Articles, Cellular/Molecular

M1 Muscarinic Receptors Inhibit L-type Ca2+ Current and M-Current by Divergent Signal Transduction Cascades

Liwang Liu, Rubing Zhao, Yan Bai, Lee F. Stanish, James E. Evans, Michael J. Sanderson, Joseph V. Bonventre and Ann R. Rittenhouse
Journal of Neuroscience 8 November 2006, 26 (45) 11588-11598; https://doi.org/10.1523/JNEUROSCI.2102-06.2006

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Abstract

Ion channels reside in a sea of phospholipids. During normal fluctuations in membrane potential and periods of modulation, lipids that directly associate with channel proteins influence gating by incompletely understood mechanisms. In one model, M1-muscarinic receptors (M1Rs) may inhibit both Ca2+ (L- and N-) and K+ (M-) currents by losing a putative interaction between channels and phosphatidylinositol-4,5-bisphosphate (PIP2). However, we found previously that M1R inhibition of N-current in superior cervical ganglion (SCG) neurons requires loss of PIP2 and generation of a free fatty acid, probably arachidonic acid (AA) by phospholipase A2 (PLA2). It is not known whether PLA2 activity and AA also participate in L- and M-current modulation in SCG neurons. To test whether PLA2 plays a similar role in M1R inhibition of L- and M-currents, we used several experimental approaches and found unanticipated divergent signaling. First, blocking resynthesis of PIP2 minimized M-current recovery from inhibition, whereas L-current recovered normally. Second, L-current inhibition required group IVa PLA2 [cytoplasmic PLA2 (cPLA2)], whereas M-current did not. Western blot and imaging studies confirmed acute activation of cPLA2 by muscarinic stimulation. Third, in type IIa PLA2 [secreted (sPLA2)]−/−/cPLA2−/− double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M-current over-recovery after washout of muscarinic agonist. Our findings support a model of M1R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.

Introduction

L-type Ca2+ (L-) channels are found throughout the nervous system, where they participate in many aspects of electrical activity, e.g., integrating synaptic input, encoding information in action potentials, and coordinating electrical activity at the cell membrane with underlying biochemical and transcriptional events (West et al., 2002; Marshall et al., 2003). Because L-channels are concentrated in cell bodies (Hell et al., 1993), their modulation by neurotransmitters may profoundly affect these processes. A well studied model of voltage-independent modulation is found in superior cervical ganglion (SCG) neurons, where M1 muscarinic receptor (M1R) agonists inhibit L-current (Suh and Hille, 2005). Although the changes in channel gating are understood (Mathie et al., 1992), the slow acting signal transduction pathway by which M1Rs modulate L-channel activity remains uncharacterized.

This lack of information on L-current modulation contrasts the wealth of information known about M1R inhibition of two other currents in SCG neurons, the K+ current M-current and the N-type Ca2+ (N-) current. The first steps in the cascade involve M1Rs coupling to Gq-like G-proteins (Haley et al., 2000) and phospholipase C (PLC) (Wu et al., 2002; Liu and Rittenhouse, 2003; Delmas and Brown, 2005; Suh and Hille 2005). Whether additional enzymes are required for M- and N-current modulation is controversial. Two models have been developed to explain the molecular events downstream of PLC leading to channel inhibition. The first model hypothesizes that loss of phosphatidylinositol-4,5-bisphosphate (PIP2), normally associated with M- and N-channels, decreases channel activity (Suh and Hille, 2002; Wu et al., 2002; Ford et al., 2003; Zhang et al., 2003; Gamper et al., 2004). Our alternative model for N-current modulation proposes that inhibition requires generation of free fatty acid by phospholipase A2 (PLA2). This model is supported by our findings that exogenously applied arachidonic acid (AA) mimicked muscarinic agonists and that antagonizing PLA2 with oleyloxyethyl phosphorylcholine (OPC) or limiting free endogenous AA minimized whole-cell N-current modulation (Liu and Rittenhouse, 2003). However, OPC did not alter inhibition in perforated-patch recordings (Gamper et al., 2004), raising uncertainty about PLA2 involvement. Moreover, whether L-current modulation requires PLA2 remains untested. The conflicting interpretations of these findings underscore the importance of determining whether the same signaling cascade mediates inhibition of Ca2+ currents and M-current and whether the pathway entails simply a loss of PIP2 (Suh and Hille, 2002) or requires additional phospholipid metabolism by PLA2 (Liu and Rittenhouse, 2003).

To clarify these issues, the present study used pharmacological, biochemical, imaging, and genetic approaches to first investigate whether PLA2 participates in L-current modulation. Here we report that L-current is inhibited by M1Rs coupled to Gq/11, PLC/PIP2, group IVa PLA2 [cytoplasmic PLA2 (cPLA2)], and a free fatty acid, probably AA. We also tested whether M-current modulation requires PLA2 and found that loss of PLA2 activity dramatically slows M-current recovery from inhibition but exerts no effect on inhibition. Thus, L-current inhibition and M-current recovery use the same signaling cascade, whereas M-current inhibition requires only a decrease in PIP2 levels. This unanticipated divergence in M1R-mediated modulation of L- and M-current inhibition may contribute to neuronal plasticity.

Materials and Methods

Neonatal rat SCG neuron preparation.

Dissociated sympathetic neurons were obtained from SCG of 1- to 4-d-old Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) following the methods of Liu and colleagues (Liu et al., 2001; Liu and Rittenhouse, 2003). Cells were incubated at least 4 h before initiating whole-cell recording experiments and used within 12 h to avoid recording from cells with processes.

Adult SCG neuron preparation for electrophysiology and imaging.

cPLA2−/− mice were created by homologous recombination in J1 embryonic stem cells derived from 129/Sv mice and then introduced into C57BL/6J mice (Bonventre et al., 1997). The C57BL/6J × 129/Sv mice carry a spontaneous mutation that results in loss of secreted PLA2 (sPLA2). Thus, these mice are naturally sPLA2(−/−), whereas the cPLA2−/− mice are a PLA2 double-knock-out and referred to as s/cPLA2(−/−). Mice (8–16 weeks old) were decapitated, and their SCGs were removed and desheathed in Earle's balanced salt solution (EBSS) (Sigma, St. Louis, MO). Each ganglion was then cut into several pieces, transferred into a 25 cm2 culture flask containing 5 ml of EBSS, 0.5 mg/ml trypsin (Worthington Biochemicals, Freehold, NJ), 1 mg/ml collagenase D (Roche Applied Science, Indianapolis, IN), 0.1 mg/ml DNaseI (Roche Applied Science), 3.6 g/L glucose, and 10 mm HEPES, and incubated at 34°C in a 5% CO2/95% O2 gassed, shaking water bath. After 1 h, the flask was shaken vigorously to release cell somata from ganglion fragments or dissociated by trituration with a P5000 Pipetman (Gilson, Middleton, WI). The dissociation was stopped by adding 5 ml of modified Eagle's medium (MEM) (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 4 mm glutamine, and 100 IU/ml penicillin–100 μg/ml streptomycin. Cells were pelleted by centrifuging at 500 × g for 5 min. For electrophysiological studies, the resulting pellet was resuspended in DMEM supplemented as for neonatal SCG neurons. Dissociated cells from the equivalent of one SCG were plated on poly-l-lysine-coated glass coverslips and incubated in Falcon (Franklin Lakes, NJ) 35 mm dishes at 37°C in a 5% CO2 environment. SCG neurons were used within 12 h. For imaging studies, the pellet was gently resuspended in MEM, and a half ganglion was aliquoted into one 35 mm poly-l-lysine-coated glass-bottom Petri dish (number 1.5; MatTek, Ashland, MA) and incubated at 37°C in a 5% CO2 environment.

Electrophysiological methods.

Standard whole-cell recording methods and configuration were using following Liu and Rittenhouse (2003). Peak current amplitudes were measured 15 ms after the start of the test pulse. For experiments in which the long-lasting tail current was measured, a second cursor was placed ∼13 ms after the test pulse.

The external solution for recording whole-cell Ca2+ currents contained the following (in mm): 135 N-methyl-d-glucamine-Asp, 10 HEPES, 20 Ba2+ acetate, and 0.0005 tetrodotoxin (TTX) (293 mOsm). The pipette solution contained the following (in mm): 123 Cs-Asp, 10 HEPES, 0.1 bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), 5 MgCl2, 4 ATP, (Sigma), and 0.4 GTP (Sigma) (264 mOsm). The external solution used to record M-current contained the following (in mm): 160 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 8 glucose. The pipette solution contained the following (in mm): 175 KCl, 5 MgCl2, 5 HEPES, 0.1 BATPA, 4 ATP, and 0.4 GTP.

As required for each experiment, drugs were added to the bath. Nimodipine (Miles, New Haven, CT), (+)-202-791 [S(+)-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid isopropyl ester] (a gift from Sandoz, Basel, Switzerland), FPL 64176 (2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester) (FPL) [Research Biomedicals (Natick, MA) or Sigma], 7,7-dimethyl-5,8-eicosadienoic acid (DEDA) (Sigma), OPC (Calbiochem, La Jolla, CA), and U-73122 (1-[6[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) (Biomol, Plymouth Meeting, PA) were prepared as stock solutions in 100% ethanol and diluted with bath solution to a final ethanol concentration of <0.17% The maximal final concentration of ethanol had no significant effect on whole-cell peak or long-lasting tail currents. Stock solutions of AA (NuCheck, Elysian, MN) were kept under nitrogen at −80°C in sealed glass vials. Bovine serum albumin (BSA) (fraction V, heat shock, fatty acid ultra free; Roche Applied Science) was added directly to the bath solution. Stock solutions of oxotremorine-M (Oxo-M) (Tocris Cookson, Ellisville, MO), the Gq antagonist GPantagonist-2A (GPant2A) (Biomol), the mamba snake toxin MT-7 (Peptides International, Osaka, Japan), TTX (Sigma), and ω-conotoxin GVIA (ω-CgTX) (Bachem, Torrance, CA) made up in double distilled water were diluted at least 1000 times to their final concentration with bath solution. Oxo-M was used at a final concentration of 10 μm. Antagonists of the AA signaling cascade were used at concentrations shown previously to have just maximal inhibition in either the SCG or other cell systems. Supramaximal concentrations were avoided when possible to minimize cross-reactivity. IgG (1:1000 or 6.5 μg/ml; Calbiochem) or antibodies (Abs) selective for Gαq/11 (1:1000 or 2.2 μg/ml; Calbiochem), Gαq (1:1000 or 2.2 μg/ml; Calbiochem), group IIa PLA2 (sPLA2; 1:250 or 0.8 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), or group IVa PLA2 (cPLA2; 1:250 or 0.18 μg/ml; 1:500 or 0.09 μg/ml; Cell Signaling Technology, Beverly, MA) were included in the pipette solution.

Gas chromatography–mass spectrometric analysis of AA release from SCG.

Adult rat SCGs were preincubated for 20 min at 37°C in 500 μl of EBSS in a shaking water bath gassed with 5% CO2/95% O2. 5,8,11,14-Eicosatetraynoic acid (30 μm; Sigma) was included in all EBSS solutions to block metabolism of AA. Ganglia were incubated for 10 min in the absence or presence of 0.5 mg/ml BSA. After stimulation, the incubation medium was immediately frozen in liquid nitrogen. Lipids were extracted from each sample by sequentially adding 1.5 ml of methanol, chloroform, and 0.88% KCl. The lower phase was removed, dried under nitrogen, and redissolved in 100 μl of chloroform/methanol (1:2). An aliquot (25 μl) was further processed for gas chromatography–mass spectrometric (GC–MS) analysis by converting the free fatty acids to their pentafluorobenzyl (PFBz) esters. That is, each sample had 100 ng of d3-phytanic acid added as an internal standard, was dried, redissolved in acetonitrile/triethylamine/pentafluorobenzyl bromide (50:10:5), and allowed to react at room temperature (22–24°C) for 15 min. Acetonitrile (200 μl) and n-hexane (1.0 ml) were added to each sample and mixed vigorously for 1 min. The hexane upper phase was removed, evaporated to dryness, and redissolved in 200 μl of n-hexane for analysis by negative ion electron capture chemical ionization (NCI) GC–MS analysis. GC–MS analyses were performed with a Waters Associates (Milford, MA) Quattro-II triple quadrupole mass spectrometry system equipped with an Agilent (Palo Alto, CA) 6890 gas chromatograph and Agilent 7683 autosampler. These analyses used a 30 × 0.25 mm inner diameter fused silica capillary column with a 0.25 μm DB-5 stationary phase coating (Agilent) and with helium as the carrier gas at 0.7 ml/min. Each sample (1 μl) was injected (injector, 300°C) in the splitless mode with the column at 185°C. After a 1 min hold at 185°C, the column temperature was programmed at 20°C/min to 240°C and then to 280°C at 5°C/min. Elution of the fatty acid PFBz esters was monitored by NCI GC–MS analysis with methane as the moderator gas at 4 e−4 mbar and the ion source at 150°C. Full scan spectra were acquired from m/z 223–345 with a cycle time of 0.5 s.

Reverse transcription-PCR analysis.

RNA from 4-d-old SCG was isolated using Trizol (Invitrogen) according to the instructions of the manufacturer. Total RNA was resuspended in DEPC-treated water. Reverse transcription (RT) was performed using Omniscript RT kit (Qiagen, Alameda, CA) according to the instructions of the manufacturer. Group IVa PLA2 transcripts were detected by PCR. A master mix contained 1.25 U of Taq polymerase, 1× Mg-free buffer, 1.5 mm MgCl2, 200 mm dNTP mix (all from Promega, Madison, WI), and 0.24 μm forward and reverse primers (Invitrogen). Rat-specific primer sequences used were as follows: forward, 5′ ATG CTA ATG GCC TTG GTG AG 3′; and reverse, 5′ CTT GGC CTT GCA GAA AAG TC 3′. Mouse-specific primers used were as follows: forward, 5′ GGA AGC GAA CGA GAC ACT TC 3′; and reverse, 5′ CGA CTC ATA CAG TGC CTT CAT CAC 3′. RT at 10% was used as a DNA template per PCR reaction. After an initial denaturation step at 94°C for 5 min, samples were amplified using 40 cycles of 94°C for 30 s, 56°C for 45 s, and 72°C for 45 s. Samples were loaded onto agarose gels (1.8%); PCR products were separated by electrophoresis and visualized with ethidium bromide.

Oxo-M stimulation of adult SCG: Western blot analysis of phosphorylated cPLA2.

Pairs of adult ganglia were preincubated for 30 min at 37°C in EBSS gassed with 5% CO2/95% O2, as described previously (Rittenhouse and Zigmond, 1999). One ganglion was stimulated with 10 μm Oxo-M for 6 min. The contralateral ganglion served as an unstimulated control. After treatment, ganglia were homogenized on ice in lysis buffer containing 0.15 m NaCl, 5 mm EDTA, pH 8.0, 1% Triton X-100, 10 mm Tris-Cl, pH 7.4, and a protease inhibitor cocktail (Roche Diagnostics, Penzberg, Germany). The suspension was sonicated on ice for 15 s and incubated on ice for 30 min. Nuclear and cellular debris was removed by centrifugation at 14,000 × g for 10 min at 4°C. After determining protein content (RC/DC protein assay; Bio-Rad, Hercules, CA), lysates were loaded on either an 8 or 10% SDS-polyacrylamide gel. Proteins were separated by electrophoresis for 1 h and transferred to polyvinylidene difluoride membranes (Bio-Rad) using standard procedures.

Membranes were blocked overnight in PBS containing 5% (w/v) nonfat dry milk. Blots were probed for at least 1 h with Abs to either cPLA2 (1:1000; Cell Signaling Technology) or to cPLA2 phosphorylated at serine 505 (P-cPLA2) (1:250; Cell Signaling Technology), washed, treated for 1 h with horseradish peroxidase (HRP)-conjugated secondary Abs (diluted 1:15,000; Bio-Rad), and rewashed. Membranes were developed using immuno-star HRP chemiluminescent kit (Bio-Rad) and exposed to film (Eastman Kodak, Rochester, NY). Membranes probed for cPLA2 were stripped with 0.2N NaOH and reprobed with anti-β-actin Abs (1:10,000; Sigma). Membranes probed for P-cPLA2 were reprobed with cPLA2 Abs (1:250) as loading controls. After films were scanned with Fluor-S MultiImager (Bio-Rad), the density of the bands was analyzed with NIH ImageJ133 (http://rsb.info.nih.gov/ij/). Because equal numbers of ganglia were taken for control and stimulation, small differences in protein amount were corrected by normalizing the P-cPLA2 signal to the cPLA2 signal.

Immunocytochemistry of P-cPLA2.

Immunocytochemistry was performed to detect changes in P-cPLA2 (Ser505) after stimulation with Oxo-M. Adult SCG neurons were cultured at low density at 37°C in MEM as above, for at least 16 h in a gridded chamber slide (Nagle Nunc, Rochester, NY) pretreated with poly-l-lysine. Under these conditions, little axonal outgrowth occurred, minimizing any potential synaptic or paracrine interactions among cells. Each chamber received a different experimental treatment. After stimulation, the chambers were removed, which allowed simultaneous processing of all cell groups for immunocytochemistry (22–24°C). Each slide was washed with PBS (two times for 5 min) and fixed with 100% acetone for 10 min, followed by three washes for 5 min with PBS. To identify principle neurons, cells were exposed to PBS containing 10% of normal goat serum for 60 min, followed by a 60 min exposure to 1:200 rabbit anti-tyrosine hydroxylase (TH) (Cell Signaling Technology) diluted in DakoCytomation (Carpinteria, CA) Antibody Diluent. Cells were washed with PBS (three times for 5 min) and incubated for 60 min in the dark with 1:200 Alexa Fluor 555 secondary anti-rabbit Ab (Invitrogen), diluted in DakoCytomation Antibody Diluent. After exposure to the secondary Ab, cells were again washed with PBS (three times for 5 min). The fixation and immunostaining protocols were repeated using 1:5000 rabbit anti-P-cPLA2 (Cell Signaling Technology), followed by 1:200 Alexa Fluor 488 secondary anti-rabbit Ab. Slides were washed with distilled water and coated with the aqueous mounting medium Prolong Gold Antifade reagent (Invitrogen). Fluorescent images were visualized using a Nikon (Tokyo, Japan) Optiphot microscope and captured using Axio Vision 4.4 software (Zeiss, Oberkochen, Germany). Fluorescence intensity of each immunopositive cell was quantitated using NIH ImageJ133.

Confocal imaging of PLA2 activity.

Dissociated SCG neurons from sPLA2−/− or s/cPLA2−/− mice were loaded with 1-O-(6-Dabcyl-amino-hexanoyl)-2-O-(6-[12-BODIPY-dodecanoyl]amino-hexanoyl)-sn-3-glyceryl-phosphatidylcholine (DBPC), a generous gift from Tyler Rose and Glenn Prestwich (Center for Cell Signaling, University of Utah and Echelon Biosciences, Salt Lake City, UT). A stock solution of DBPC (100 μg/100 μl chloroform) was prepared as described previously (B-7701 product information; Invitrogen), divided into 12 μl samples, dried with nitrogen, and stored at −80°C. Aliquots were redissolved in 12 μl of chloroform, mixed with 44 μl of phosphatidylserine (PS) (2 mg/ml chloroform; Avanti Polar Lipids, Alabaster, AL), and redried under nitrogen. DBPC–PS liposomes were kept in a desiccated environment (−20°C) in the dark. Liposomes were rehydrated with 1 ml of PBS, sonicated for 15 min on ice, and used within a few hours of preparation. Cells cultured in Matek dishes were preincubated in HBSS (Invitrogen) for 30 min. An aliquot (200 μl) of HBSS (total of 300 μl) was replaced with DBPC-labeled liposome and lightly mixed, yielding a final DBPC concentration of 0.01 μg/μl. Cells were incubated for 40 min at 37°C and washed three times with HBSS to remove any adherent liposomes. Cells were then viewed on a custom-built, video-rate confocal microscope (Sanderson and Parker, 2003) with a 40× objective lens. An excitation wavelength of 488 nm was used, and emission spectra were collected with long-pass filters at 515 nm (Perez and Sanderson, 2005). After recording time 0 images, cells were stimulated with Oxo-M or treated with HBSS for unstimulated control images. For each time point, images were collected at room temperature at 30 frames/s for 1 s using Video Savant (IO Industries, London, Ontario, Canada) and directly written to a personal computer. Each set of 30 images was averaged to create an image for time intervals ranging from 1 to 9 min and analyzed using NIH ImageJ133.

Statistical analysis.

Data are expressed as the mean or percentage change ± SEM. Statistical significance was determined by either a two-way Student's t test for two means or a two-tailed paired t test. p < 0.05 was considered significant.

Results

M1Rs, Gq, and PLC mediate slow pathway inhibition of M- and L-currents

To determine whether L-current inhibition by the slow pathway shares the same initial steps as those for M- and N-currents, M1Rs, Gq, and PLC were blocked individually with selective antagonists (Fig. 1A). Cells were exposed to the muscarinic agonist Oxo-M plus each antagonist while L-current was monitored by two methods. First, preincubating cells with 1 μm ω-CgTX for at least 20 min minimized N-current. Under these conditions, the majority of the remaining current is L-current; thus, the ω-CgTX-insensitive current is referred to as L-current (for an additional explanation, see supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Under these conditions, Oxo-M inhibited L-current 45 ± 6.6% (n = 12) (Fig. 1B,C). When MT-7, a selective mamba toxin antagonist of M1Rs, was included in the bath, Oxo-M no longer significantly inhibited the current (Fig. 1B,D). Second, including the L-channel agonist FPL in the bath elicited a long-lasting tail current comprising entirely L-current (Liu et al., 2001). Under these conditions, reversible muscarinic inhibition of the long-lasting tail current occurred but was also lost with MT-7 (Fig. 1E–G). These findings demonstrate that M1Rs mediate muscarinic inhibition of L-current. When Abs selective for Gαq/11 (Fig. 1B,G) or Gαq (Fig. 1B) were included in the pipette solution as functional antagonists of Gq, L-current inhibition by Oxo-M decreased. In contrast, dialyzing neurons with non-immunized IgG (Fig. 1B) did not affect Oxo-M inhibition of L-current. GPant2A, a selective peptide antagonist of Gq G-proteins (Fig. 1G,H), and U-73122, a selective inhibitor of PLC (Fig. 1B,G,I), also antagonized Oxo-M-induced inhibition of L-current (for controls, see supplemental Fig. 2, available at www.jneurosci.org as supplemental material).

Figure 1.
Figure 1.

M1Rs, Gq, and PLC mediate inhibition of L-current by the slow pathway. A, Schematic illustrates the experimental strategy used to delineate the initial steps in the M1R signal transduction cascade. In this and subsequent figures, currents were modulated with 10 μm Oxo-M. The M1R-selective mamba snake toxin MT-7, the Gq antagonist GPant2A, Gαq/11 and Gαq Abs, and the PLC inhibitor U-73122 were tested for their ability to minimize current inhibition by Oxo-M. B–D, N-channels were blocked by pretreating SCG neurons with 1 μm ω-CgTX for at least 30 min. B, Summary of average percentage inhibition of L-current after Oxo-M treatment (hatched bar) or Oxo-M plus inhibitor (filled bars). *p < 0.05, **p < 0.01, ***p < 0.001 compared with no inhibitor, using a two-way t test for two means; n = 4–11 recordings per group. Example time course and selected sweeps (inset) illustrate current inhibition by Oxo-M under control conditions (C) and in the presence of 100 nm MT-7 (D). Fast tail currents of selected sweeps (C, D) have been truncated. E–I, L-current was isolated by a second method. Including the nondihydropyridine L-channel agonist FPL (1 μm) in the bath elicited a large, long-lasting tail current made up entirely of L-channel activity but only a modest increase of 20 ± 5% in peak current. E, Oxo-M reversibly inhibited peak and long-lasting tail currents over time. F, Locations of peak (1) and long-lasting tail (2) current measurements are shown in example sweeps taken from E. G, Summary of average percentage inhibition of the long-lasting tail current after Oxo-M treatment (hatched bar) or Oxo-M plus inhibitor (filled bars). **p < 0.01 compared with no antagonist using a two-way t test for two means; n = 4–11 recordings per group. H, Examples of tail currents before FPL (Con), with FPL and GPant2A (10 μm), and ∼1 min after Oxo-M in the continued presence of FPL and GPant2A (Oxo-M). I, Inhibition of long-lasting tail currents was reduced in the presence of 2.5 μm U-73122. Con, Control.

Wortmannin blocks recovery of M- but not L-current inhibition

The above data and previous work of others indicate that M1Rs (Shapiro et al., 1999), Gq (Haley et al., 2000) and PLC, and therefore PIP2 breakdown, are required for L-current modulation by the slow pathway similar to previous studies on whole-cell sympathetic Ca2+ currents (Wu et al., 2002), native N-current (Liu and Rittenhouse, 2003), and native and recombinant M-current (Suh and Hille, 2002). If the same signaling cascade modulates L- and M-currents, they should respond similarly to additional manipulations of the M1R signaling cascade. When PIP2 synthesis is blocked with a high concentration of wortmannin (10–50 μm), which antagonizes both phosphatidylinositol 3 kinase (IC50 of 5 nm) and phosphatidylinositol 4 (PI4) kinase (IC50 of 100 nm) (Nakanishi et al., 1995), M-current remains inhibited after washout of Oxo-M (Suh and Hille, 2002; Ford et al., 2003; Zhang et al., 2003). We also found that blocking PIP2 synthesis with 10 μm wortmannin prevented recovery of M-current inhibition in adult SCG neurons (Fig. 2A,B,D). When we repeated this experiment with ω-CgTX-treated cells, wortmannin alone had no effect on L-current (p > 0.10 when comparing control currents with wortmannin; n = 4; data not shown). Oxo-M-induced L-current inhibition in the presence of wortmannin was normal. Moreover, inhibition of L-current reversed after ∼1 min of washout (Fig. 2C,D).

Figure 2.
Figure 2.

Wortmannin blocks recovery of M-current but not L-current. A, Bath application of Oxo-M reversibly inhibits M-current in adult SCG neurons. Inset, Selected traces from the recording. B, Wortmannin (10 μm) blocked M-current recovery from inhibition. Inset, Selected traces from the recording. C, In contrast, L-current inhibition in neonatal SCG neurons reversed in the presence of wortmannin. Inset shows selected traces from the recording. The fast tail currents have been truncated. D, Summary of L- and M-current inhibition by Oxo-M and their recovery in the presence of wortmannin. Under control conditions, Oxo-M inhibited M-current 72 ± 5% (n = 6). Inhibition (hatched bars) was readily reversible, with only 9.6 ± 6.5% inhibition remaining after ∼1 min of washing (filled bars). In contrast, although the presence of wortmannin did not significantly change M-current inhibition by Oxo-M (58 ± 7%; n = 4), M-current remained inhibited by 62 ± 7.6% after 3 min of wash. Oxo-M-induced L-current inhibition in the presence of wortmannin was normal (30 ± 6%; n = 6) in ω-CgTX-treated cells (p > 0.17 when comparing percentage current inhibition of untreated to wortmannin-treated cells using a two-tailed t test for two means). Inhibition readily reversed, with 0.1 ± 3.6% remaining after ∼1 min of washout. **p < 0.01 compared with inhibition by Oxo-M using a two-tailed paired t test. Con, Control.

L- but not M-current inhibition requires PLA2 activity

The difference in L- and M-current sensitivities to wortmannin was unexpected. This difference may be attributable to L-current having a higher affinity for PIP2. If so, a small amount of PIP2 synthesis in the presence of wortmannin might account for full recovery. However, this mechanism seems unlikely because the concentration of wortmannin used is 100 times greater than the IC50 for PI4 kinase. A second mechanism might explain our findings. Although L- and M-current inhibition by Oxo-M share initial steps in the signal transduction pathway, the pathways diverge after PIP2 metabolism by PLC. This hypothesis is supported by our previous findings that N-current modulation by the slow pathway appears to require PLA2 in addition to PLC (Liu and Rittenhouse, 2003).

To resolve whether inhibition of L- and/or M-current also requires PLA2, we first used a pharmacological approach to examine PLA2 participation in L-current inhibition. Four PLA2 antagonists were tested for their ability to block L-current inhibition (Fig. 3A). Under the conditions used, none of them alone significantly altered control current levels (data not shown). First, 4-bromophenacyl bromide (BPB) was examined for its ability to block the inhibition of long-lasting tail currents by Oxo-M. In the presence of FPL and 20 μm BPB, Oxo-M inhibition of long-lasting tail current was essentially eliminated (data not shown). However the presence of BPB also significantly reduced muscarinic inhibition of the peak current to 14 ± 4% (p < 0.05; n = 4), suggesting that BPB may be disrupting not only the slow pathway but also the membrane-delimited pathway.

Figure 3.
Figure 3.

PLA2 antagonists minimize L-current but not M-current inhibition by Oxo-M. A, Schematic illustrating experimental protocol to test effects of selective PLA2 antagonists, DEDA (IC50 of ∼20 μm) and OPC (IC50 of 6 μm), on L-current modulation. B–F, When FPL was included in the bath to enhance L-current, Oxo-M-induced inhibition of the long-lasting tail current was lost in the presence of either 100 μm DEDA (B, C) or 10 μm OPC (E, F). D, Summary of the average percentage inhibition of the long-lasting tail current after Oxo-M treatment (hatched bar) or Oxo-M plus various PLA2 inhibitors (filled bars): DEDA (100 μm; 20 ± 4%); OPC (10 μm; −8 ± 6%). **p < 0.01, ***p < 0.001 compared with no inhibitor, using a two-way t test for two means; n = 4–8 recordings per group. G, When cells were preincubated in ω-CgTX to block N-channel activity, Oxo-M-induced inhibition of L-current was minimized in the presence of OPC. Inset, Selected sweeps from the experiment; fast tail currents have been truncated. H, Oxo-M inhibited M-current over time and in individual sweeps (inset) in the presence of OPC. I, Summary of Oxo-M-induced inhibition of peak L-current (n = 10) in ω-CgTX-treated cells and of M-current (n = 5) in the presence of 10 μm OPC. Con, Control.

Therefore, we tested whether AACOCF3 [arachidonyl trifluoromethylketone; Biomol], a second PLA2 antagonist, could antagonize the actions of Oxo-M on ω-CgTX-treated neurons. When AACOCF3 (50 μm) was dialyzed into cells for 5 min, Oxo-M inhibited the current by only 10 ± 5% (n = 6). Because these conditions did not allow us to manipulate the compound along with other bath-applied antagonists, we tested two additional antagonists (Fig. 3B–D). In the presence of DEDA (100 μm), inhibition of the long-lasting tail current by Oxo-M was significantly reduced to 19 ± 4% (p < 0.01; n = 8), whereas inhibition of the peak current remained (32 ± 5%; data not shown). In addition, DEDA decreased the inhibition of long-lasting tail current by Oxo-M when a different L-channel agonist, (+)-202–791 (1 μm), was used (p < 0.05; n = 5; data not shown), indicating that DEDA was not simply disrupting the actions of FPL.

Last, in the presence of OPC (10 μm), long-lasting tail current inhibition by Oxo-M was lost (Fig. 3D–F), whereas Oxo-M inhibited peak current by 39 ± 12% (p < 0.02 compared with unstimulated levels; n = 4). To verify the loss of L-current modulation in the presence of OPC, L-current was isolated by pretreating cells with ω-CgTX. When these cells were exposed to OPC, Oxo-M inhibited whole-cell current <10% (Fig. 3G,I). In contrast, OPC did not affect L-current inhibition by AA (Fig. 4A–D). These results indicate that OPC alone does not affect L-current and does not act nonselectively upstream of PLA2 to disrupt either M2R inhibition of N-current or general G-protein functioning nor act downstream of PLA2 to block L-current inhibition by AA. Using a similar protocol, we found that Oxo-M in the presence of OPC inhibited M-current normally, but inhibition reversed by only 17 ± 5% (n = 5) after 2 min of wash compared with 62 ± 6% (n = 6; p < 0.001) in control recordings (Fig. 3H,I). These findings support a hypothesis in which L- and M-currents may be inhibited by divergent pathways.

Figure 4.
Figure 4.

AA may participate in L-current inhibition by the slow pathway. A, Schematic illustrates putative sites of action for OPC, BSA, and AA. B, Example traces of AA-induced inhibition of long-lasting tail currents. C, Example traces of AA-induced inhibition of long-lasting tail currents in the presence of 10 μm OPC. D, Summary of the effects of 5–10 μm AA in the absence (n = 6) or presence (n = 6) of OPC. AA inhibited both the long-lasting tail current (50 ± 7%; n = 6) and peak current (43 ± 6%; n = 6), similar to its effects in the absence of OPC; *p < 0.05 compared with control current levels when using a two-tailed paired t test. E, Model by which BSA antagonizes M1R modulation of channels. Control, After its liberation from phospholipids, AA diffuses from the inner to the outer lipid layer of the cell membrane. BSA, BSA binds free AA, facilitating its movement from the outer bilayer into the bath, consequently lowering AA levels in the bilayer and cytoplasm. AA binds to BSA well within several seconds, potentially fast enough to minimize any actions of AA liberated by M1R stimulation (Kanterman et al., 1990). F, The presence of BSA increased the amounts of AA found in the incubation medium of adult SCG stimulated with Oxo-M for 10 min. ***p < 0.001 compared with no BSA in the bath; n = 5 ganglia per group when using a two-way t test for two means. G, In the presence of 1 μm FPL and 0.5 mg/ml BSA, Oxo-M rapidly and reversibly inhibited peak current, but inhibition of the long-lasting tail current was lost. H, Selected sweeps from G. I, Summary of the effects of BSA (n = 6); p > 0.05 when comparing control (Con) with BSA group.

Limiting endogenous AA minimizes L-current inhibition

Because L-current inhibition by Oxo-M requires PLA2 and AA can inhibit L-current, we examined the relationship between Oxo-M and AA inhibition of L-current in three experiments. First, we documented that AA, like Oxo-M, inhibits FPL-induced long-lasting tail currents (Fig. 4A,B,D), as shown previously for peak L-current (Liu and Rittenhouse, 2000, 2003). Second, we found that the activation kinetics of L-currents were similar in the presence of either Oxo-M or AA and did not significantly change from those in FPL. For kinetic measurements, cells were preincubated with 1 μm ω-CgTX for at least 20 min to block N-current. In the continued presence of FPL (1 μm), cells were exposed to Oxo-M for 2 min. Oxo-M was washed out and 5 μm AA was introduced to the bath in the continued presence of FPL. The activation kinetics of L-current were measured for each condition from a composite of six traces. Current activation was best described by two time constants (τ1 = 0.75 ± 0.07 ms; τ2 = 8.31 ± 0.90 ms; n = 9). The fast and slow time constants did not significantly change in the presence of Oxo-M (τ1 = 0.68 ± 0.08 ms; τ2 = 7.98 ± 1.06 ms; n = 8) or AA (τ1 = 0.54 ± 0.10 ms; τ2 = 9.33 ± 1.12 ms; n = 7). Last, when Oxo-M was added after AA (10 μm), the long-lasting tail current was not inhibited further (n = 3; data not shown), indicating non-additivity of inhibition. The results from these three experiments are consistent with Oxo-M and AA using the same signal transduction pathway to inhibit L-current.

If release of endogenous AA mediates L-current modulation rather than decreased membrane levels of PIP2, then exposing cells to BSA to lower the availability of AA should minimize L-current inhibition (Fig. 4E). Alternatively, if loss of PIP2 is sufficient for L-current modulation as has been proposed for M-current (Suh and Hille, 2002), BSA should either leave inhibition intact or increase it by removing PIP2 breakdown products. To test these possibilities, we included BSA in the bath and examined L-current inhibition by Oxo-M. Extracellular BSA rapidly binds AA (Spector, 1975), effectively scavenging free AA and other biologically active fatty acids that are present at the interface between the extracellular fluid and SCG nerve cell membranes (Fig. 4E). To demonstrate the effectiveness of this strategy, we incubated SCG with Oxo-M in the presence or absence of BSA and measured AA levels in the medium. AA levels were >10-fold higher in the presence of BSA than in its absence (Fig. 4F). Moreover, AA was undetectable (<0.1 ng) in BSA-containing medium that had not been exposed to an SCG (data not shown), demonstrating that the detected AA originated in the SCG. When BSA was included in the bath in whole-cell experiments, Oxo-M inhibition of the long-lasting tail current was lost (Fig. 4G–I). However, the peak current was significantly inhibited (31 ± 5%; p < 0.001; n = 6) because of the membrane-delimited pathway (Fig. 4G), indicating a selective loss of slow pathway modulation. These findings cannot explain a model in which L-channel activity decreases when PIP2 dissociates from channels. Instead, our findings support a model in which AA is liberated from the membrane by Oxo-M stimulation of PLA2. AA then acts either directly or indirectly to inhibit L-channel activity.

cPLA2 is required for L-current inhibition by Oxo-M

We sought to identify the PLA2 responsible for L-current modulation. PLA2s generally fall into three categories: (1) secreted; (2) cytoplasmic and Ca2+ independent; or (3) cytoplasmic and Ca2+ dependent. Group IVa PLA2 (cPLA2), a cytoplasmic and Ca2+-dependent PLA2, was the likely phospholipase participating in the slow pathway for several reasons, most notably cPLA2 preferentially hydrolyzes AA, versus other fatty acids found in the sn-2 position of phospholipids (Dennis, 1997). We detected cPLA2 mRNA and protein in SCG using RT-PCR and Western blot analysis, respectively (Fig. 5A,B). Because phosphorylation of serine 505 by a number of kinases (e.g., protein kinase C and extracellular signal-regulated kinases 1 and 2) contributes to acute activation of cPLA2 (Lin et al., 1992, 1993; Gijon et al., 1999), we tested whether muscarinic stimulation altered the levels of serine 505 phosphorylation as a measure of activation. When adult SCG were stimulated with Oxo-M for 6 min, Western blot analysis showed that cPLA2 phosphorylation increased approximately twofold (Fig. 5C,D) with no change in the level of total cPLA2.

Figure 5.
Figure 5.

cPLA2 antibody reduces L-current inhibition by Oxo-M. A, RT-PCR amplification of cPLA2 cDNAs. Total RNA isolated from different cells was used in RT-PCR reactions to amplify cPLA2 cDNA. Rat-specific primers yielded a fragment of 340 bp for 4-d-old SCG, adult SCG, and rat kidney cells. Kidney cells were used as a rat-specific cPLA2 positive control. Mouse-specific primers yielded a fragment of 476 bp for immortalized kidney mesangial cell line (Mess+/+); these cells were used as a mouse-specific cPLA2 positive control. RT Con (no homogenate) and DNA Con (no RT product) yielded no bands. B, Western blot analysis of neonatal rat SCG homogenates demonstrates that the cPLA2 Ab (1:1000) recognizes a single band at ∼105 kDa. β-Actin Ab (1:10,000) served as a loading control. C, Oxo-M significantly increased cPLA2 phosphorylation at serine 505 (P-cPLA2) in adult SCG. Ganglia were stimulated with Oxo-M for 5 min, with their contralateral SCG serving as unstimulated control. Blots were probed with Ab to P-cPLA2, stripped, and reprobed with Ab to cPLA2. D, Summary of Western blot analysis (n = 9 blots). For each lane, P-cPLA2 band density was normalized to that of the cPLA2 band. *p < 0.05, Oxo-M compared with unstimulated controls, using a two-way t test for two means. E, Immunofluorescence of phosphorylated cPLA2 in adult SCG neurons. Cells were preincubated in MEM in the absence (left column) or presence (right column) of MT-7 (100 nm) for 1 h at 37°C. Half of the cells served as unstimulated controls (top row), and the remaining cells were exposed to 10 μm Oxo-M (bottom row) for 10 min. Scale bar, 10 μm. After the stimulation, cells were immediately fixed and probed with primary Abs (for details, see Materials and Methods). Exposing cells only to secondary Ab yielded no cPLA2-positive neurons (data not shown). F, Summary of average immunofluorescence intensity (pixel units). Control, 80 ± 3.3 (n = 42 cells); Oxo-M, 115 ± 3.7 (n = 58 cells); MT-7 plus Oxo-M, 65 ± 1.7 (n = 68 cells); MT-7, 81 ± 4.3 (n = 59 cells). *p < 0.05 compared with controls, MT-7 plus Oxo-M, or MT-7 groups. All cells analyzed were TH positive (data not shown). G, Cells dialyzed with cPLA2 Ab exhibit reduced L-current inhibition by Oxo-M over time. H, In contrast, when cells were dialyzed with sPLA2 Ab, Oxo-M inhibited the current. I, Summary of the effects of PLA2 Abs on Oxo-M-induced inhibition of L-current. cPLA2 Ab at 1:200 to 20 ± 4%; n = 8. *p < 0.05 compared with control, using a two-way t test for two means. †p < 0.05 compared with inhibition in cells dialyzed with sPLA2 Ab (41 ± 6%; n = 9), using a two-way t test for two means. J, Dialyzing cPLA2 Ab into cells minimized long-lasting tail current inhibition by Oxo-M. Peak current still exhibited inhibition, most likely attributable to N-current modulation by the membrane-delimited pathway. K, Consistent with membrane-delimited inhibition, inhibited currents exhibited slowed activation kinetics observed in individual sweeps taken from J. L, Summary of peak and long-lasting tail current inhibition by Oxo-M (n = 6). *p < 0.05 compared with FPL level, using a two-tailed paired t test. Con, Control.

Because the Western blot results demonstrated muscarinic stimulation of cPLA2 phosphorylation, we tested whether stimulation of M1Rs increased cPLA2 phosphorylation in SCG neurons. Figure 5E documents that the Ab used in the Western blot analysis to detect phosphorylated cPLA2 immunoreacted with dissociated adult SCG neurons, indicating the presence of cPLA2 in dissociated rat SCG neurons. All neurons positive for TH were cPLA2 positive (data not shown). SCG neurons, exposed to Oxo-M, displayed increased immunofluorescence (Fig. 5F). In contrast, exposing cells to Oxo-M in the presence of MT-7 blocked the increase in fluorescence, whereas MT-7 alone caused no change in fluorescence intensity. These findings demonstrate that M1R stimulation accounts for the acute increase in cPLA2 phosphorylation.

These findings indicated that Oxo-M acutely activates cPLA2 in SCG. Therefore, we tested whether cPLA2 is required for L-current modulation. Abs to cPLA2 were dialyzed into ω-CgTX-treated SCG neurons via the patch pipette for 7–8 min. Oxo-M was then bath applied, and current inhibition was measured at +10 mV. Under these conditions, L-current inhibition by Oxo-M decreased significantly (Fig. 5G,I). In contrast, when cells were dialyzed with Abs to group IIa PLA2 (sPLA2), Oxo-M inhibited L-current normally (Fig. 5H,I). We confirmed a role for cPLA2 by monitoring the long-lasting tail current in the presence of FPL (Fig. 5J–L). Under these conditions, the cPLA2 Ab minimized inhibition of long-lasting tail current to <10%. In these same recordings, Oxo-M inhibited peak current by 40 ± 7% (n = 6), consistent with intact membrane-delimited inhibition of N-current.

Fatty acid release and L-current inhibition by Oxo-M are lost in SCG neurons lacking cPLA2

If M1R modulation of L-current requires cPLA2, its absence should block the ability of Oxo-M to stimulate AA release from SCG neurons and subsequent L-current modulation. Taking a genetic approach, we used SCG neurons from C57BL/6J × 129/Sv mice lacking cPLA2 to study Oxo-M-stimulated AA release and L-current inhibition. As with rat SCG neurons, wild-type mouse SCG neurons normally express cPLA2 (Hornfelt et al., 1999). Interestingly, both the C57BL/6J and 129/Sv mouse strains have a naturally occurring background mutation in group IIa PLA2 (sPLA2) (Bonventre et al., 1997), a PLA2 stimulated by effectors downstream from cPLA2 (Murakami et al., 2000). Thus, C57BL/6J × 129/Sv cPLA2 null mice are consequently double mutant animals (sPLA2−/−/cPLA2−/−) and are designated as s/cPLA2−/−. In contrast, littermate “control” mice are referred to as sPLA2−/−.

To determine whether SCG neurons from s/cPLA2−/− adult mice exhibit decreased PLA2 activity, we performed a fluorescence dequenching assay for PLA2 using a fluorescently labeled phospholipid (Feng et al., 2002) detected with confocal imaging methods. In sPLA2−/− SCG neurons exposed to Oxo-M, total cell fluorescence increased over time but decreased in unstimulated sPLA2−/− neurons (Fig. 6A,B). In s/cPLA2−/− SCG neurons, fluorescence decreased over time in both unstimulated and Oxo-M-treated neurons (Fig. 6A,C). The majority of the decrease is most likely attributable to photobleaching of the BODIPY moiety (see Materials and Methods). Figure 6D shows that Oxo-M-stimulated fluorescence rapidly increased over time in sPLA2−/− neurons, and, at 6 min, fluorescence was 40% greater than that of s/cPLA2−/− cells. These findings along with our cPLA2 phosphorylation and AA release studies show that Oxo-M-mediated activation of cPLA2 increases free AA in SCG neurons within a time frame that could account for Oxo-M-induced decreases in L-current.

Figure 6.
Figure 6.

Oxo-M stimulates fatty acid release in sPLA2−/− but not in s/cPLA2−/− SCG neurons. PLA2 activity was monitored using an engineered phosphatidylcholine molecule, DBPC. DBPC has a BODIPY-labeled fluorescent moiety attached to a fatty acid in the sn-2 position. A Dabcyl moiety, attached to the fatty acid in the sn-1 position, quenches the fluorescence of BODIPY. During cleavage, fluorescence of the BODIPY-labeled fatty acid is dequenched and detected as emission at 515 nm (Feng et al., 2002). Cells were preincubated for 40 min with DBPC to allow its uptake and equilibration among membrane compartments and washed to remove excess DBPC, and fluorescence was measured. A, Examples of fatty acid fluorescence in sPLA2−/− and in s/cPLA2−/− mouse SCG neurons. Con, 0 min of stimulation; Oxo-M, 6 min exposure to Oxo-M. B, C, Time course of the changes in fluorescence for control or Oxo-M-stimulated sPLA2−/− (B) and s/cPLA2−/− (C) neurons. Fluorescence intensity was normalized to the 0 min time point. B, †p < 0.001 (two-tailed paired t test) compared with 0 min of Oxo-M. *p < 0.05, ***p < 0.001 compared with corresponding time point for control cells, using a two-way t test for two means. C, †p < 0.001 for control and Oxo-M-treated neurons compared with 0 min using a two-tailed paired t test. Fatty acid fluorescence of control and Oxo-M-treated neurons did not differ at any time point. D, Time course of fold change in fluorescence in stimulated compared with unstimulated neurons (n = 10–20 cells per group). Con, Control.

If AA, liberated by activated cPLA2, participates in L-current modulation by the slow pathway, inhibition of L-current should be reduced in s/cPLA2−/− SCG neurons. The slow pathway in mouse SCG neurons has similar characteristics to that in rat SCG neurons (Haley et al., 2000). However, mouse SCG neurons express not only L- and N-channels but also P/Q- and “R-”channels (Martinez-Pinna et al., 2002). Therefore, we examined modulation of L-current by testing whether inhibition of FPL-induced long-lasting tail currents was reduced in transgenic SCG neurons. Oxo-M inhibited the long-lasting tail current in sPLA2−/− SCG neurons (Fig. 7A,D). In contrast, SCG neurons from conspecific s/cPLA2−/− littermates exhibited no significant current inhibition by Oxo-M. However, Oxo-M significantly inhibited peak current, demonstrating selective loss of slow pathway modulation (Fig. 7D). The amplitudes of long-lasting tail currents did not differ in s/cPLA2−/− and sPLA2−/− neurons (Fig. 7C), indicating no inherent change in basal L-current between the two genetic backgrounds. Moreover, application of 5 μm AA inhibited current in ω-CgTX-treated s/cPLA2−/− neurons (Fig. 7D), demonstrating that L-current sensitivity to AA remained intact. These findings indicate that L-current inhibition by the slow pathway requires cPLA2.

Figure 7.
Figure 7.

L-current inhibition by Oxo-M is reduced in s/cPLA2−/− SCG neurons. A, Oxo-M inhibited both peak (56 ± 3%) and long-lasting tail (31 ± 5%) currents (n = 14) in sPLA2−/− SCG neurons. B, In contrast, Oxo-M inhibited peak current by 42 ± 3% but long-lasting tail current by only 6 ± 4% (n = 18) in s/cPLA2−/− neurons from conspecific littermates. Inhibition of peak current appears to be attributable to the membrane-delimited pathway, indicating that it remains active in transgenic neurons. C, FPL enhanced long-lasting tail currents to a similar magnitude in sPLA2−/− and s/cPLA2−/− SCG neurons. D, Summary of the effects of Oxo-M on peak and long-lasting tail currents in the presence of FPL. **p < 0.01, ***p < 0.001, s/cPLA2−/− (solid bars) compared with sPLA2−/− (open bars) SCG neurons using a two-way t test for two means. Hatched bar, Peak current inhibition by 10 μm AA in ω-CgTX-treated neurons. Con, Control.

In contrast, M-current inhibition was not significantly different between sPLA2−/− and s/cPLA2−/− SCG neurons (Fig. 8A,B). However, washout of inhibition was impaired in s/cPLA2−/− neurons, similar to the OPC experiments (Fig. 3H). Inhibition in s/cPLA2−/− SCG neurons recovered just 13 ± 8% (n = 3) compared with 46 ± 8% in sPLA2−/− neurons (n = 5; p < 0.05) after 60 s of washing (Fig. 8D). To test whether the decreased rate of recovery observed in s/cPLA2−/− neurons was attributable to a lack of liberated free fatty acid, we tested whether M-current in sPLA2−/− neurons exhibited normal inhibition and washout when BSA was included in the bath. Under these conditions, Oxo-M inhibited current normally; however, M-current recovered from inhibition significantly less than in the absence of BSA, suggesting that generation of AA by cPLA2 participates in M-current recovery from inhibition.

Figure 8.
Figure 8.

M-current inhibition by Oxo-M is normal in s/cPLA2−/− SCG neurons. Bath application of Oxo-M inhibited M-current in sPLA2−/− (A), s/cPLA2−/− (B), and BSA-treated sPLA2−/− (C) SCG neurons from C57BL/6J × 129/Sv mice. Insets, Selected traces taken from the recording. D, M-current inhibition by Oxo-M was not significantly different (p > 0.2) among sPLA2−/− (61 ± 6%; n = 7), sPLA2−/− plus BSA (63 ± 3%; n = 7), and s/cPLA2−/− (57 ± 4%; n = 6) neurons. M-current recovery was significantly greater in sPLA2−/− SCG neurons (*p < 0.03) than in BSA-treated sPLA2−/− or in s/cPLA2−/− SCG neurons. There was no significant difference in recovery among s/cPLA2−/− and BSA-treated sPLA2−/− SCG neurons (p > 0.4). Con, Control.

Discussion

This study compared the roles of PLA2, AA, and PIP2 in L- and M-current inhibition by muscarinic agonists. Inhibition of L-current by Oxo-M was minimized by blocking M1Rs, Gq, or PLC with toxins, Abs, and/or selective antagonists. These findings are consistent with a model in which M1R modulation of L-, M-, and N-current involves the same second-messenger pathway (Shapiro et al., 2001; Suh and Hille, 2002, 2005; Wu et al., 2002). However, antagonizing PIP2 synthesis with wortmannin, at a concentration that blocked recovery of M-current inhibition (Suh and Hille, 2002; Ford et al., 2003; Zhang et al., 2003), did not block recovery of L-current inhibition. These unexpected findings suggested that L-current was modulated differently from M-current.

We used biochemical, imaging, pharmacological, and genetic approaches to determine whether further phospholipid metabolism is required for L-current modulation. Our Western blot and immunocytochemical findings support a model in which Oxo-M acutely stimulates cPLA2 phosphorylation, leading to acute enzyme activation and increased AA release from cell membranes. Consistent with phosphorylation acutely activating cPLA2, our imaging studies show that Oxo-M stimulates fatty acid liberation in cPLA2-containing (sPLA2−/−) but not in s/cPLA2−/− neurons. Pharmacologically antagonizing PLA2, dialyzing cPLA2 Abs into cells, or using s/cPLA2−/− neurons significantly decreased L-current inhibition by Oxo-M, indicating a requirement for cPLA2. In particular, our finding that L-current inhibition by Oxo-M is lost in SCG neurons that do not express cPLA2 is hard to interpret other than by concluding that cPLA2 is required for modulation. The s/cPLA2−/− mouse is well characterized (Bonventre et al., 1997). To date, s/cPLA2−/− mice are known to differ biochemically from wild-type mice in one respect: they have decreased levels of cyclooxygenase-2 (COX-2) mRNA and protein (Bosetti and Weerasinghe, 2003). Decreased COX-2 in s/cPLA2−/− SCG neurons is unlikely to account for the loss of L-current inhibition because we found no evidence that AA metabolism is required for L-current inhibition (Liu et al., 2001; Liu and Rittenhouse, 2003). Thus, we propose a pathway in which M1R coupling to Gq/11 activates breakdown of PIP2 by PLC. PLC and PLA2 likely participate in the pathway sequentially because PLC activity also leads to protein kinase C and mitogen-activated protein kinase activation, followed by subsequent phosphorylation of cPLA2 at Ser505 (Lin et al., 1992, 1993; Gijon et al., 1999). In turn, cPLA2 liberates AA from membrane phospholipids, which directly or indirectly alters channel activity. Future studies are needed to determine the nature of the relationship between PLC and cPLA2.

The possibility that PLA2 participates in the slow pathway has been examined and rejected by other laboratories based on negative pharmacological findings. Gamper et al. (2004) reported that OPC did not affect the ability of Oxo-M to inhibit current recorded in the perforated-patch configuration from SCG neurons. This result differed from our finding that OPC significantly decreased M1R inhibition of N-current (Liu and Rittenhouse, 2003), a discrepancy that was attributed by Gamper et al. to differences in recording conditions. However, they did not report alternative experimental approaches or positive controls documenting active OPC. A second study examined the role of PLA2 in L-current inhibition in HEK 293 cells transfected with M1R rat CaV1.2c, α2δ, and rabbit β2a (Bannister et al., 2002). Muscarinic stimulation robustly inhibited whole-cell current. When PLC or PLA2 activity was antagonized with U-73122 or quinacrine, respectively, M1R-stimulated inhibition was unaffected (Bannister et al., 2002). Interestingly, we previously found that quinacrine as high as 1 mm did not inhibit purified cPLA2, whereas 100 μm DEDA, the concentration used in our patch-clamp experiments, antagonized cPLA2 activity by 80% (Kim and Bonventre, 1993). No positive controls were presented in the Cav1.2c study; thus, it is unclear whether conditions were suboptimal for the antagonists. A more interesting possibility is that M1Rs could modulate CaV1.2c differently than wild-type L-current in SCG neurons, which is thought to arise from CaV1.3 (Lin et al., 1996). At present, however, we know of no sequence variability that might explain these differences. Nevertheless, these studies raise interesting questions as to whether all types of L-channels, including splice variants (Safa et al., 2001; Liao et al., 2004), express the same critical residues to mediate channel inhibition by the slow pathway.

Our model involving AA participation in slow pathway inhibition of L-current is supported by a growing list of findings. (1) AA mimics inhibition of L-channel activity by Oxo-M recorded in whole-cell and cell-attached patch configurations (Mathie et al., 1992; Liu and Rittenhouse, 2000, 2003; Liu et al., 2001). (2) The kinetics of Oxo-M-inhibited L-currents are statistically indistinguishable from those in the presence of AA. (3) AA occludes L-current inhibition by Oxo-M. (4) Decreasing the availability of free AA by including BSA in the bath antagonized L-current inhibition by Oxo-M. If loss of PIP2 decreased channel activity, BSA should not have affected or should have increased the magnitude of current inhibition because BSA binds fatty acids and effectively removes them from solution, promoting further metabolism of PIP2. It is hard to reconcile the decrease in Oxo-M-induced L-current inhibition in the presence of BSA with a model proposing that current is inhibited when PIP2 dissociates from channels.

How AA inhibits L-current is unknown, but several theories have been proposed. AA may mediate M1R signaling by activating additional downstream molecules such as kinases (Gailly et al., 1997; Liao et al., 2004) or phosphatases, as has been proposed for AA-mediated inhibition of T- and cardiac L-currents (Petit-Jacques and Hartzell, 1996; Zhang et al., 2000) and AA-mediated M-current enhancement (Yu, 1995). This model does not require a loss of PIP2 bound to channels. In a second scenario, AA could occupy a critical position around the channel, competing with PIP2 for the same site of interaction (Liu et al., 2004), as has been proposed for some K+ channels (Rogalski and Chavkin, 2001). A third possibility is that AA acts directly on the channel at a site distinct from PIP2. This model has been proposed for Kv currents in which PIP2 binding to A-type Kv channels eliminates N-type fast inactivation. In contrast to the actions of PIP2, AA promotes a rapid form of inactivation, converting non-inactivating, delayed rectifier Kv currents into fast-inactivating, A-type Kv currents (Oliver et al., 2004). These findings document a critical, stabilizing, structural role for both AA and PIP2 interactions with K+ channels at distinct sites. Our analysis of unitary L-channel activity found that AA increased the percentage of null sweeps, consistent with stabilizing either a closed or inactivated state (Liu and Rittenhouse, 2000). At the whole-cell level, AA increased holding potential-dependent inactivation (Liu et al., 2001), suggesting an increase in C-inactivation. The opposing actions of PIP2 (Wu et al., 2002; Gamper et al., 2004) and AA on Ca2+ currents (Liu and Rittenhouse, 2000; Liu et al., 2001; Zhang et al., 2003; Talavera et al., 2004) appear remarkably similar to those for Kv currents, suggesting that their actions may be conserved across a number of voltage-gated ion channel families, including Ca2+ channels.

These conserved actions of PIP2 and AA may extend to M-current in which M-channel availability to open increases with PIP2 (Zhang et al., 2003). However, M-current is not inhibited by AA nor does PLA2 activity participate in M-current inhibition by M1Rs, as demonstrated previously in NG108 cells (Robbins et al., 1993) and here in SCG neurons. We examined whether OPC or BSA antagonized M-current inhibition by Oxo-M. Neither agent changed inhibition compared with control conditions. These findings were confirmed in transgenic studies; Oxo-M inhibited M-current similarly in sPLA2−/− and s/cPLA2−/− SCG neurons. We interpreted these differences as indicators that a diverging M1R signaling cascade differentially inhibits L- and N-current from M-current. Moreover, our unanticipated findings that OPC, BSA, or the absence of cPLA2 greatly attenuated M-current recovery from inhibition indicated a role for cPLA2 and AA in recovery from the inhibitory process. A role for AA in regulating M-current is supported by the finding that exogenously applied AA enhances M-current in frog sympathetic neurons (Villarroel, 1994; Yu, 1995).

Together, these results indicate that M-current inhibition requires only PIP2 breakdown, whereas L- and N-current inhibition and M-current recovery also require generation of a fatty acid, probably AA, by cPLA2. These differences indicate that AA uses additional, novel mechanisms to regulate M-current compared with Ca2+ channels and other Kv channel activity (Oliver et al., 2004). Moreover, this signaling cascade appears remarkably similar to the PLA2-sensitive pathway responsible for M-current over-recovery observed after washing out of agonist in other cell types (Robbins et al., 1993; Villarroel, 1994; Yu, 1995). Mechanistically, these unexpected findings suggest that, to observe the effects of AA on recovery, sufficient PIP2 must be present in the membrane and/or associated with the channels. Our model of M1R-meditated channel modulation thus broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity. At the cellular level, the consequence of channel modulation by M1R stimulation is to depolarize membranes and increase action potential firing in sympathetic (Brown and Adams, 1980) and cortical (Hamilton et al., 1997; Potier and Psarropoulou, 2004) neurons. These findings indicate that the slow pathway acts within a neural network to regulate the particular excitability state of a neuron. Such a process is thought to be involved in the release of modulatory transmitters from cortical projections to regulate attention and working memory (Goldman-Rakic, 1999; Mechawar et al., 2000).

Footnotes

  • This work was supported by National Institutes of Health (NIH) Grants NS34195 (A.R.R.) and DK38452 (J.V.B.) and a Grant-In-Aid and an Established Investigator Award from the American Heart Association (A.R.R.). We acknowledge NIH Grant NS29632 to Glen Prestwich for the development of DBPC. Steve Farber, Glen Prestwich, and Taylor Rose provided advice in developing the imaging protocol. We thank Joshua J. Singer, Jim Hamilton, and Alan Kleinfeld for helpful discussions about fatty acid movement across membranes and their effects on ion channels and Trevor Shuttleworth for early advice on phospholipase inhibitors. We thank Eileen O'Leary for care of and genotyping the transgenic mice. We also thank Claire Baldwin, John F. Heneghan, Tora Mitra, Mandy L. Roberts, and John Walsh for critically reading various versions of this manuscript.

  • Correspondence should be addressed to Dr. Ann R. Rittenhouse, Program of Neuroscience, Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. ann.rittenhouse{at}umassmed.edu

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M1 Muscarinic Receptors Inhibit L-type Ca2+ Current and M-Current by Divergent Signal Transduction Cascades
Liwang Liu, Rubing Zhao, Yan Bai, Lee F. Stanish, James E. Evans, Michael J. Sanderson, Joseph V. Bonventre, Ann R. Rittenhouse
Journal of Neuroscience 8 November 2006, 26 (45) 11588-11598; DOI: 10.1523/JNEUROSCI.2102-06.2006
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