Inositol Triphosphate-Mediated Ca²⁺ Signals Direct Purinergic P2Y Receptor Regulation of Neuronal Ion Channels

Abstract

Purinergic P2Y receptors are one of four types of G_q/11-coupled receptors in rat superior cervical ganglia (SCG) sympathetic neurons. In cultured SCG neurons, purinergic and bradykinin suppression of I_M were similar in magnitude and somewhat less than that by muscarinic agonists. The effects of the P2Y receptor agonist UTP on neuronal excitability and discharge properties were studied. Under current clamp, UTP increased action potential (AP) firing in response to depolarizing current steps, depolarized the resting potential, decreased the threshold current required to fire an AP, and decreased spike-frequency adaptation. These effects were very similar to those resulting from bradykinin stimulation and not as profound as from muscarinic stimulation or full M-current blockade. We then examined the P2Y mechanism of action. Like bradykinin, but unlike muscarinic, purinergic stimulation induced rises in intracellular [Ca²⁺]_i. Tests using expression of IP₃“sponge” or IP₃ phosphatase constructs implicated IP₃ accumulation as necessary for purinergic suppression of I_M. Overexpression of wild-type or dominant-negative calmodulin (CaM) implicated Ca²⁺/CaM in the purinergic action. Both sets of results were similar to bradykinin, and opposite to muscarinic, suppression. We also examined modulation of Ca²⁺ channels. As for bradykinin, purinergic stimulation did not suppress I_Ca, unless neuronal calcium sensor-1 (NCS-1) activity was blocked by a dominant-negative NCS-1 construct. Our results indicate that P2Y receptors modulate M-type channels in SCG cells via IP₃-mediated [Ca²⁺]_i signals in concert with CaM and not by depletion of phosphatidylinositol-4, 5-biphosphate. We group purinergic P2Y and bradykinin B₂ receptors together as having a common mode of action.

Introduction

The M-type K⁺ current is a prime regulator of neuronal excitability wherever its constituent Kv7 (KCNQ) gene products are expressed (Cooper and Jan, 2003). M-current suppression thus causes increased action potential (AP) firing and bursting, whereas its augmentation inhibits neuronal discharge (Wang et al., 1998; Peretz et al., 2005; Gamper et al., 2006; Yue and Yaari, 2006; Zaika et al., 2006). Although the critical regulatory action of transmitters on M-type channels centers on binding to membrane phosphatidylinositol 4,5-bisphosphate (PIP₂), two major modes of inhibition of M-type channels by stimulation of G_q/11-coupled receptors have emerged that use partly distinct, and partly shared, intracellular mechanisms. Both involve activation of phospholipase C (PLC) and hydrolysis of PIP₂ but differ in the use of IP₃-mediated release of Ca²⁺ from stores, subsequent [Ca²⁺]_i signals and calmodulin action (Delmas and Brown, 2005). Four G_q/11-coupled receptors have been characterized in superior cervical ganglion (SCG) sympathetic neurons: muscarinic M₁, angiotensin AT₁, bradykinin (BK) B₂, and purinergic P2Y. Muscarinic and angiotensin stimulation do not elicit [Ca²⁺]_i signals and suppress M current by PIP₂ depletion (Shapiro et al., 1994; Suh and Hille, 2002; Zhang et al., 2003; Zaika et al., 2006). Conversely, bradykinin suppression involves Ca²⁺/CaM binding to the channels (Gamper and Shapiro, 2003), which may lower their affinity for PIP₂, but such B₂ receptor stimulation does not strongly lower global PIP₂ abundance (Gamper et al., 2004; Winks et al., 2005; Gamper and Shapiro, 2007; Hughes et al., 2007). High-threshold Ca_V2 channels are also sensitive to membrane PIP₂ abundance and are inhibited during PIP₂ depletion (Wu et al., 2002; Gamper et al., 2004). Consistent with specificity among G_q/11-coupled receptors for inducing PIP₂ depletion, stimulation of M₁ and AT₁, but not B₂, receptors suppresses endogenous I_Ca and heterologously expressed K_ir3 current in SCG cells (Gamper et al., 2004; Winks et al., 2005).

Purinergic receptors, classified as P2X ionotropic and P2Y metabotropic, are found throughout the nervous system (Burnstock, 2006). Although the P2Y₁, P2Y₂, and P2Y₆ subtypes have been described in mouse and rat sympathetic ganglia (Vartian et al., 2001; Calvert et al., 2004), P2Y₆ seems to be primary in rat SCG (Bofill-Cardona et al., 2000), whereas the P2Y₁ subtype dominates in mammalian CNS (Moore et al., 2001). In many neuronal types, P2Y receptor stimulation depresses M current via PLC activation, but the intracellular mechanism used from that point on appears intriguingly divergent. In the rat SCG cells also studied here, the action involves intracellular Ca²⁺ signals (Bofill-Cardona et al., 2000) but does not in frog ganglia (Stemkowski et al., 2002). Moreover, in rat hippocampus, the P2Y₁ receptor-mediated suppression of I_M is not associated with [Ca²⁺]_i signals and is suggested to be via PIP₂ depletion (Filippov et al., 2006). In this work, we systematically investigate the action of P2Y receptor stimulation on neuronal excitability and discharge properties of rat SCG cells and its mode of action on the endogenous I_M and I_Ca in those cells. The purinergic signals are compared with those from muscarinic and bradykinin stimulation and show the purinergic action to increase neuronal excitability via IP₃-mediated [Ca²⁺]_i signals.

Materials and Methods

SCG neuron culture and transduction.

Sympathetic neurons were isolated from the SCG of 7- to 14-d-old male rats (Sprague Dawley) and cultured for 2–4 d. Rats were anesthetized with halothane and decapitated. Neurons were dissociated using methods of Bernheim et al. (1991), plated on 4 × 4 mm glass coverslips (coated with poly-l-lysine) and incubated at 37°C (5% CO₂). Fresh culture medium containing nerve growth factor (50 ng/ml) was added to the cells 3 h after plating. In some experiments, pertussis toxin (PTX) (100 ng/ml) was added to the culture medium. For exogenous expression of cDNA constructs in SCG neurons, we used the PDS-1000/He biolistic particle delivery system (“gene gun”; Bio-Rad, Hercules, CA), as described recently (Gamper and Shapiro, 2006). Transfection efficiency was assumed to be determined by the random distribution of fired gold particles and was up to 10% of cultured neurons.

Perforated-patch electrophysiology.

Pipettes were pulled from borosilicate glass capillaries (1B150F-4; World Precision Instruments, Sarasota, FL) using a Flaming/Brown micropipette puller P-97 (Sutter Instruments, Novato, CA) and had resistances of 1–4 MΩ when filled with internal solution and measured in standard bath solution. Membrane current was measured with pipette and membrane capacitance cancellation, sampled at 5 ms or 200 μs, and filtered at 1 or 2.5 kHz (for I_M and I_Ca, respectively) by an EPC-9 amplifier and PULSE software (HEKA/InstruTech, Port Washington, NY). In all experiments, the perforated-patch method of recording was used with amphotericin B (600 ng/ml) in the pipette (Rae et al., 1991). Amphotericin was prepared as a stock solution as 60 mg/ml in DMSO. In these experiments, the access resistance was typically 10 MΩ 5–10 min after seal formation. Cells were placed in a 500 μl perfusion chamber through which solution flowed at 1–2 ml/min. Inflow to the chamber was by gravity from several reservoirs, selectable by activation of solenoid valves (Warner Instruments, Hamden, CT). Bath solution exchange was essentially complete by <30 s. Experiments were performed at room temperature.

M currents in SCG cells were studied by holding the membrane potential at −25 mV and applying a 500 ms hyperpolarizing pulse to −60 mV every 3 s. M-current amplitude was measured at −60 mV from the decaying time course of the deactivating current sensitive to the M-channel-specific blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone] (Zaczek et al., 1998). UTP, BK, oxotremorine-M (oxo-M), and XE991 were used at concentrations of 10 μm, 250 nm, 10 μm, and 50 μm, respectively. To evaluate the amplitude of I_Ca, cells were held at −80 mV, and 15 ms depolarizing steps to 5 mV were applied every 5 s. The amplitude of I_Ca was usually defined as the inward current sensitive to Cd²⁺ (100 μm). The external solution used to record M currents contained the following: 150 mm NaCl, 5 mm KCl, 2 mm CaCl₂, 1 mm MgCl₂, 10 mm glucose, 10 mm HEPES, and 500 nm tetrodotoxin, pH 7.4 with NaOH. For I_Ca recordings, KCl was reduced to 2.5 mm, and CaCl₂ was increased to 5 mm. The pipette solution for voltage-clamp experiments contained the following (in mm): 150 KCl/CsCl, 5 MgCl₂, and 10 HEPES to record I_M/I_Ca. For current-clamp experiments, the pipette solution contained the following (in mm): 20 KCl, 100 K-acetate, 5 MgCl₂, and 40 HEPES. Data are presented as mean ± SEM. Statistical tests were performed using paired t test or unpaired t test when appropriate.

Imaging.

Fluorescent microscopy was performed with an inverted Nikon (Tokyo, Japan) Eclipse TE300 microscope with an oil-immersion 40×/1.30 numerical aperture objective. A Polychrome IV monochromator (T.I.L.L. Photonics, Martinsreid, Germany) was used as the excitation light source, and a FURA2 71000 filter cube (Chroma Technology, Brattleboro, VT) was used for fura-2 imaging. SCG neurons were bath loaded with fura-2 AM (2 μm) for 30 min at 37°C in the presence of pluronic acid (0.01%). Cells were excited alternatively at 340 and 380 nm (50–200 ms every 2 s), the fluorescence emission was collected by an IMAGO 12-bit cooled CCD camera, and images were stored/analyzed with TILLvisION 4.0 software. These fura-2 signals were not calibrated because of inherent difficulties in calibrating esterified indicator dyes (Zhou and Neher, 1993).

Computer modeling.

Simulations were performed using NEURON version 5.9 (Hines and Carnevale, 2001) on a Macintosh G4 computer (Apple Computers, San Jose, CA). The passive properties of a single compartment model (50 μm diameter) were chosen to reflect the input resistance and charging time constant of SCG neurons to hyperpolarizing current steps (specific membrane resistivity, 50,000 Ωcm²; specific capacitance, 1 μF/cm²). Four voltage-gated and two Ca²⁺-dependent conductances (available for download through ModelDB at http://senselab.med.yale.edu) were inserted into the model. A fast voltage-gated Na⁺ conductance (nahh.mod) (Traub et al., 1991) was inserted with a density of 5 mS/cm². Threshold was adjusted by shifting the voltage dependence of the forward and backward activation rate constants by +20 and +10 mV, respectively. Spike repolarization was achieved with a delayed rectifier K⁺ conductance (kdr.mod) (Migliore et al., 1995) at a density of 0.3 mS/cm² (E_K of −80 mV). A high-threshold, non-inactivating voltage-gated Ca²⁺ conductance (cal.mod) (Migliore et al., 1995) at a density of 2 mS/cm² was inserted with E_Ca determined by the Goldman–Hodgkin–Katz equation. Intracellular calcium dynamics were modeled including both rapid buffering and Ca²⁺ pumps (cadifus.mod). Two Ca²⁺-dependent K⁺ conductances were included in the model. A BK-type Ca²⁺-dependent conductance (cagk.mod) at a density of 0.1 mS/cm² contributed mostly to spike repolarization, whereas an SK-type Ca²⁺-dependent conductance (g_SK of 3.9 · [Ca²⁺] mS/cm²) contributed mostly to spike-frequency adaptation (SFA). Finally, the M-channel conductance (g_M) (im.mod) (Migliore et al., 1995) was added with a density of 0.0175 mS/cm². It was assumed that the M current was active at rest and therefore contributed to the resting potential of the cell. Therefore, the equilibrium potential of the leak conductance was determined with no M current resulting for an initial resting potential (V_rest) of −50 mV. When g_M was active, V_rest in the model became −55 mV.

DNA constructs.

Plasmids containing wild-type (wt) or dominant-negative (DN) vertebrate CaM were given to us by Trisha Davis (University of Washington, Seattle, WA). DN CaM has an alanine substitution in each of the four Ca²⁺-binding EF hands (D20A, D56A, D93A, and D129A). DN neuronal calcium sensor-1 was given to us by Andreas Jeromin (Mt. Sinai Hospital, Toronto, Ontario, Canada). The IP₃ sponges consist of residues 224–604 of the rat type 1 IP₃ receptor containing either the R441Q (“high-affinity sponge”) or K508A (“low-affinity sponge”) mutations, made by PCR amplification and given to us by Llewelyn Roderick (Babraham Institute, Cambridge, UK).

Reagents.

The following reagents were used: UTP, TTX, NGF, bradykinin, oxo-M, and collagenase type I (Sigma, St. Louis, MO); fura-2 AM (Invitrogen, Carlsbad, CA); DMEM, fetal bovine serum, and penicillin/streptomycin (Invitrogen); amphotericin B and PTX (Calbiochem, La Jolla, CA); and XE991 (Tocris Bioscience, Ellisville, MO).

Results

We first evaluated the effect of purinergic stimulation on the M current in cultured SCG neurons and compared it with that of muscarinic and BK stimulation. Such a comparison between the effects of stimulating these receptor types was made throughout this study, because the muscarinic and bradykinin actions are the prototypes for the two described mechanisms of G_q/11-mediated suppression of I_M, the first being via PIP₂ depletion and second via IP₃, [Ca²⁺]_i signals, and CaM (Delmas and Brown, 2005). Cells were studied under perforated-patch voltage clamp, and agonists were applied by bath perfusion. Guided by previous work on P2Y receptor-mediated modulation of M current (I_M) in these cells (Boehm, 1998; Bofill-Cardona et al., 2000), we chose to use UTP as the purinergic agonist in this study. UTP is not an agonist of the ionotropic P2X receptors in the SCG (Calvert and Evans, 2004). Application of UTP caused a significant suppression of I_M in approximately one-half and BK in approximately three-fourths of the neurons examined, which we assume to reflect that proportion of neurons that express UTP-sensitive P2Y or BK receptors, respectively. There were no morphological differences between cells that did or did not respond to UTP. For those neurons that responded to UTP with a suppression of I_M >5%, we compared the response with those from bradykinin and the muscarinic agonist oxo-M. Application of UTP, BK, or oxo-M suppressed I_M by 54 ± 5% (n = 6), 62 ± 6% (n = 6), and 81 ± 6% (n = 6), respectively (Fig. 1). These values are consistent with those reported previously, although the extent of the purinergic suppression is slightly greater than that seen previously (Bofill-Cardona et al., 2000).

Figure 1.

Purinergic stimulation suppresses M current in SCG neurons. Purinergic inhibition of M current recorded from cultured SCG neurons using the pulse protocol depicted in the inset. A, Plotted are normalized M-current amplitudes, quantified as the deactivating time-dependent relaxations at −60 mV from pulses given every 3 s. UTP (10 μm), BK (250 nm), oxo-M (10 μm), or XE991 (50 μm) were bath applied during the periods shown by the bars and representative current traces shown below. B, Bars show mean inhibitions of I_M by UTP, BK, or oxo-M.

Purinergic and bradykinin stimulation increases neuronal excitability and decreases spike-frequency adaptation

Given the widely demonstrated control of neuronal excitability by M-current activity (Jones et al., 1995; Gu et al., 2005; Peretz et al., 2005; Peters et al., 2005; Shen et al., 2005; Gamper et al., 2006; Vervaeke et al., 2006; Yue and Yaari, 2006; Zaika et al., 2006), we next assessed the effects of purinergic stimulation on AP firing in our preparation of cultured SCG cells. We also systematically evaluated the effects of BK stimulation, whose actions on neuronal discharge have only been briefly described (Jones et al., 1995). Using perforated-patch current-clamp recording, we evaluated both the active and passive properties of the neurons in response to current steps in the presence or absence of UTP and BK. We compared the effects of UTP and BK with the well described M-current-mediated effects of muscarinic agonists or M-channel blockade. PTX was not used, meaning that G_q/11-mediated actions are not isolated in these experiments. Because our initial voltage-clamp experiments suggested that only approximately half of the neurons express the P2Y receptor, we triaged the cells studied under current clamp so as to characterize only P2Y receptor-expressing neurons. For each neuron studied, the resting potential (V_rest) was monitored for 10 min before application of any agonist. During application of UTP, if V_rest changed outside of the range seen during that 10 min control period, we assumed that neuron to express P2Y receptors, and the cell was studied further. As for the voltage-clamp experiments, approximately half of the neurons satisfied that criterion.

Shown in Figure 2 are representative voltage recordings in response to positive or negative current steps. In control, a 100 pA current injection elicited a short train of APs that terminated well before the end of the current step (Fig. 2A). Application of UTP caused a reversible increase in the number of APs elicited by the 100 pA current step, consistent with suppression of M current (Fig. 2B,C). Subsequent application of BK also caused a reversible increase in AP firing that was slightly greater than that caused by UTP (Fig. 2D,E). Finally, the response to muscarinic stimulation was a spike train that was nearly as intense as that caused by total blockade of M channels by the selective blocker XE991 (Fig. 2F,G). In control, the number of APs elicited by the 2 s, 100 pA depolarizing current was 4.1 ± 1.2 (n = 14). Application of UTP, BK, oxo-M, or XE991 increased the number of such APs to 7.8 ± 0.9 (p < 0.01; n = 9), 8.7 ± 0.8 (p < 0.01; n = 11), 20.2 ± 0.8 (p < 0.001; n = 12), or 22.0 ± 1.8 (p < 0.001; n = 12), respectively. There was not a significant change in the responses to hyperpolarizing current steps, ruling out an effect of the agonists on inward conductances such as I_h.

Figure 2.

Purinergic stimulation increases action potential firing in SCG neurons. Cultured SCG neurons were studied under perforated-patch current clamp, and voltages were recorded from a family of current pulses (inset). Voltage sweeps are shown in control (A), after UTP (10 μm) (B), first wash (C), after BK (250 nm) (D), second wash (E), after oxo-M (10 μm) (F), or after XE991 (50 μm) (G).

The effect of receptor stimulation on V_rest was quantified as an initial indictor of control over excitability (Fig. 3A). In control, V_rest was −56.2 ± 0.6 mV (n = 14). Application of UTP or BK modestly depolarized V_rest to −53.7 ± 0.4 mV (p < 0.01; n = 9) and −53.1 ± 0.6 mV (p < 0.01; n = 11), respectively. In some experiments, we tested the effect of BK applied first to rule out any residual effect of previous UTP application, and, in those cells, the initial V_rest was −55.7 ± 0.3 mV, which was depolarized by BK to −53.4 ± 0.1 mV (p < 0.01; n = 12), similar to the effect of BK in cells in which UTP was applied first. Application of oxo-M induced a stronger depolarization of V_rest, to −50.9 ± 0.4 mV (p < 0.01; n = 12), nearly as much as that induced by XE991 (−50.0 ± 0.6 mV; p < 0.01; n = 12). We further quantified the effects of stimulation of G_q/11-coupled receptors on excitability by examining the relationship between injected current and AP firing (Fig. 3B,C). In control, the amplitude of the current required to reach threshold for AP generation (AP_thresh) was 28.8 ± 0.4 pA (n = 20). Application of UTP or BK modestly decreased AP_thresh to 25.9 ± 1.7 pA (p < 0.05; n = 9) and 24.1 ± 2.1 pA (p < 0.05; n = 11), respectively. Application of oxo-M reduced AP_thresh more strongly, to 22.1 ± 1.7 pA (p < 0.01; n = 12), nearly as much as from full M-channel blockade by XE991 (21.1 ± 2.3 pA; p < 0.01; n = 12). Evaluated over a range of current amplitudes, UTP and BK modestly, but significantly, increased the number of elicited APs at all amplitudes of injected current (Fig. 3C). The effect of muscarinic stimulation was stronger and was nearly as great as that produced by XE991. In summary, purinergic and BK stimulation both suppressed I_M by 50–60%, and both modestly, but significantly, increased somatic excitability to a similar degree.

Figure 3.

Control of somatic excitability by regulation of M current. Bars show summarized data of the resting potential (A) and the threshold current required for firing of action potentials (B) for SCG neurons in control, after UTP (10 μm), BK (250 nm), oxo-M (10 μm), or XE991 (50 μm). C, Plotted are the number of APs elicited during a 2 s depolarizing current injection over a range of current amplitudes, during the indicated conditions. *p < 0.05; **p < 0.01.

Many neurons, including ganglia cells, exhibit SFA, characterized by the slowing or cessation of AP firing during a maintained stimulatory input. M current has been shown to be a prime contributor to SFA because of its accumulating conductance as the slowly activating M channels turn on during a spike train (Lawrence et al., 2006; Vervaeke et al., 2006; Zaika et al., 2006). We evaluated the effect of P2Y or BK receptor stimulation on SFA during a train of spikes using two related measures: (1) the relationship between interspike interval and spike number, and (2) spike-train duration (Fig. 4A). Spike trains elicited by 100 pA stimulatory currents were analyzed from SCG neurons studied under perforated-patch current clamp. In control, the instantaneous interspike interval rapidly lengthened during the short spike train, which terminated after 0.4 ± 0.1 s (n = 14). In the presence of UTP or BK, the slope of the interspike interval versus spike number relationship was significantly more shallow, and the spike-train duration was lengthened to 1.2 ± 0.1 s (p < 0.01; n = 9) and 1.3 ± 0.1 s (p < 0.01; n = 11), respectively. In accord with its greater effect on I_M and on AP firing, muscarinic stimulation more greatly dampened SFA, resulting in a very shallow relationship between interspike interval and spike number, and a spike-train duration that was lengthened to 1.8 ± 0.1 s (p < 0.001; n = 12). Complete blockade of M channels by application of XE991 made that relationship only slightly shallower and resulted in a spike train that lasted throughout the length of the 2 s stimulatory pulse.

Figure 4.

Effects on spike-frequency adaptation and comparison with a computer model of neuronal discharge. Plotted are the instantaneous interspike interval as a function of spike number (A1) and spike-train duration (A2) from spike trains elicited by a 100 pA stimulus applied to SCG neurons under current clamp, in control, after UTP (10 μm), BK (250 nm), oxo-M (10 μm), and XE991 (50 μm). In B1 are shown the simulated curves of interspike interval versus spike number predicted by a neuronal discharge model over the indicated range of fractional M-current activity. In B2 are plotted the simulated dependence of spike-train duration on fractional M-current activity. The observed suppressions of M current by the compounds used in this paper are marked on the abscissa and the predicted spike-train durations on the ordinate for comparison with the data in A2. **p < 0.01, ***p < 0.001.

In a recent paper, we used a multi-conductance neuronal model to successfully predict the effects on AP firing and SFA of suppression of M-channel conductance by angiotensin II (Zaika et al., 2006). The model program, NEURON version 5.7 (Hines and Carnevale, 2001), contains four voltage-gated and two Ca²⁺-dependent conductances, including g_M (Borg-Graham, 1991; Migliore et al., 1995). In the study by Zaika et al. (2006), we included only a BK conductance as a K_Ca type, but here we also include an SK conductance in the simulation. The values of the six conductances were adjusted to best correlate with the observed behavior of the neurons (for details, see Materials and Methods). In the model, full activity of both SK- and M-type conductances resulted in a response to a depolarizing current of only several APs, but turning off of M current (at first leaving the SK current intact) changed the response to an uninterrupted spike train exhibiting only minimal SFA (data not shown), similar to what we observed (Fig. 2A,G). In Figure 4B are shown the predictions of the model for the interspike interval versus spike number relationship and spike-train duration over a range of fractional g_M activation. We examined these model data to see whether the effects on SFA correlate with the degree of suppression of I_M for the agonists used in this study. In comparing Figure 4, A₁ and B₁, one can see that the predicted interspike interval versus spike number relationships at 100, 60, 20, and 0% g_M qualitatively match the experimentally observed relationships for control, UTP ≈ BK, oxo-M, and XE991, under which conditions the fractional I_M is 100, 38–46, 18, and 0%, respectively. Indicated on the model predictions of spike-train durations are the measured suppressions of I_M and the predicted spike-train durations as a result (Fig. 4B₂). For control, UTP, BK, oxo-M, and XE991, the predicted spike-train durations are ∼0.4, 1.25, 1.35, 1.65, and 1.8 s, which qualitatively agree with the experimentally observed values (Fig. 4A₂).

With the SK conductance included, the model prediction of spike-train duration versus g_M includes an inflection in the curve at ∼10% g_M, which the model ascribes to an oscillation in the membrane potential from sequential SK-induced hyperpolarization, turning off of M current, depolarization that produces rises in [Ca²⁺]_i via influx through Ca²⁺ channels, and cycling back to the turning on of SK channels. We observed such membrane oscillations of 2–3 mV in many of our current-clamp recordings, although this phenomenon was not studied further. The reduction of SFA produced by muscarinic stimulation, as measured by the interspike interval versus spike number relationship, was somewhat greater than that predicted by M-current suppression alone, especially at spike numbers >15, in which the model predicts an upward inflection as opposed to the flat relationship actually observed. This is likely attributable to concurrent suppression of I_Ca by M₁ and M₄ receptor stimulation and consequential reduction in K_Ca conductances. Indeed, systematic reduction of I_SK in the model reduced SFA for later spikes of a given train. As a result, when g_M was 20% of control, reducing I_SK or I_Ca resulted in SFA that approached that of 0% g_M, as observed experimentally.

P2Y receptor stimulation induces intracellular Ca²⁺ signals and requires IP₃ for suppression of I_M

In SCG neurons, stimulation of bradykinin B₂, but not muscarinic M₁ nor angiotensin AT₁, receptors induce [Ca²⁺]_i signals from IP₃-gated stores (Delmas et al., 2005). Although all three receptor types couple to G_q/11 and activate phospholipase C, it is hypothesized that colocalization of B₂, but not M₁ or AT₁, receptors with IP₃ receptors in “microdomains” accounts for this selectivity for eliciting [Ca²⁺]_i signals (Delmas and Brown, 2002; Delmas et al., 2002). Previous work has demonstrated P2Y receptor-mediated suppression of I_M to be via G_q/11 and to require PLC activity (Bofill-Cardona et al., 2000; Stemkowski et al., 2002). We thus began our investigation of the intracellular mechanism linking P2Y receptor stimulation with suppression of I_M by asking whether UTP elicits [Ca²⁺]_i signals in SCG neurons. [Ca²⁺]_i changes were observed using fura-2 loaded into the cells via the bath solution as the AM ester. We tested the ability of UTP, as well as BK and oxo-M, to provoke Ca²⁺ signals. Only cells in which the resting 340/380 nm ratio was <0.3 were used in these measurements. Although the cells were not voltage clamped, the bath contained TTX to eliminate the possibility of [Ca²⁺]_i rises caused by AP spiking and Ca²⁺ influx through Ca_V channels attributable to agonist-induced depolarization of V_rest. Shown in Figure 5A is a representative experiment from two adjoining SCG neurons. The 340/380 nm ratio is shown pseudocolored using the indicated scale. Muscarinic stimulation with oxo-M did not result in a measurable [Ca²⁺]_i signal, but subsequent application of UTP caused an obvious and reversible rise in [Ca²⁺]_i that was not as large as that induced by BK. In such experiments, the resting 340/380 nm ratio was 0.21 ± 0.01 (n = 20), and the rises in the 340/380 nm ratio evoked by application of oxo-M, UTP, and BK were 0.016 ± 0.002 (n = 6), 0.10 ± 0.02 (n = 7), and 0.31 ± 0.03 (n = 15), respectively (Fig. 5B). Our data indicate that, like bradykinin but unlike muscarinic agonists (Cruzblanca et al., 1998), purinergic P2Y receptor agonists induce rises in intracellular Ca²⁺ in sympathetic ganglia cells.

Figure 5.

Purinergic stimulation induces intracellular Ca²⁺ signals. Shown in A is a representative experiment from two SCG neurons loaded with fura-2 dye from the bath as the AM ester. Shown are pseudocolor 340/380 nm ratio images of fura-2 fluorescence from two adjoining neurons in control and during 2 min applications of oxo-M (10 μm), UTP (10 μm), and BK (250 nm). The pseudocolor scale runs from blue (low [Ca²⁺]) to red (high [Ca²⁺]). Plotted is the 340/380 nm ratio during the experiment for the topmost neuron (white asterisk). The agonists were applied during the indicated times. Bars in B are the summarized results of these experiments.

P2Y receptor stimulation requires IP₃ and functional CaM for suppression of I_M

We then probed for the involvement of IP₃ in UTP-induced suppression of I_M. Our first test used a construct containing a fusion protein of enhanced green fluorescent protein (EGFP) and point mutants of the IP₃-binding domain in the N-terminal of the rat type 1 IP₃ receptor. A protein consisting only of residues 224–604 has an affinity for IP₃ in the range of 92 pm and the R441Q mutant 45 pm, some 1000-fold higher than the native IP₃ receptor, whereas the K508A mutant reduces the affinity to only 340 nm. The R441Q, but not the K508A, mutant has been shown to be an effective sponge of IP₃ when overexpressed in cells, preventing accumulation of IP₃ in the cytoplasm during PLC activation and any downstream [Ca²⁺]_i signal (Uchiyama et al., 2002). Transfection of SCG neurons was performed using a biolistic particle delivery device (gene gun), using recently described procedures (Gamper and Shapiro, 2006), and the neurons were studied the following day under perforated-patch voltage clamp. Figure 6, A and B, shows experiments on neurons transfected with either the K508A low-affinity sponge or the R441Q high-affinity sponge. Whereas the cell overexpressing the low-affinity sponge displayed robust and reversible suppressions of I_M by UTP, BK, and oxo-M, the cell overexpressing the high-affinity sponge responded to both UTP and BK with very little suppressions of I_M, but the response to oxo-M was unaltered. These data are summarized in Figure 6C (left). In neurons transfected with the low-affinity sponge, the suppressions of I_M by UTP, BK, and oxo-M were 54 ± 9% (n = 5), 57 ± 8% (n = 6), and 78 ± 6% (n = 6), respectively. In neurons transfected with the high-affinity sponge, the suppressions of I_M by UTP, BK, and oxo-M were 25 ± 5% (p < 0.01; n = 5), 26 ± 6% (p < 0.01; n = 8), and 69 ± 15% (n = 8). There was no effect on tonic I_M densities of sponge expression (Fig. 6C, right). In neurons transfected with the K508A sponge or the R441Q sponge, tonic I_M densities were 1.0 ± 0.2 and 0.9 ± 0.2 pA/pF (n = 6 and 8), respectively. As another test to determine which receptor types require IP₃ accumulation for suppression of I_M, we assayed the effects of overexpression of a type 1 IP₃-5′phosphatase (IP₃-P), whose activity is the first step in IP₃ degradation. Overexpression of IP₃-P results in rapid dephosphorylation of IP₃ molecules as soon as they are made, preventing its accumulation in the cytoplasm (Hirose et al., 1999). As for the IP₃ sponge experiments, the constructs were transfected into SCG neurons using the gene gun and studied under perforated-patch voltage clamp. Figure 7, A and B, shows experiments on a control neuron transfected only with EGFP or a neuron cotransfected with EGFP-tagged IP₃-P. Whereas the control neuron displayed robust and reversible suppressions of I_M by UTP, BK, and oxo-M, the neuron overexpressing IP₃-P responded to both UTP and BK with suppressions of I_M that were very weak, but the response to oxo-M was still large.

Figure 6.

A high-affinity IP₃ sponge blocks purinergic inhibition of M current. Plotted are normalized M-current amplitudes, quantified as before, from neurons transfected with the N-terminal IP₃ type 1 receptor (NT IP₃R₁) K508A low-affinity sponge (A) or the N-terminal IP₃R₁ R441Q high-affinity sponge (B) using the gene gun and studied using perforated-patch recording. UTP (10 μm), BK (250 nm), oxo-M (10 μm), or XE991 (50 μm) were bath applied during the periods shown by the bars. Representative current traces during the experiments are shown in the insets. C, Bars show summarized inhibitions by UTP, BK, or oxo-M (left) or tonic I_M densities (right) for cells transfected with the low-affinity (K508A) or the high-affinity (R441Q) sponges. **p < 0.01.

Figure 7.

Purinergic suppression of I_M requires cytosolic IP₃ and functional CaM. Plotted are normalized M-current amplitudes quantified as before, from SCG cells transfected using the gene gun with EGFP only (A), type 1 IP₃-5′phosphatase (B), DN CaM (C), or wt CaM (D). UTP (10 μm), BK (250 nm), oxo-M (10 μm), or XE991 (50 μm) were bath applied during the periods shown by the bars. Representative current traces during the experiments are shown in the insets. Bars in E show summarized data from these experiments for I_M suppression by UTP, BK, and oxo-M (left) or tonic I_M densities (right). *p < 0.05; **p < 0.01; ***p < 0.001.

Along with the involvement of IP₃-mediated [Ca²⁺]_i signals in suppression of I_M by bradykinin is the use of CaM as the Ca²⁺ sensor of M channels. Much like the role of CaM in Ca²⁺-dependent inactivation of Ca_V channels (Halling et al., 2006) and activation of SK-type K⁺ channels (Maylie et al., 2004), CaM binds to M-type channels and inhibits them on [Ca²⁺]_i signals, perhaps by reducing the affinity of PIP₂ for the channels (Brown et al., 2007). Given the role of IP₃ in the purinergic action shown here, we therefore tested for the involvement of CaM in UTP-mediated suppression of I_M. SCG neurons were transfected with either wt CaM or a DN CaM that has an Asp-to-Ala mutation in all four Ca²⁺-binding sites, rendering it unable to bind Ca²⁺ (Geiser et al., 1991). If [Ca²⁺]_i signal sensing by CaM mediates purinergic inhibition of M channels, then we predict overexpression of DN CaM to block the suppression because channels will be bound by CaM molecules that cannot sense [Ca²⁺]_i, and we would predict overexpression of wt CaM to amplify tonic Ca²⁺/CaM modulation because many channels would be bound by Ca²⁺/CaM at resting [Ca²⁺]_i (Gamper and Shapiro, 2003). Figure 7C shows an experiment on a neuron overexpressing DN CaM. In this cell, the suppression of I_M in response to application of UTP or BK was negligible but that from oxo-M application was robust. In Figure 7D, an experiment on a neuron overexpressing wt CaM is shown, in which the responses to UTP and BK were very small and that from oxo-M application was somewhat attenuated.

The suppressions of I_M in these four groups of cells are summarized in Figure 7E (left). In neurons transfected only with EGFP, the suppressions of I_M by UTP, BK, and oxo-M were 58 ± 8% (n = 7), 61 ± 6% (n = 9), and 80 ± 5% (n = 9), indistinguishable from nontransfected cells. However, in cells transfected with IP₃-P, the suppressions of I_M by UTP and BK, but not oxo-M, were severely blunted, with responses to UTP, BK, and oxo-M of 22 ± 8% (p < 0.01; n = 5), 23 ± 7% (p < 0.01; n = 6), and 71 ± 9% (n = 6), respectively. Likewise, in cells transfected with DN CaM, only the suppression of I_M by oxo-M remained intact. Thus, the responses to UTP, BK, and oxo-M were 18 ± 5% (p < 0.01; n = 5), 20 ± 4% (p < 0.01; n = 5), and 82 ± 7% (n = 5), respectively. The fractional suppressions of I_M by UTP and BK were also sharply reduced and that by oxo-M somewhat attenuated in cells overexpressing wt CaM. In those cells, the responses to UTP, BK, and oxo-M were 21 ± 5% (p < 0.01; n = 4), 23 ± 6% (p < 0.01; n = 4), and 50 ± 12% (p < 0.05; n = 4), respectively. The results from the IP₃-P and DN CaM experiments are consistent with our scheme of M-channel regulation, in which there is little tonic modulation at rest via IP₃ or Ca²⁺/CaM signals. Thus, there should be little effect on tonic I_M densities of lowering resting [IP₃] by transfection of the IP₃-P or occlusion of tonic CaM action by DN CaM overexpression. Conversely, overexpression of wt CaM might greatly amplify tonic Ca²⁺/CaM inhibition because the IC₅₀ of the sensitivity of KCNQ2/3 channels for [Ca²⁺]_i is ∼70 nm, in the range of resting [Ca²⁺]_i, and many more channels will now be tonically bound by Ca²⁺/CaM (Gamper and Shapiro, 2003). Indeed, this is what we observed (Fig. 7E, right). In neurons transfected with EGFP only, IP₃-P, or DN CaM, tonic I_M densities were very similar: 1.2 ± 0.3, 1.1 ± 0.2, and 1.2 ± 0.2 pA/pF (n = 9, 6, and 5), respectively. However, in cells overexpressing wt CaM, the tonic I_M density was reduced by more than half, to 0.5 ± 0.2 pA/pF (p < 0.01; n = 4). Moreover, the reduction of tonic I_M currents in those cells is similar to that produced by BK or UTP, as if Ca²⁺/CaM-mediated suppression is now tonically strong, consistent with our hypothesis.

The attenuation of muscarinic suppression of I_M in cells overexpressing wt CaM was surprising and somewhat different from the unaffected action of oxo-M in such cells reported previously (Gamper and Shapiro, 2003). One possible reason for the effect involves myrisotylated alanine-rich C kinase substrate (MARCKS) protein that binds to both PIP₂ and Ca²⁺/CaM in a manner in which binding of the latter is thought to favor unbinding of the former (McLaughlin and Murray, 2005). At low [Ca²⁺]_i, MARCKS can sequester much PIP₂ and then release it on [Ca²⁺]_i rises. Thus, overexpression of wt CaM may greatly increase the fraction of MARCKS proteins bound by Ca²⁺/CaM, resulting in increased tonic [PIP₂]. Similar to the effect of increasing tonic [PIP₂] by overexpression of phosphatidyl-4-phosphate 5-kinase [PI(4)5-kinase], muscarinic suppression of I_M would then be reduced (Winks et al., 2005). It has been suggested that Ca²⁺/CaM inhibits M channels by reducing their affinity for PIP₂ (Brown et al., 2007; Gamper and Shapiro, 2007). A second, related, explanation is that overexpression of wt CaM may result in many channels unbinding PIP₂, again resulting in increased tonic [PIP₂] in the vicinity of the channels.

In summary, the IP₃-P and IP₃ sponge data indicate that P2Y receptor-mediated suppression of I_M requires cytosolic IP₃ accumulation. They also provide more evidence for previous conclusions suggesting M-current suppression by BK, but not oxo-M, to also use an IP₃-mediated signal (Cruzblanca et al., 1998). The blockade of purinergic and BK inhibitions in cells overexpressing DN CaM is consistent with those agonists acting via [Ca²⁺]_i signals in concert with CaM, whose actions are now occluded by the overexpressed DN CaM bound to the channels. Overexpression of wt CaM likely results in most channels already bound by Ca²⁺/CaM and tonic currents already suppressed before any receptor stimulation, with little additional IP₃/[Ca²⁺]_i-dependent suppression observed. Our data suggest that P2Y receptors in SCG neurons, like B₂ and unlike M₁ receptors, modulate M current through CaM action.

P2Y receptor stimulation does not suppress I_Ca unless neuronal Ca²⁺ sensor-1 activity is blocked

Two groups have shown Ca_V2 channels to be regulated by plasma-membrane PIP₂ and its depletion to underlie M₁ receptor-mediated inhibition (Wu et al., 2002; Gamper et al., 2004). Although both muscarinic and bradykinin stimulation provoke robust PLC-mediated PIP₂ hydrolysis in SCG neurons, the former, but not the latter, inhibits PIP₂-sensitive voltage-gated Ca²⁺ channels in those cells (Gamper et al., 2004) (but see Lechner et al., 2005). We suggested this differential action on the Ca²⁺ current (I_Ca) to be attributable to receptor-specific depletion of PIP₂, arising from concurrent stimulation of PIP₂ synthesis by bradykinin, but not muscarinic, agonists. In this model, bradykinin, but not muscarinic, stimulation produces IP₃-mediated [Ca²⁺]_i signals, resulting in acceleration of phosphatidylinositol-4-kinase (PI4)-kinase activity by neuronal Ca²⁺ sensor-1 (NCS-1) (Delmas et al., 2005). Given the similar mode of action of P2Y and B₂ receptors on M current seen in this study, we therefore tested the action of purinergic stimulation on I_Ca in SCG cells. As for bradykinin, we found purinergic stimulation to cause little suppression of I_Ca. Figure 8A shows an example of a neuron studied under perforated-patch voltage clamp under conditions that isolate I_Ca. Because stimulation of P2Y receptors has been reported to also activate G_o/i-mediated signaling pathways (Filippov et al., 2003), SCG cultures were treated overnight with pertussis toxin to isolate G_q/11-coupled receptor signaling (Beech et al., 1992). Application of neither UTP nor BK caused an appreciable suppression of I_Ca, whereas oxo-M produced a robust response in the same cell. In response to application of UTP, BK, or oxo-M, I_Ca was suppressed by 4.5 ± 1.0% (n = 7), 6.2 ± 0.8% (n = 7), and 52.1 ± 4.2% (n = 7), respectively (Fig. 8D, control). This result suggests that, as for B₂ receptor stimulation, P2Y receptor stimulation does not appreciably lower PIP₂ abundance. We then tested for the involvement of NCS-1-mediated stimulation of PIP₂ synthesis by transfecting a DN mutant that cannot bind Ca²⁺ (Koizumi et al., 2002). In that scenario, cells overexpressing DN NCS-1 should have [Ca²⁺]_i-dependent stimulation of PIP₂ synthesis aborted, allowing PIP₂ abundance to fall during P2Y or BK stimulation and I_Ca to be suppressed. Neurons were transfected either with enhanced yellow fluorescent protein (EYFP) only or with EYFP-tagged DN NCS-1. Figure 8, B and C, show examples from both groups of neurons. In the cell transfected only with EYFP, UTP and BK caused very little suppression of I_Ca, but oxo-M induced robust suppression, as in Figure 8A. However, in the cell transfected with DN NCS-1, UTP and BK now induced a significant suppression of I_Ca, as did oxo-M. These data are summarized in Figure 8D. In neurons transfected with EYFP only, application of UTP, BK, and oxo-M suppressed I_Ca by 5.8 ± 3.6% (n = 5), 8.2 ± 3.2% (n = 5), and 53.2 ± 5.7% (n = 5). In neurons transfected with DN NCS-1, the suppressions were 31.4 ± 4.7% (p < 0.01; n = 4), 36.6 ± 6.7% (p < 0.01; n = 4), and 59.7 ± 8.3% (n = 4), respectively. There was no significant effect on tonic I_Ca current amplitudes of NCS-1 overexpression. For nontransfected neurons or those transfected with EYFP only or with NCS-1, I_Ca densities were 44.4 ± 4.1, 50.4 ± 6.1, and 52.9 ± 3.2 pA/pF (n = 7,5,4), respectively. These results are in accord with our model of selective depletion of PIP₂ by muscarinic, but not purinergic or bradykinin, stimulation. Thus, we suggest that stimulation of both P2Y and B₂ receptors concurrently stimulate PIP₂ synthesis, at least in part, via NCS-1 action, compensating for PIP₂ depletion by PLC activity. With NCS-1 action blocked by overexpression of a DN form, [PIP₂] is allowed to fall during stimulation of either receptor, and stimulation of either now induces significant suppression of I_Ca.

Figure 8.

Purinergic and bradykinin stimulation do not suppress I_Ca unless NCS-1 activity is blocked. Plotted are normalized I_Ca amplitudes from nontransfected SCG neurons (A) or those transfected using the gene gun with EYFP only (B), or EYFP-tagged DN NCS-1 (C) and studied using perforated-patch recordings. SCG cultures were treated overnight with PTX (100 ng/ml) to block G_o/i-mediated actions. UTP (10 μm), BK (250 nm), oxo-M (10 μm), or CdCl₂ (100 μm) were bath applied during the periods shown by the bars. Representative current traces are shown in the insets. D, Bars show summarized data for suppressions of I_Ca by UTP, BK, and oxo-M for the three groups of cells. **p < 0.01.

Discussion

We suggest that the action of G_q/11-coupled receptors can be divided into two fundamental modes that have in common PLC activation and hydrolysis of PIP₂. The first mode is based on the widening discovery that many ion channels are regulated by a membrane PIP₂ abundance that can be strongly lowered by PLC (Suh and Hille, 2005). This mechanism seems particularly established for M₁ receptor suppression of M current in sympathetic ganglia and likely for AT₁ receptors as well (Shapiro et al., 1994; Zaika et al., 2006; Suh and Hille, 2007). Both receptor types share the lack of provoked IP₃-mediated [Ca²⁺]_i signals and dual inhibition of both M-type K⁺ and Ca²⁺ channels. The second mode is used by G_q/11-coupled receptors that do provoke such [Ca²⁺]_i signals, which then activate multiple Ca²⁺-binding signaling proteins. The bradykinin B₂ receptor of ganglia cells is the best described in this category. Because of concurrent stimulation of PIP₂ synthesis by bradykinin (Xu et al., 2003) via [Ca²⁺]_i-dependent activation of NCS-1 and acceleration of PI4-kinase activity, PIP₂ levels do not appreciably fall, M channels are inhibited instead by Ca²⁺/CaM action, and Ca²⁺ channels are little affected (Gamper et al., 2004; Winks et al., 2005; Brown et al., 2007). However, is this action in SCG cells unique to bradykinin? Our work here suggests not and places the purinergic P2Y receptors of these cells in the category using the second mode along with the B₂ receptor. For both, the action of IP₃ receptors is required, CaM functions as the Ca²⁺-sensor mediating suppression of M current, and Ca²⁺ channels are not modulated. Figure 9 summarizes the bipartite division of G_q/11-coupled receptor mechanisms hypothesized in sympathetic ganglia cells.

Figure 9.

Schematic representation of the two modes of G_q/11-coupled receptor mechanisms in SCG neurons. Both pathways involve activation of G_q/11 G-proteins, PLC, and PIP₂ hydrolysis. The “mode 1” pathway suppresses M current (I_M) and Ca²⁺ current (I_Ca) attributable to depletion of PIP₂. Mode 1 does not elicit [Ca²⁺]_i signals. The “mode 2” pathway does elicit [Ca²⁺]_i signals and concurrently stimulates PIP₂ synthesis via NCS-1. Released Ca²⁺ binds to NCS-1 and to CaM. Ca²⁺/CaM binds to M channels, resulting in their inhibition.

Along with muscarine and several peptides, purinergic agonists were soon identified as regulators of neuronal excitability (Siggins et al., 1977) via depression of M current (Adams et al., 1982; Lopez and Adams, 1989; Tokimasa and Akasu, 1990) in frog sympathetic neurons. The mechanism of action of neuronal P2Y receptors on I_M has been controversial and may depend on precise neuronal type or receptor isoform. Previous work in the rat SCG cells studied here, in which the dominant subtype is likely P2Y₆, suggested the requirement for [Ca²⁺]_i signals (Bofill-Cardona et al., 2000), consistent with our results, but, in frog sympathetic neurons in which the subtype is not clear, the PIP₂-depletion mode is suggested (Stemkowski et al., 2002). Recently, P2Y₁ receptor-mediated suppression of M current in hippocampus was studied, and the lack of [Ca²⁺]_i signals seen during purinergic stimulation in those cells points toward a PIP₂-depletion mechanism there as well. It seems that the basic division between (Filippov et al., 2006) modes of action hinges on the ability of an agonist to provoke [Ca²⁺]_i signals during receptor stimulation, and the critical factor for induction of such [Ca²⁺]_i release has been suggested to be colocalization of certain plasma-membrane receptors with endoplasmic reticulum-membrane IP₃ receptors (Delmas et al., 2004). Thus, if IP₃ is produced in the microdomain of the IP₃ receptor, [Ca²⁺]_i is the relevant signal, and downstream Ca²⁺-dependent actions of CaM and NCS-1 are initiated; if not, PIP₂ depletion is the mode of action. Perhaps the divergent results in different cells among different P2Y receptor subtypes predict differential localization of receptor isoforms in microdomains containing IP₃ receptors. Alternatively, different neuronal types may use different membrane protein organizations. Although we do not observe suppression of I_Ca by bradykinin unless the NCS-1 pathway is blocked (Gamper et al., 2004; this study), Lechner et al. (2005) observe such an inhibition that they ascribe to depletion of PIP₂. The most likely reason for the discrepancy is differential tonic PIP₂ levels, perhaps caused by the different medium/growth conditions used by the two laboratories (Chuang et al., 2001).

Filippov and colleagues, using SCG cells as a vehicle for heterologous expression of four different P2Y receptor subtypes, observed an primarily voltage-dependent and PTX-sensitive suppression of I_Ca and a wholly PTX-insensitive suppression of I_M by UTP (Filippov et al., 1998, 1999, 2000, 2003). The depression of I_Ca seen in those studies had all the hallmarks of the fast “willing-reluctant” type of modulation mediated by G_o/i-coupled receptors in many neuronal types (Hille, 1994) and none of the characteristics of the slower G_q/11-mediated pathway typified by M₁ receptors that we ascribe to depletion of PIP₂ (Gamper et al., 2004). We here found purinergic stimulation of PTX-treated neurons to likewise normally have little effect on I_Ca. The suppression of I_M in the papers cited above seems similar to that observed by stimulation of native P2Y receptors (Bofill-Cardona et al., 2000; this study), although additional mechanistic information from the heterologous-expression studies is not available. As in the case here using native receptors, stimulation of expressed P2Y₂ receptors increased AP firing, consistent with closure of M channels (Filippov et al., 1998). Interestingly, Filippov and colleagues observed little modulation by UTP of either type of channel in nontransfected neurons (which would use native receptors), whereas we observe suppression of I_M in approximately half the SCG cells studied. Perhaps our use of younger rats (1–2 weeks), and slightly longer culture (2–3 d), provides the explanation. Because colocalization of P2Y₆ receptors with IP₃ receptors may underlie the mechanistic specificity described in this study, we appreciate the opportunity to study the actions of the native receptors.

We suggest that stimulation of either B₂ or P2Y receptors does not appreciably deplete membrane PIP₂ attributable, at least in part, to NCS-1-mediated stimulation of PIP₂ synthesis. Such agonist stimulation of PIP₂ synthesis is by now widely documented and has been suggested to be often obligatory to account for the mass of IP₃ produced during PLC activation (Loew, 2007). Independent evidence for receptor-specific depletion of PIP₂ comes from the tubby probe that binds PIP₂, but not IP₃ (Santagata et al., 2001). Translocation of tubby was minor during bradykinin stimulation in the same cells in which muscarinic translocation was large, but, during acute blockade of PI4-kinase, the translocations were equal (Hughes et al., 2007). In our scheme, blockade of NCS-1 or PI4-kinase activity allows [PIP₂] to fall and I_Ca to be suppressed. This scenario presents us with a conundrum, however, because the prevention of IP₃ accumulation by overexpression of the IP₃ sponge or the IP₃-P should prevent the [Ca²⁺]_i signals that not only activate CaM but also NCS-1, the molecule that we implicate in stimulation of PIP₂ synthesis. With both CaM and NCS-1 activity prevented, why should stimulation of B₂ or P2Y receptors not deplete PIP₂, as by M₁ receptor stimulation? The Hille group has shown some [Ca²⁺]_i signal (either released from stores or via Ca²⁺ influx) to be necessary for full PLC activity in tsA-201 tissue-culture cells in which overexpression of IP₃-P slowed and weakened diacylglycerol production and KCNQ2/3 channel inhibition by expressed M₁ receptors (Horowitz et al., 2005; Suh et al., 2006). However in the SCG neurons in which muscarinic suppression of M current is strongest, M₁ receptor stimulation does not produce detectable [Ca²⁺]_i rises (Beech et al., 1991) [unlike stimulation of expressed M₁ receptors in tsA-201 cells (Shapiro et al., 2000)], and we did not observe any effect on muscarinic suppression of I_M of the IP₃ sponge or IP₃-P. Two possible explanations emerge. The first is receptor-specific acceleration of PI(4)5-kinase activity as well, which “virtual cell” modeling suggests is required for compensation of PIP₂ levels in the face of PLC activity (Xu et al., 2003; Loew, 2007). Thus, whereas BK and P2Y receptor triggering may depress M current primarily through IP₃-mediated [Ca²⁺]_i signals, leading to blockade of I_M suppression when IP₃ accumulation is prevented, the receptor-stimulated acceleration of PIP₂ synthesis may partly be via [Ca²⁺]_i signals and partly through receptor-specific stimulation of PI(4)5-kinase via an, as yet, unknown pathway. The second possibility is receptor-specific turn on of store-operated Ca²⁺ entry that might require coupling of G-protein-coupled receptors and IP₃ receptor but not IP₃ (Kiselyov et al., 2005). Other aspects of this issue have been discussed recently (Gamper and Shapiro, 2007).

In our comparison among agonists, the effects on excitability generally correlated with degree of M-channel inhibition, although the increase in firing by UTP and BK seemed qualitatively weaker than that by oxo-M. Our finding that neither purinergic nor bradykinin simulation normally suppress I_Ca give a hypothesis why this may be so. Simultaneous inhibition of M channels from G_q/11-coupled M₁ receptor stimulation and of Ca²⁺ channels from combined M₁ and G_o/i-coupled M₄ receptor stimulation (Hille, 1994) predict a synergistic decrease in K⁺ current braking action by the reduced I_M combined with the reduced K_Ca conductances from suppression of Ca²⁺ influx, especially likely given the strong role ascribed to the SK channels in controlling the medium afterhyperpolarization (Kawai and Watanabe, 1986; Davies et al., 1996). It is likely that additional mechanisms make a contribution to SFA in these neurons as well. We look forward to a more complete model of simultaneous modulation of multiple ion channels by the same receptor and activation by ionotropic and metabotropic receptors by the same agonist (e.g., acetylcholine, ATP, and glutamate) to understand the multifaceted potential of neurotransmitter-mediated regulation of neuronal function.