⁺ current (I_KV), because: (a) delayed rectifier K⁺ currents are essential to sustain tonic firing of action potentials (McCrossan et al., 2003; Melnick et al., 2004), as muscarinic stimulation does in SCG neurones; (b) in SCG neurones, I_KV significantly contributes to both the late spike repolarization and the early fast spike afterhyperpolarization (Marsh and Brown, 1991), and its transient genetic elimination prolongs action potentials, thereby reducing the probability of tonic firing (Malin and Nerbonne, 2002); (c) it has been reported that muscarinic agonists modulate delayed rectifier K⁺ currents from central neurones (Zhang et al., 1992; ffrench-Mullen et al., 1994). In this paper, we report that the muscarinic agonist oxotremorine-M (Oxo-M) enhanced the amplitude of I_KV, with a pharmacological profile that suggested the involvement of an M₂-like mAChR. Furthermore, some properties of this novel signalling pathway are characterized.

Methods

Cell culture

Experiments were mostly carried out on cultured SCG neurones taken from juvenile (2- to 4-week old) Wistar rats, although some current recordings were carried out in older animals (10 weeks old, 210 g weight). Rats were quickly anesthetized with chloroform and decapitated. Superior cervical ganglia were quickly removed, cleaned from non-neuronal tissue and placed for 18 min in modified Hank's solution containing papain (20 U ml⁻¹ at 37°C). Thereafter, papain was replaced by a mixture of collagenase I (1.6 mg ml⁻¹) and dispase II (5 mg ml⁻¹) and further incubated for 14 min, with gentle pipette trituration every 7 min to increase cell dissociation. The acutely dissociated cells were suspended twice in Dulbecco's modified Eagle's medium (DMEM), centrifuged and plated on 4 × 4 mm glass chips (coated with poly-L-lysine). Cells were incubated for 10 h at 37°C (5% CO₂) with DMEM supplemented with 10% of heat-inactivated foetal bovine serum (FBS), before current recording. For some experiments, the acutely dissociated neurones were directly placed in the recording chamber for immediate patch-clamping.

Electrophysiology

Whole-cell patch-clamp recordings of I_KV or M-current were made with a List EPC-7 amplifier and seals were obtained with patch pipettes having 1–2 MΩ resistance. The resulting steady series resistance was 3–4 MΩ, 2 min after seal breakthrough. The cell membrane capacitance was measured by integrating the capacitive current generated during a 10 mV depolarizing step, from a holding of −80 mV. The experiments were carried out in SCG neurones with a mean membrane cell capacitance of 73±3.4 pF (range: 47–88 pF). Voltage command pulses and current records were generated and acquired (sampling rate, 5 kHz) using a 12-bit interface (Indec Systems Inc., Sunnyvale, CA, USA). Membrane currents were low-pass filtered at 1 kHz and analysed using BASIC-FASTLAB (Indec Systems) and Sigma Plot (SPSS Inc.). The M-current was fully activated by setting the holding potential (V_h) at −25 mV and further deactivated by 500 ms pulses from −25 to −60 mV, every 4 s. The amplitude of M-current and its percentage of inhibition were measured as described (Cruzblanca et al., 1998). For I_KV isolation, neurones were held at −50 mV to fully inactivate the I_A K⁺ current (Wang and McKinnon, 1995) and Cd²⁺ (200 μM) was added to the external solution to block the voltage-activated Ca²⁺ currents and the Ca²⁺-activated K⁺ currents. Thereafter, I_KV was elicited by 100 ms command pulses from −50 to 40 mV, in steps of 10–20 mV. I_KV was quantified from leakage subtracted records, as the difference between the average of a 10 ms segment of basal current and the averaged outward current recorded during the last 5 ms of the command step. Thus, current density (pA pF⁻¹) was calculated by dividing the amplitude of I_KV (pA) and the cell membrane capacitance (pF). Modulation of I_KV was measured as the change in its current density at 0 mV. Membrane voltages were corrected on line for a −2 mV junction potential (standard internal solution).

Solutions

The composition of the modified Hank's solution was (in mM): 137 NaCl, 5.4 KCl, 0.44 KH₂PO₄, 0.34 NaH₂PO₄, 5 4-(2-hydroxyethyl)-1-piperazineethyl-sulphonic acid (HEPES), 5 glucose, pH=7.4. SCG cells were transferred to a recording chamber (400 μl) and bathed (2.8 ml min⁻¹) with the appropriate external solution. For M-current recording, the external solution was (in mM): 160 NaCl, 2.5 KCl, 5 CaCl₂, 1 MgCl₂, 10 HEPES, 8 glucose and 0.5 μM tetrodotoxin (TTX). The pH was adjusted to 7.4 with NaOH. For I_KV recording, Cd²⁺ (200 μM) was added to the external saline. Solution changes were accomplished in <10 s and the experiments were carried out at 25°C.

The composition of the standard pipette internal solution was (in mM): 175 KCl, 5 MgCl₂, 5 HEPES, 0.1 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 3 K₂-ATP (adenosine 5′-triphosphate), 0.1 Na-GTP, 0.08 leupeptin (pH=7.4). For experiments requiring cell dialysis with a test molecule (adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP)) from the patch pipette, we waited for 5 min dialysis before beginning the K⁺ current recordings. To avoid systematic bias, control and test measurements were alternated within each set of experiments.

Statistical analysis

Average results are given as the mean±s.e.m. and sample means were compared using Student's t-test. Differences between means were considered significant at P<0.05.

Materials

Reagents were obtained as follows: pertussis toxin (PTX) (List Biological Labs., Campbell, CA, USA); collagenase I, poly-L-lysine, HEPES, Na-GTP, pirenzepine, himbacine, linopirdine, Oxo-M, bethanechol (Sigma, St Louis, MO, USA); BAPTA (Molecular Probes, Eugene, OR, USA); papain, dispase II, leupeptin and K₂-ATP (Roche Diagnostics GmbH, Mannheim, Germany); DMEM and heat-inactivated FBS (GIBCO/Invitrogen Co, Carlsbad, CA, USA); TTX and AMP-PNP (Calbiochem, La Jolla, CA, USA). Stock solutions of AMP-PNP and Na-GTP were prepared in water and stored at −20°C.

Results

The muscarinic agonist Oxo-M enhances the amplitude of I_KV

In our preparations, by setting the V_h at −50 mV and bathing the cells with a TTX- and Cd²⁺-containing external solution, it was possible to block the Na⁺ and Ca²⁺ inward currents and to eliminate the contribution of the A-type and Ca²⁺-activated K⁺ currents, revealing a delayed-like outward current named here as I_KV (see inset in Figure 1a). I_KV was activated at membrane voltages more positive than −40 mV (Figure 1c) and was significantly more sensitive to external 4-AP than tetraethylammonium (TEA) (500 μM TEA or 4-AP suppressed I_KV tail current by 29±2% and 56±4%, respectively; n=6). To assess whether I_KV is under regulation by mAChRs, the cultured SCG neurones were bathed with 10 μM of the muscarinic agonist Oxo-M. As shown in Figure 1, Oxo-M produced an increase of I_KV that was more noticeable at membrane potentials more positive than −20 mV (Figure 1a and b). For instance at 0 mV, Oxo-M increased the amplitude of I_KV from 4.1 to 5.5 nA (Figure 1c). For comparison purposes among cells, the enhancement of I_KV was normalized to the cell membrane capacitance. Thus, in eight cells Oxo-M increased I_KV current density by 16.1±1.4 pA pF⁻¹ from a non-stimulated basal level, at 0 mV, of 35±3.6 pA pF⁻¹. In most of the cells tested (60%), there were neither changes on the holding current elicited at −50 mV nor on the leakage current generated by a 10 mV hyperpolarizing command pulse, from a V_h of −60 mV (control, 33±4 pA; Oxo-M, 31±4 pA). In the rest of the cells (40%), the enhancement of I_KV occurred in parallel with a small inward shift (67±11 pA) of the holding current and with a reproducible, but not statistically significant, increase of the leakage current from 40±4.4 to 48±4.6 pA.

Figure 1.

The muscarinic agonist Oxo-M enhances the amplitude of I_KV. I_KV records elicited from −40 to +40 mV (V_h=−50 mV), taken before (a) and during (b) Oxo-M (10 μM) exposure. The inset in (a) shows the superposition of the membrane currents recorded at 0 mV when a cultured SCG neurone was bathed with the normal external solution (Control saline) and during addition of TTX (500 nM) plus Cd²⁺ (200 μM); the dashed line indicates the zero current level. (c) I–V relationship taken before (open circles) and during (filled circles) Oxo-M bath application. (d) Superimposition of I_KV traces generated in an acutely dissociated SCG neurone from a juvenile rat, recorded before (Control) during Oxo-M exposure (Oxo-M) and after 2 min of the agonist removal (Wash).

It may be argued that the muscarinic enhancement of I_KV reported here could be induced by the culturing process or that modulation of I_KV could be mediated by a developmental process that disappears during the adult age. To assess for these possibilities, the effect of Oxo-M was tested on acutely dissociated SCG cells from juvenile rats (2–4 weeks old) and on cultured cells from adult (10 weeks old) animals. Stimulation of mAChRs resulted in a slowly reversible enhancement of I_KV, both in acutely dissociated juvenile SCG cells (Figure 1d) and cultured SCG neurones from adult rats as well. The mean increase in I_KV current density was 11.7±1.5 pA pF⁻¹ (n=5) for juvenile recently dissociated SCG cells and 17.4±2 pA pF⁻¹ (n=6) for cultured neurones from 10-week old rats.

The enhancement of I_KV reflects a novel muscarinic signalling pathway operating in SCG neurones

The modulation of I_KV was further characterized by clarifying whether its enhancement reflected the activity of a novel muscarinic pathway or whether it was a space-clamp artefact generated by the parallel strong muscarinic inhibition of the M-current (Adams et al., 1982; Marrion et al., 1989). To address this question, some experiments were carried out in the presence of linopirdine, a relatively selective M-channel blocker (Wang et al., 1998). As expected, linopirdine (10 μM) practically abolished M-current (Figure 2a, upper records). Nevertheless, in the presence of linopirdine, Oxo-M still enhanced I_KV (Figure 2a, bottom records). In summary, the increments of I_KV current density in the absence or presence of linopirdine were 16.1±1.4 (n=8) and 12.5±1.6 pA pF⁻¹ (n=5), respectively, a difference not statistically significant. Similar results were found when Ba²⁺ was used to suppress M-current (Adams et al., 1982). Application of Ba²⁺ (4 mM) reduced the amplitude of I_KV by only 12.8±1.5% (n=9) (cf. Adams et al., 1982), however, the residual I_KV was still enhanced by Oxo-M (Figure 1b). Thus, Oxo-M enhanced I_KV by 16.9±1.7 pA pF⁻¹ (n=6) in control cells, whereas in the presence of Ba²⁺, the enhancement of current density was 12.1±1.6 pA pF⁻¹ (n=9).

Figure 2.

Stimulation of mAChRs produces independent modulation of M-current and I_KV. (a) Top panel, M-current deactivation records elicited before (Control) and during bath application of 10 μM of the M-channel blocker linopirdine (Linop). (a) Bottom panel, records of I_KV elicited before (Control) and during Oxo-M (10 μM) superfusion (Oxo-M); linopirdine was present throughout the experiment as indicated by the wide bar. The dashed line on current records indicates the zero current level. (b) The symbols represent the amplitude of I_KV measured every 4 s, before and when Ba²⁺ and Oxo-M were applied as indicated by the horizontal bars. Note that Ba²⁺ reduced I_KV; nevertheless, the residual outward current was enhanced by Oxo-M. Lower plots show the time course of the muscarinic-induced modulation of M-current (c) and I_KV (d). Inhibition of M-current was calculated as 1−[(M-current (Oxo-M)/M-current (control)], whereas the enhancement of I_KV was measured as [I_KV (Oxo-M)−I_KV (Control)]/I_KV (Control). Current records were taken every 4 s and Oxo-M (10 μM) was applied during periods as indicated by the horizontal bars. For comparison, note the slower enhancement of I_KV and its partial recovery 96 s after Oxo-M was removed.

Furthermore, the time course of the enhancement of I_KV was relatively slower than that of the inhibition of M-current. For instance, a 20 s bath application of Oxo-M was enough to attain the maximum effect on M-current (Figure 2c), whereas the maximum enhancement of I_KV required at least 60 s (Figure 2d). The time course of the I_KV enhancement was described by a single exponential having a mean time constant of 20.4±3.9 s (n=6), whereas the corresponding time constant for M-current inhibition was only 3.6±0.5 s (n=6). Moreover, it was observed that recovery of I_KV modulation was slow with a mean time constant of 76.2±5.4 s (n=6).

The M₁-antagonist pirenzepine does not disrupt the modulation of I_KV

In SCG neurones, the M₁ receptor underlies the suppression of M-current (Hamilton et al., 1997) and the relatively selective M₁-antagonist pirenzepine (pK_B=8.2; Ellis, 2002) blocks the effects of muscarinic agonists on M-current (Marrion et al., 1989; Bernheim et al., 1992). In order to assess whether the M₁ receptor underlies the modulation of I_KV, cells were treated with 100 nM pirenzepine 2 min before and throughout the Oxo-M challenge. Initially, modulation of M-current was used as a positive control to verify the potency of pirenzepine in our experimental conditions. M-current suppression by Oxo-M at 200 nM (a concentration close to its reported IC₅₀, 300 nM; Bernheim et al., 1992) was 43±2% (n=11) but, in the presence of pirenzepine, this effect of Oxo-M was significantly reduced (n=10; Figure 3a and b). Conversely, pirenzepine did not prevent the enhancement of I_KV elicited by Oxo-M (Figure 3c and d).

Figure 3.

The M₁-antagonist pirenzepine disrupts the modulation of M-current, while leaves intact the muscarinic enhancement of I_KV. Representative traces of M-current (a) and I_KV (c) from non-treated neurones (No PZ) and those incubated with 100 nM pirenzepine (PZ). For each panel, recordings were taken before (1) and during (2) bath application of 200 nM Oxo-M and the dashed line indicates zero current level. Bar plots show the mean (±s.e.m.) Oxo-M-induced inhibition of M-current (b) and mean enhancement of I_KV current density (d) in control cells (solid bars) and in neurones treated with PZ (open bars). In PZ-treated cells only the muscarinic inhibition of M-current was significantly (^**P<0.01) attenuated from 43±2 to 19.3±2.3%. Oxo-M increased I_KV current density by 11.6±1.7 and 11.4±1.2 pA pF⁻¹ in No PZ- and PZ-treated cells, respectively. The numbers above bars indicate the numbers of cells tested for each condition.

The M_2/4-receptor antagonist himbacine disrupts the modulation of I_KV

The M_2/4-antagonist himbacine (pK_B=8.1–8.2; Ellis, 2002) has been successfully used to distinguish between M₁- and M_2/4-mediated signalling pathways. Therefore, to test whether modulation of I_KV is mediated by an M₂-like receptor, cells were treated with himbacine (100 nM) 2 min before and during Oxo-M bath application. For these experiments, the M₁-mediated modulation of M-current was checked again because himbacine antagonizes M₁-receptors with a lower potency (pK_B=7.1) than it does for M_2/4 mAChRs (Ellis, 2002) and because 100 nM himbacine is expected to produce some effect on M-current modulation (Bernheim et al., 1992). Indeed, in control neurones the suppression of M-current by 200 nM Oxo-M was 44.9±2.6% (n=6), whereas in himbacine-treated cells the suppression was slightly lower (35.9±1%, n=6). On the other hand and in contrast to pirenzepine, himbacine significantly attenuated the muscarinic enhancement of I_KV (Figure 4a, b).

Figure 4.

The muscarinic modulation of I_KV has a pharmacological M_2/4-profile. (a) Effect of Oxo-M on I_KV in a non-treated cell (No HIMB) and in a neurone incubated with the M_2/4-antagonist himbacine (HIMB). For each panel, I_KV was elicited before (1) and during (2) bath application of 200 nM Oxo-M. (b) Mean (±s.e.m.) increase of I_KV current density in control cells (solid bar) and in neurones treated with HIMB (open bar); HIMB significantly (^**P<0.01) reduced the muscarinic enhancement of I_KV from 10.4±1 to 4.3±0.5 pA pF⁻¹ (c) Effect on M-current of the sequential application of the muscarinic agonists bethanechol (BeCh) and Oxo-M; note that BeCh was applied for a longer period than Oxo-M, as indicated by the horizontal bars. (d) The mean suppression of M-current by BeCh (solid bar) and Oxo-M (open bar) were 22.2±6.1 and 79.6±2%, respectively; a difference statistically significant (^**P<0.01). (e) BeCh (solid bar) and Oxo-M (open bar) applied at the same concentration (10 μM) enhanced I_KV current density by 10.9±1 and 13.3±2.3 pA pF⁻¹, respectively. For all bar plots, the numbers on columns indicate the numbers of cells tested.

So far, the muscarinic modulation of I_KV has been tested with the unspecific agonist Oxo-M. To confirm that an M₂-like mAChR subtype underlies the enhancement of I_KV, the effect of the agonist bethanechol was tested because this agonist selectively activated the M₂-mediated inhibition of I_CaN in neonatal SCG neurones (Liu and Rittenhouse, 2003). In our preparations of SCG neurones, bethanechol (10 μM) produced a small inhibition of M-current, as compared with the large effect generated by Oxo-M (Figure 4c and d). There was no significant difference in the potency between these muscarinic agonists (both at 10 μM) when they were tested on the modulation of I_KV (Figure 4e).

PTX does not block the muscarinic modulation of I_KV

In many cell types, the M₂ and M₄ mAChRs couple to and signal through PTX-sensitive G proteins (Ellis, 2002). To assess whether modulation of I_KV is sensitive to PTX, cells were incubated overnight with 500 ng ml⁻¹ of that toxin. Surprisingly, PTX did not prevent the enhancement of I_KV elicited by 10 μM Oxo-M (Figure 5a). Summarizing, in non-treated neurones the muscarinic agonist increased current density by 17.9±2.5 pA pF⁻¹ (n=8), whereas in PTX-treated cells the increment was 16.7±1.7 pA pF⁻¹ (n=10), a difference not statistically significant (P<0.05%). To validate these results, we tested the well-known PTX sensitivity of the α₂-adrenoceptor-mediated inhibition of I_CaN, which is voltage-dependent and mediated by G_o/i (Delmas et al., 1999). The voltage-dependent inhibition of I_CaN was revealed with two 10 ms depolarizing test pulses from a V_h of −80 to +10 mV, interposed by a 25-ms depolarization to +125 mV. In the absence of agonist, each +10 mV pulses generated identical Ca²⁺ currents as shown in Figure 5b (C₁, C₂). Application of noradrenaline (NA) reduced I_CaN, although this effect was partially reversed by the +125 mV depolarization, facilitating I_CaN during the second test pulse (left panel in Figure 5b). As expected, PTX attenuated both the inhibitory effect of NA on I_CaN and current facilitation that results from the transient removal of its voltage-dependent inhibition (right panel in Figure 5b).

Figure 5.

PTX does not prevent the muscarinic modulation of I_KV. (a) Representative delayed rectifier K⁺ and (b) Ca²⁺ current records from non-treated neurones (No PTX) and those cells incubated overnight with 500 ng ml⁻¹ of PTX. Note that PTX did not block the muscarinic enhancement of I_KV (traces were taken before (1) and during (2) bath application of 10 μM Oxo-M). (b) Typical I_CaN traces elicited by a two-pulse protocol (see text) in a control cell (left panel) and in a PTX-incubated neurone (right panel); I_CaN records were elicited before (C₁ and C₂) and during adrenergic stimulation (NA). As expected, PTX reduced the voltage-dependent inhibition of I_CaN. The dashed lines are the zero current level.

ATP is required for muscarinic modulation of I_KV

It is now well accepted that the activity of ion channels can be modulated by the activation of protein kinases linked to second-messenger systems (Hilgemann, 1997). Given that ATP is commonly used by protein kinases, we tested whether modulation of I_KV required the presence of ATP. Thus, cells were dialysed for ∼8 min with a standard internal solution containing the non-hydrolysable ATP analogue, AMP-PNP, instead of ATP. Figure 6a shows the Oxo-M response in a cell dialysed with the standard ATP-containing pipette solution. The long-term dialysis procedure, by itself, did not prevent the muscarinic-induced enhancement of I_KV. In contrast, in a cell dialysed with AMP-PNP, the muscarinic enhancement of I_KV was absent (Figure 6b). Similar results were found in two more paired experiments.

Figure 6.

Muscarinic modulation of I_KV is mediated by an ATP-dependent process. Representative effect of Oxo-M on I_KV in a neurone dialysed with the ATP-containing pipette standard internal solution (a) and in a cell where ATP was replaced with its non-hydrolysable parent compound, AMP-PNP (b). The ATP-analogue readily blocked the muscarinic enhancement of I_KV, as are shown in the current records and plots describing the value of current density (pA pF⁻¹) measured every 4 s, before (open circles) and during Oxo-M challenge (filled circles and horizontal bars). Similar results were found in two more paired experiments.

Discussion and conclusions

Our experiments have shown that stimulation of mAChRs induced a robust enhancement of I_KV in cultured or acutely dissociated SCG neurones from juvenile rats (Figure 1). This effect did not result from the improvement of the space-clamp conditions, secondary to the strong muscarinic inhibition of M-current, because: (a) the enhancement of I_KV by Oxo-M remained practically unaltered when M-channels were blocked either by linopirdine or Ba²⁺ (Figure 2); (b) the kinetic for muscarinic inhibition of M-current was, at least, five times faster than the time course of I_KV modulation (Figure 2); (c) the enhancement of I_KV was noticeable at membrane voltages more positive than −20 mV (Figure 1), a voltage range where M-channels reached their maximal activation (Wang et al., 1998). Therefore, it is suggested that the enhancement of I_KV reflects a novel muscarinic pathway operating in rat sympathetic SCG neurones.

Given that these nerve cells mostly express the M₁, M₂ and M₄ receptor subtypes (Brown et al., 1995), the M₁-antagonist pirenzepine and the M_2/4-antagonist himbacine were used to pharmacologically characterize the mAChR that underlies the modulation of I_KV. The use of these muscarinic antagonists has been reported to be a valuable pharmacological tool to identify the subtypes of mAChR that modulate different SCG endogenous ion currents including the M-current, I_CaN and the Ca²⁺-activated chloride current (Marrion et al., 1989; Bernheim et al., 1992; Marsh et al., 1995). Here, it was found that the enhancement of I_KV was insensitive to pirenzepine, whereas this M₁-antagonist reduced the inhibition of M-current by ∼50% (Figure 3). In contrast, the enhancement of I_KV was significantly reduced by himbacine (Figure 4). In agreement with these results, the relatively selective M_2/4-agonist bethanechol (Constanti, 2003; Liu and Rittenhouse, 2003) showed an ability to enhance I_KV, comparable to that of Oxo-M (Figure 4). Taken together, these results suggest that an M₂-like mAChR is responsible for the muscarinic modulation of I_KV. However, we cannot distinguish between the M₂ and M₄ because himbacine antagonizes both subtypes with similar potency (pK_B for M₂=8.1 and pK_B for M₄=8.2) (Ellis, 2002). Further genetic studies are required to identify the exact M₂-like receptor subtype that mediates the modulation of I_KV.

It is generally accepted that M₂-like receptors prefer to couple to G proteins of the PTX-sensitive G_i family (Ellis, 2002). Indeed, in SCG neurones M₂/M₄ subtypes couple to G_oA, the most abundant isoform of Gα_o expressed in these cells, to induce the Gβγ-mediated and voltage-dependent inhibition of I_CaN (Beech et al., 1992; Delmas et al., 1998). Surprisingly, PTX did not affect the muscarinic enhancement of I_KV (Figure 5) suggesting that in SCG cells the M₂-like receptor couples to a PTX-insensitive G protein as well. Such potential divergence at the level of G proteins has been reported for M₂ receptors co-expressed with Ca_V2.3 channels in human embryonic kidney cells. In this heterologous expression system, the agonist carbachol produces a rapid and PTX-sensitive inhibition, followed by a slow and PTX-insensitive stimulation of the Ca²⁺ current (Meza et al., 1999). Moreover, G-protein-activated, inwardly rectifying, K⁺ (GIRK) currents also show a biphasic PTX-sensitive and -insensitive modulation by M₂ receptors, when receptors and GIRK channel subunits are co-expressed in Chinese hamster ovary K1 cells (Bünemann et al., 2000). For I_KV modulation, the most plausible candidate might be G_z because: (a) G_z is the unique PTX-insensitive member that belongs to the G_i family; (b) SCG neurones express G_z and its overexpression in PTX-treated cells rescued the voltage-dependent inhibition of I_CaN by α₂-adrenergic or somatostatin receptors, thereby indicating that G_z is able to link G_i-coupled receptors to ion channels (Jeong and Ikeda, 1998). Whatever the nature of the PTX-insensitive G protein might be, it stimulated an ATP-dependent, slow signalling pathway to modulate I_KV.

Muscarinic modulation of delayed rectifier K⁺ currents has been reported to occur in some central neurones as well. However, the effect depends on the cell type because in CA1 hippocampal neurones, like in SCG cells, stimulation of mAChRs potentates a voltage-activated sustained K⁺ current (Zhang et al., 1992), whereas in hypothalamic neurones a I_KV is inhibited (ffrench-Mullen et al., 1994). It is possible that the muscarinic enhancement of I_KV seen both in CA1 and SCG neurones reflects the modulation of either Kv2.1 or Kv2.2 subunits, because they mostly contribute to the delayed rectifier currents generated in these neurone types (Murakoshi and Trimmer, 1999; Malin and Nerbonne, 2002). In agreement with this suggestion, I_KV had a pharmacological profile consistent with the involvement of Kv2.1 subunits. For instance, (a) like rat cloned Kv2.1 channels (Chandy and Gutman, 1995), I_KV had a greater sensitivity to 4-AP than to TEA; (b) it was reported that 500 μM of 4-AP blocked homomeric Kv2.1 currents by 50% (Chandy and Gutman, 1995) and this concentration of 4-AP applied to the SCG cells reduced I_KV by ∼55%; (c) 500 μM 4-AP practically abolished the enhancement of I_KV by Oxo-M or angiotensin II (unpublished observations).

An effect of muscarine on action potential shape was not observed in microelectrode recordings from adult rat or bullfrog sympathetic neurones (Brown and Constanti, 1980; Adams et al., 1982). The data presented in this study would have predicted an effect. This apparent discrepancy is not due to the different ages among the animals used or to the cell culture process per se, because: (a) it was found that Oxo-M enhanced I_KV, both in cultured SCG neurones from juvenile (2–4 weeks old) or adult (10 weeks old) rats; (b) in SCG cells from juvenile rats, the enhancement of I_KV was similar both in cultured and fresh dissociated cells. There is evidence from SCG cells that a delayed rectifier K⁺ current (I_K(DR)) with a pharmacological profile similar to that of I_KV, significantly contributes to the early fast spike afterhyperpolarization (Marsh and Brown, 1991). Thus, it might be expected that the effect of Oxo-M on I_KV would cause an increase of the fast spike afterhyperpolarization. This had been reported previously (see Figure 8 in Brown and Constanti, 1980). It is possible that the final effect of muscarinic agonists on single action potential shape would be difficult to discern, because of specific roles that each voltage- and Ca²⁺-activated K⁺ currents have in shaping the SCG action potential. For instance, when I_KV was blocked in bullfrog sympathetic neurones its contribution to the action potential was only revealed (prolonged action potential duration and reduced fast afterhyperpolarization) when the Ca²⁺-activated K⁺ currents I_C and I_AHP were also blocked (Goh et al., 1989). In SCG cells, I_C and I_AHP are mediated by BK- and SK-type K⁺ channels that are activated by Ca²⁺ entry through L-type and N-type Ca²⁺ channels, respectively (Davies et al., 1996). Given that BK- and SK channels contribute to spike repolarization and afterhyperpolarization, respectively, their indirect suppression secondary to the well-known muscarinic inhibition of the L- and N-type Ca²⁺ currents (Bernheim et al., 1992; Shapiro et al., 1999), could mask the effect of the muscarinic enhancement of I_KV on spike repolarization (faster) and afterhyperpolarization (larger amplitude), thereby resulting in no noticeable changes in single action potential morphology. So, the impact of each of these K⁺ currents on the action potential repolarization and fast afterhyperpolarization may depend on the corresponding K⁺ channel-type density (Malin and Nerbonne, 2002) and the strength and duration of their muscarinic modulation.

It is concluded that in SCG neurones from juvenile and adult rats, an M₂-like muscarinic receptor couples to a PTX-insensitive G protein and to an ATP-dependent pathway to modulate the delayed rectifier current I_KV. The muscarinic enhancement of I_KV must be taken into consideration in order to understand better how mAChRs acting on different ion channels regulate sympathetic excitability.

Conflict of interest

The author states no conflict of interest.

Abbreviations

AMP-PNP

adenosine 5′-(β,γ-imido)triphosphate

I_CaN

N-type Ca²⁺ current

I_KV

delayed rectifier K⁺ current

mAChRs

muscarinic acetylcholine receptors

NA

noradrenaline

Oxo-M

oxotremorine-M

PTX

pertussis toxin

SCG

superior cervical ganglion