Identification of a signaling cascade that maintains constitutive δ-opioid receptor incompetence in peripheral sensory neurons

AKAP facilitates PKA-dependent GRK2 phosphorylation

In immortalized cells, PKA activation stimulates phosphorylation of numerous proteins, including the direct phosphorylation GRK2 at Ser-685 (24). Although PKA phosphorylation of GRK2 does not enhance GRK2 catalytic activity, it facilitates GRK2 modulation of GPCRs by targeting GRK2 to the plasma membrane.

We sought to determine whether this mechanism occurs in peripheral sensory neurons. To determine whether PKA activation leads to direct PKA phosphorylation of GRK2, we utilized a phosphorylation site-specific antibody for GRK2 at Ser-685 (Fig. 2, A and B). Serum-starved rat TG cultures were stimulated with 8-Br-cAMP (10 μm; 5 min), followed by homogenization and crude fractionation. Next, GRK2 was isolated from cytosol and membrane lysates by immunoprecipitation. Stimulation by 8-Br-cAMP significantly increases PKA-dependent GRK2 phosphorylation by 278.00 ± 80.42% in the cytosol, whereas PKA stimulation resulted in a trend toward increased PKA-dependent phosphorylation by 147.80 ± 58.43% at the plasma membrane (Fig. 2, A and B; p = 0.0259 (A) and p = 0.0647 (B), n = 3 independent trials, unpaired student's t test). Compared with vehicle-treated cells, 8-Br-cAMP treatment also resulted in a trend toward increased GRK2 translocation to the plasma membrane by 61.60 ± 22.35% (Fig. 2C, p = 0.0511, n = 3 independent trials, unpaired Student's t test). This experiment recapitulates what was found in immortalized cells and reveals that stimulation of PKA enhances PKA-dependent phosphorylation in the cytosol and, albeit just beyond significance, may have similar effects at the plasma membrane as a result of translocation of GRK2 in rat primary sensory neurons.

Open in a separate window

Figure 2.

AKAP facilitates PKA-dependent GRK2 phosphorylation and targets GRK2 to PM. A and B, crude cytosolic (CYTO) and PM fraction immunoprecipitations from TG cultures serum-starved for 18 h and treated with vehicle (black) and 8-Br-cAMP (green; 10 μm, 5 min). C, additional crude membrane samples were collected to examine GRK2 expression at the PM. D and E, crude cytosolic and PM fraction immunoprecipitations from TG cultures treated in mock fashion (black) or with AKAP siRNA (gray) and serum-starved for 4 h. F, additional crude membrane samples were collected to examine GRK2 expression at the PM. G, crude PM fraction immunoprecipitation from naive TG from WT AKAP (black) mice and AKAP KO (red) littermates. H, additional crude membrane samples were collected to look at GRK2 expression at the PM. n = 3 independent trials; mean ± S.E. (error bars); *, p < 0.05; ns, not significant; unpaired Student's t test.

AKAP scaffolding of PKA indirectly regulates GRK2 membrane translocation in immortalized cells, which requires PKA to be tethered to the plasma membrane (24). In cultured rat TG lysates, that AKAP co-immunoprecipitates with PKA RII (23). However, it has yet to be determined whether AKAP can indirectly regulate PKA-dependent phosphorylation and translocation of GRK2 in primary sensory neurons. For this experiment, we harvested and fractionated serum-starved rat TG cultures that had been transfected either in mock fashion or with AKAP siRNA. Knockdown of AKAP did not reduce PKA-dependent GRK2 phosphorylation in the cytosol (Fig. 2D, p = 0.3292, n = 3 independent trials, unpaired Student's t test). However, AKAP knockdown significantly reduces membrane GRK2 phosphorylation at Ser-685 by 39.32 ± 15.48% and is accompanied by a reduction in GRK2 membrane targeting by 44.22 ± 18.71% (Fig. 2, E and F; p = 0.0441 (E) and p = 0.0560 (F), n = 3 independent trials, unpaired Student's t test). Similarly, crude membrane TG lysates from AKAP KO mice have 31.10 ± 9.47% less PKA-dependent GRK2 phosphorylation than WT littermates (Fig. 2G, p = 0.0304, n = 3 independent trials, unpaired Student's t test). Importantly, GRK2 targeting to the plasma membrane in AKAP KO TG is significantly reduced by 26.75 ± 9.54% compared with WT littermates (Fig. 2H, p = 0.0485, n = 3 independent trials, unpaired Student's t test). These experiments agree with the aforementioned study in immortalized cells and demonstrate that AKAP expression is crucial to constitutive PKA-dependent phosphorylation of GRK2 targeted to the membrane in rat peripheral sensory neurons.

PKA phosphorylation of GRK2 drives association with DOR

Under non-inflammatory conditions, DOR is chronically bound to GRK2, which renders the receptor dormant or unresponsive to agonist stimulation in peripheral sensory neurons (11). In immortalized cells, phosphorylation by PKA facilitates GRK2 translocation to the plasma membrane to negatively modulate GPCR signaling (24). Given that PKA-dependent phosphorylation dictates plasma membrane translocation of GRK2 (Fig. 2), we hypothesized that PKA-dependent phosphorylation of GRK2 may underlie the constitutive association between DOR and GRK2 at the plasma membrane in primary sensory neurons. To test this, we utilized site-directed mutagenesis. Because site-specific mutants were used rather than a dominant-negative mutant that would antagonize wild-type GRK2, the abundant native kinase was first knocked down with previously validated GRK2 siRNA (11) (75.9 ± 1.8 and 78.8 ± 1.5% endogenous GRK2 reduction for GRK2 siRNA and FITC-GRK2 siRNA, respectively). Following knockdown of endogenous GRK2, we nucleofected either empty vector, wild-type GRK2, GRK2-S685D (phosphomimetic), or GRK2-S685G (phosphodeficient) cDNA into TG cultures. Following overnight serum starvation, cells were harvested, fractionated, and processed for co-immunoprecipitation studies.

Following GRK2 silencing and empty vector introduction, a small amount of native GRK2 co-immunoprecipitates with plasma membrane DOR. When wild-type GRK2 is reconstituted, constitutive GRK2 association with DOR increases to 149.78 ± 61.52% above levels in cells nucleofected with empty vector. Introduction of the phosphomimetic mutant, GRK2-S685D, yields a comparable 154.52 ± 34.23% increase in GRK2 that co-immunoprecipitates with DOR at the plasma membrane. In contrast, introduction of the phosphodeficient mutant, GRK2-S685G, produced a small, non-significant increase of 33.90 ± 2.96% above levels in GRK2-silenced TG nucleofected with empty vector (Fig. 3A; *, p ≤ 0.05; ANOVA summary, p = 0.0111; n = 4 independent trials; one-way ANOVA with Bonferroni post hoc test). These data demonstrate that in peripheral sensory neurons, PKA phosphorylation of GRK2 drives the constitutive association between DOR and GRK2 at the plasma membrane.

Open in a separate window

Figure 3.

PKA phosphorylation of GRK2 enhances association with DOR in primary sensory neurons of the periphery. A, TG cultures were treated with GRK2 siRNA followed by nucleofection of EV (light gray), wild-type GRK2 (dark gray), phosphomimetic GRK2-S685D (dark green), or phosphodeficient GRK2-S685G (light green) cDNA. Following 4-h serum starvation, crude plasma membrane fractions were collected for co-immunoprecipitation and probed for DOR association with GRK2. n = 4 independent trials; mean ± S.E. (error bars); *, p < 0.05; ns, not significant; one-way ANOVA with Bonferroni post hoc test. B and C, cumulative traces and quantification of DPDPE (1 μm) inhibition of KCl (50 mm)-evoked Ca²⁺ influx in capsaicin-sensitive DRG treated with GRK2 siRNA followed by co-nucleofection of GFP with EV, GRK2, GRK2-S685D, or GRK2-S685G cDNA and 2-h serum starvation. n = 29–33 DRG/group, mean ± S.E.; ***, p < 0.005; ns, not significant; one-way ANOVA with Bonferroni post hoc test.

To build on this, we next carried out a functional Ca²⁺ imaging assay in peripheral sensory neurons to determine whether PKA-dependent phosphorylation of GRK2 governs DOR incompetence. We measured opioid inhibition of voltage-gated Ca²⁺ channels (VGCCs) elicited by KCl-evoked Ca²⁺ transients in cultured sensory neurons, which is a validated method that has been used by multiple groups to quantify DOR activity in a heterogeneous neuronal population (10, 11, 27). In contrast to patch-clamp electrophysiology, this method circumvents inevitable user bias in determining whether to include a cell that does not respond to a DOR agonist due to lack of receptor expression versus lack of functional receptor competence. Furthermore, it avoids the limitations of requiring gene-targeted receptor-fusion protein introduction into cells that do not typically express DOR, which carries with it potential changes to normal receptor activity, interaction partners, and/or biochemistry.

We previously reported that small to medium capsaicin (CAP; 1 μm)-sensitive DRG neurons transfected with FITC-GRK2 siRNA produce significant DOR-mediated inhibition of VGCCs compared with mock-treated cells that have little to no DOR activity in the presence of a DOR agonist (11). To test whether PKA-dependent phosphorylation of GRK2 underlies functional DOR incompetence, we used siRNA-mediated knockdown of GRK2 followed by co-nucleofection of GFP with empty vector, wild-type GRK2, GRK2-S685D, or GRK2-S685G cDNA. Recapitulating published population findings in primary cultures, GFP-positive CAP-sensitive DRG transfected with FITC-GRK2 siRNA and empty vector produce a robust response to the DOR agonist [d-Pen^2,5]enkephalin (DPDPE; 1 μm), which inhibits KCl (50 mm)-evoked Ca²⁺ transients by 28.10 ± 4.23% in CAP-sensitive DRG (Fig. 3, B and C). Reintroduction of wild-type GRK2 or nucleofection of GRK2-S685D cDNA similarly antagonizes DPDPE inhibition of KCl-evoked Ca²⁺ transients to 5.48 ± 2.74 and 3.96 ± 1.87%, respectively. However, nucleofection of GRK2-S685G cDNA following GRK2 knockdown results in 25.18 ± 2.92% DPDPE inhibition of KCl-evoked Ca²⁺ transients, similar to DRG transfected with FITC-GRK2 siRNA and empty vector cDNA (Fig. 3, B and C; ***, p < 0.005 for GRK2 and GRK2-S685D compared with both empty vector and GRK2-S685G; ANOVA summary, p < 0.0001; n = 29–33 DRG/group; one-way ANOVA with Bonferroni post hoc test). Taken together, data presented herein identify that Ser-685 is an important site for GRK2 maintenance of functional DOR incompetence in primary sensory neurons.

Animals

Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200–250 g were used for biochemistry and Ca²⁺ imaging in this study. Additional studies in AKAP WT and KO littermates with a C57/B16 background (37) were carried out for immunohistochemistry and biochemistry. Animals were housed in clean cages with a 12-h light/dark cycle for 1 week with food and water ad libitum before use.

Generation of PKA phosphorylation site-specific GRK2 mutants

Bovine GRK2 cDNA (generous gift from Dr. Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia, PA) was used for single-point mutational creation of GRK2-S685D (phosphomimetic) and GRK2-S685G (phosphodeficient) in pcDNA3 vector. For GRK2-S685D, a single point mutation changing Ser-685 to aspartic acid was introduced by PCR using the primers 5′-CCA GCG CGG CGA TGC CAA CGG CCT CTG-3′ (forward) and 5′-CAG AGG CCG TTG GCA TCG CCG CGC TGG-3′ (reverse). For GRK2-S685G, a single point mutation changing Ser-685 to glycine was introduced by PCR using the primers 5′-CCA GCG CGG CGG TGC CAA CGG C-3′ (forward) and 5′-GCC GTT GGC ACC GCC GCG CTG G-3′ (reverse). Mutant clones were generated by QuikChange mutagenesis (Stratagene, La Jolla, CA) and then verified by DNA sequencing.

Sensory neuronal cultures

For immunohistochemistry, TG or DRG (L4–L6) were dissected bilaterally from male mice. TG/DRG were dissociated by 30-min collagenase (Worthington) treatment followed by 30-min trypsin (Sigma-Aldrich) treatment, with gentle rocking every 10 min. Cells were then resuspended in complete medium (Dulbecco's modified Eagle's medium, Invitrogen), supplemented with 10% fetal bovine serum (Invitrogen), 100 ng/ml nerve growth factor (NGF; Harlan Laboratories, Indianapolis, IN), mitotic inhibitors (Sigma), 1% penicillin/streptomycin (Invitrogen), and 1% glutamine (Sigma), and plated on poly-d-lysine-coated plates (Corning, Glendale, AZ). Cultures were maintained at 37 °C and 5% CO₂ and grown for 3 days with medium changed 2 days following culture and supplemented with NGF. On the third day post-dissection, cells were prepared for immunofluorescent staining.

For biochemistry, TG were dissected bilaterally from male rats and prepared as described above with cultures maintained at 37 °C and 5% CO₂ and grown for 5–6 days with medium changed the following day and every 2 days thereafter. TG were exclusively utilized for biochemical experiments to satisfy NIH requirements to reduce animal use in research.

For Ca²⁺ imaging, DRG dissected bilaterally at L4–L6 from male rats were dissociated by a 40-min co-treatment with collagenase (Worthington) and dispase (Sigma) with gentle rocking every 10 min. Next, cells were resuspended in complete medium and plated on poly-d-lysine/laminin-coated coverslips (BD Biosciences). Cultures were maintained at 37 °C and 5% CO₂, medium was changed the following day, and experiments were conducted within 24–48 h of initial culture.

Tissue processing and immunohistochemistry

On day 3 post-dissection, wells containing coverslips with cultured sensory neurons from AKAP WT and KO mice were aspirated and rinsed with 0.1 m phosphate-buffered saline (PBS) for three periods of 5 min each. Next, cells were fixed for 10 min in fresh filtered 4% paraformaldehyde solution. Cells were then rinsed quickly once in 0.1 m PBS and again for three periods of 5 min each. To minimize nonspecific binding, samples were incubated for 1 h in blocking solution (2% bovine-globulin (Sigma), 4% normal horse serum (Sigma), and 0.3% Triton X-100 (Fisher) in 0.1 m PBS). Next, the tissue was incubated in primary antibodies against PKA RII (Upstate Cell Signaling Solutions (Lake Placid, NY); at 1:75 dilution in blocking solution) (38) and neuronal/nerve fiber marker protein gene product 9.5 (PGP9.5; AB5898, Chemicon (Billerica, MA); at 1:100 dilution in blocking solution) (39) for 18 h in a humidifier. The following day, cells were rinsed with 0.1 m PBS and then rinsed again for three periods of 10 min each. Next, cells were placed in a dark humidifier for 60 min to incubate in corresponding species-specific secondary antibody Alexa Fluor 488 or 568 (Molecular Probes at dilutions of 1:100 and 1:50, respectively, in blocking solution) used to visualize PKA RII and PGP9.5 immunoreactivity. Cells were quickly rinsed in 0.1 m PBS, followed by an additional three periods of 10 min rinses. Finally, cells were quickly rinsed in ultrafiltered nanopure deionized water. Once dry, stained cells were coverslipped with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA) and stored at 4 °C until they were evaluated by confocal microscopy. Cells were evaluated, and images were obtained using a Nikon 90i microscope equipped with a C1si laser-scanning confocal imaging system (Nikon, Melville, NY). Confocal images are representative of three individual trials.

To analyze PKA RII distribution in each representative cell, a second-order smoothing polynomial of six individual plot profiles measured with 60° separations near the cell membrane was collected under the green filter of the RGB stack using NIH ImageJ software. For every individual plot profile measured under the green filter, a measurement was taken under the red filter to identify distribution constraints for normalization across the cell. PKA RII distribution at the plasma membrane relative to the whole cell was determined using single-pixel-wide segmented measurements selected around the edges of PGP9.5-expressing cells under the red level filter for each cell in every RGB stacked confocal image collected to remove user bias, and PKA RII average densitometry measurements were collected under the green level filter and compared with the average densitometry of the whole cell by closing the segmented line. Appropriate statistics were carried out as described in the figure legends.

Crude membrane preparation

Primary rat TG cultures were pretreated as indicated, and fresh TG from AKAP WT and KO littermates were bilaterally dissected from naive animals. For cultures, cells were harvested and homogenized in homogenization buffer (25 mm HEPES, 25 mm sucrose, 1.5 mm MgCl₂, 50 mm NaCl (pH 7.4), 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate (Sigma), 1 μg/ml pepstatin (Sigma), 1 μg/ml leupeptin (Sigma), 1 μg/ml aprotinin (Sigma), and 100 nm phenylmethylsulfonyl fluoride (PMSF; Sigma)) with 20 strokes using a Potter-Elvehjem pestle and glass homogenizer tube. Similarly, naive TG from AKAP WT and KO littermates were cut 20 times following dissection and then homogenized in homogenization buffer with 40 strokes. Homogenates were placed on ice for a 15-min incubation and then centrifuged at 1000 × g for 1 min to remove nuclei and unlysed cells from the homogenate. The resulting supernatant was centrifuged at 16,000 × g for 30 min at 4 °C to separate cell membrane proteins from cytosolic proteins. Cytosolic supernatant was separated from the pellet (crude membrane fraction), which was resuspended in 250 μl of homogenization buffer containing 1% Triton X-100 (Fisher). Total protein was quantified using Bradford assay (Sigma) prior to co-immunoprecipitation (co-IP).

PKA kinase activity assay/ELISA

Using an in vitro PKA kinase activity kit (Enzo Life Sciences, Farmingdale, NY), we measured PKA activity in crude membrane fractions from primary rat TG cultures treated in mock fashion or with AKAP siRNA or fresh TG from AKAP WT and KO littermates. The assay was performed according to the manufacturer's instructions. Following Bradford assay and quantification, 2-μg plasma membrane samples and kit standards were plated onto a precoated 96-well PKA substrate microtiter plate. In brief, ATP was used to initiate the kinase reaction for 90 min at 30 °C. After the reaction was terminated, wells were incubated with a phosphospecific substrate antibody for 60 min at room temperature, when it binds to the phosphorylated peptide substrate. A peroxidase-conjugated secondary antibody that binds the phosphospecific antibody was added for 30 min at room temperature. Finally, a tetramethylbenzidine substrate was added for 45 min at room temperature, and absorbance was measured in a microplate spectrophotometer at 450 nm. A PKA kinase activity assay performed on crude plasma membrane fractions was performed in triplicate across three independent trials for each experiment, as indicated in the corresponding figure legends.

Co-IP/IP

Total protein from either PM or cytosolic fractions was quantified using a Bradford assay (Sigma). For co-IP/IP, equal amounts of protein (125 μg) were immunoprecipitated with 1 μg of anti-GRK2 (C-15, Santa Cruz Biotechnology, Inc.) or anti-DOR (ab66317, Abcam) antiserum. GRK2 antibody specificity has been confirmed in inducible GRK2 sensory neuron knock-out animals (40), and DOR antibody specificity for immunoprecipitation has been previously vetted (41). Protein samples were eluted at 95 °C for 5 min and placed at −20 °C for future WB.

Western blot analysis

Whole cell lysates, PM, and cytosolic immunoprecipitates were resolved on SDS-PAGE (15% gels) and transferred to PVDF membrane (Millipore). Membranes were then blocked with 5% nonfat dried milk in Tris-buffered saline/Tween 20 (TBS-T: 15.35 mm Tris/HCl, 136.9 mm NaCl, pH 7.6, with 0.1% Tween 20) or 5% bovine serum albumin (Sigma) in TBS-T containing the phosphatase inhibitor sodium orthovanadate (1 μm) for phosphorylation-specific antibodies. Western blots were visualized using anti-phospho-GRK2-Ser-685 (catalog no. 12397-1, SAB Signalway Antibody), anti-GRK2 (C-15, Santa Cruz Biotechnology) (40), anti-DOR (ab66317, Abcam) (11, 41, 42), anti-caveolin-1 (N-20, Santa Cruz Biotechnology) (43), or anti-β-actin (I-19-R, Santa Cruz Biotechnology) (43), followed by appropriate horseradish-peroxidase-conjugated secondary antisera (GE Healthcare) and ECL (enhanced chemiluminescence or prime) detection following the manufacturer's protocol (GE Healthcare). Antibody specificities were verified by the manufacturers and BLAST sequence analysis and used in previous publications as indicated. Integrated density measurement values, equivalent to the product of area and mean gray value by histogram analysis, were performed using NIH ImageJ software.

siRNA transfection and cDNA nucleofection

For experiments utilizing AKAP siRNA, cells were transfected with AKAP siRNA (625 ng/10-cm plate; characterized in Ref. 23) for 24 h, and the transfection mix was replaced with complete medium. GRK2 and RKIP siRNA duplexes were transfected into cultured sensory neurons using HiPerFect (Qiagen, Valencia, CA), following the manufacturer's directions. AKAP siRNA yielded a transfection efficiency of ∼60–70%, as published previously (23).

For GRK2 siRNA/cDNA nucleofection experiments, primary TG or DRG cultures were transfected with FITC-labeled GRK2 or GRK2 siRNA (45 ng/coverslip; 20 μg/10-cm plate; characterized in Ref. 11) for 18 h, replenished with complete medium. Then 6 h following medium change, cells were nucleofected with Effectene nucleofection reagent (Qiagen) following the manufacturer's instructions, maintaining a cDNA/enhancer reagent ratio of 1:8, for 10 h. For these experiments, empty vector (EV) pcDNA3.1, GRK2 (Dr. Jeffrey L. Benovic, Thomas Jefferson University), GRK2-S685D, or GRK2-S685G cDNA (500 ng/coverslip; 4 μg/10-cm plate) were nucleofected into sensory neurons. For Ca²⁺ imaging, GFP cDNA (250 ng/coverslip) was co-nucleofected to identify positive transfection. GRK2 siRNA yielded a transfection efficiency of ∼75%, and nucleofection efficiency was ∼60%, as published previously (11). The “rescue” method for reconstituting cDNA following knockdown has been used by multiple groups in human cells (AKAP-Lbc-silenced cryopreserved human cardiomyocytes) (44), zebrafish (GRK2-silenced embryos) (45), and primary sensory neurons (RKIP-silenced TG and DRG) (11).

For AKAP overexpression experiments, DRG were nucleofected with Effectene nucleofection reagent, as described above. Following this protocol, 500 ng/coverslip EV, wild-type AKAP, AKAPΔPKA, or AKAPΔPKC cDNA was co-nucleofected with 250 ng/coverslip GFP cDNA to identify positive transfection. For overexpression of EV, wild-type AKAP, AKAPΔPKA, or AKAPΔPKC cDNA in TG or DRG cultures, 4 μg of cDNA were nucleofected onto 10-cm plates following the same protocol.

Ca²⁺ imaging

Fura-2 AM was used to image individual neurons within a population of cultured DRG following 2-h serum starvation. Next, cells were loaded with fura-2 AM (1 μm; Molecular Probes, Carlsbad, CA) with pluronic F-127 (0.04%; Molecular Probes) and incubated for 1 h at 37 °C in the dark, in standard extracellular solution (SES) containing 140 mm NaCl, 5 mm KCl, 2 mm CaCl₂, 1 mm MgCl₂, 10 mm HEPES, 10 mm d-(+)-glucose, pH 7.40. Cells were viewed on an inverted Nikon Eclipse T_i-U microscope fitted with a ×20/0.75 numerical aperture Fluor objective and imaged using the MetaFluor system for ratio fluorescence (MetaMorph, Downingtown, PA). Fluorescent images were taken alternately every 3 s with 340- and 380-nm excitation wavelengths in combination with a 510-nm emission filter with 100-ms exposure. The ratio of ΔF340/F380 was plotted for each cell versus time. Intracellular Ca²⁺ levels were analyzed as ΔF340/F380 ratios background-corrected and normalized to the initial value, R₀. Corresponding filters were used to select FITC siRNA- and/or GFP-positive DRG.

DOR activity in sensory neurons was quantified by Ca²⁺ imaging as a measure of DPDPE (1 μm; Sigma) inhibition of KCl-evoked Ca²⁺ transients in CAP-sensitive DRG (11). For positive neuronal identification, specific criteria were used and included 1) characteristic phenotype featuring bright round cell bodies with clear nuclei (26, 46); 2) sensitivity to depolarizing solution of 50 mm KCl (10, 27), and 3) sensitivity to CAP (1 μm, 25% above baseline (11); Sigma) to indicate positive sensory neuronal phenotype within a heterogeneous culture.

For each cell imaged, basal Ca²⁺ levels were recorded for 60 s. A perfusion valve controller (< 0.1 p.s.i.) and multibarrel glass pipette were used to apply 3-s exposures of 50 mm KCl at five time points: 60, 270, 740, 950, and 1440 s. The first and second exposures to KCl were given in the absence of a DOR agonist in SES bath solution. At 480 s, SES was replaced with a DPDPE in SES applied via constant perfusion (< 0.1 p.s.i.) by a multibarrel glass pipette. Prior to the third and fourth exposure to KCl, DOR agonist microinjection was terminated to reduce pressure-related artifacts. At 1160 s, SES containing DOR agonist was washed out and replaced with SES via bath perfusion (< 0.1 p.s.i.) using an ML-6 manifold apparatus prior to the fifth exposure to KCl. Finally, cells were exposed to 1 μm CAP at 1770 s to select for TRPV1-positive primary afferent neurons that can be correlated with thermal allodynia behavior.

In order to measure DPDPE inhibition of KCl-evoked Ca²⁺ influx, single-cell recording traces were used to obtain the average area under the curve, calculated as an integral over a time period (3 min) for the average cellular response to KCl in the absence or presence of a DOR agonist. The following equations were used to calculate DOR activity.

For Ca²⁺ imaging, stock solutions of DOR agonist DPDPE (1 mm) were prepared in sterile water. CAP (1 mm) stock solution was prepared in EtOH. KCl was prepared fresh in SES on the day of experimentation. All drugs and appropriate vehicles were diluted in SES.

We thank Dr. Jeffrey L. Benovic (Thomas Jefferson University, Philadelphia, PA) for gifting the GRK2 cDNA.