It is therefore consistent that c-Src inactivity, either through -arrestin2 deletion or pharmacological inhibition, affected internalization and recycling, increasing cell-surface receptors. currents were activated by depolarizing neurons from ?80 to 10 mV for 100 ms at 10 s intervals. In experiments examining constitutive inhibitory coupling to VGCCs, a two-pulse voltage protocol was used, and Ca2+ in the external solution was replaced by Ba2+ to prevent Ca2+-dependent inactivation. A depolarizing voltage step (80 ms duration) from ?80 to 80 mV preceded (by 10 ms) a voltage-step from ?80 to 10 mV (10 ms duration). The current amplitude evoked by the test pulse after an 80 mV prepulse (Tukey’s test. qPCR. Cultured DRG neurons from for 5 min at 4C. The cells were washed in ice-cold PBS containing 2% fetal bovine serum and 0.1% sodium azide (PBS/FBS/NaN3) and incubated with an anti- receptor antibody raised against the third extracellular loop, for 60 min at 4C (1:100 dilution in PBS/FBS/NaN3; Millipore, Billerica, MA). Antibodies to this region of the receptor do not label neuronal tissue lacking the receptor (Guarna et al., 2003). Thereafter, the cells were washed and incubated in the secondary antibody [allophycocyanin (APC)-conjugated rabbit IgG; 1:100; BD Biosciences] for 60 min at room temperature. After a final wash, 5000 neurons per sample were acquired on a FACScalibur flow cytometer (BD Immunocytochemistry Systems, Mountain View, CA,) and analyzed using FCS express version 3.0 (De Novo Software, Thornhill, Ontario, Canada). Flow cytometry was also used to quantify the effect of DAMGO (d-Ala2-test, significance accepted at 0.05, and are presented as mean SEM. Immunocytochemistry. DRG neurons from = 12) for = 10) and 14 5% for Tukey’s test, * 0.05 compared with inhibition of Ca2+ current amplitude by the agonist when applied to 0.05, Student’s test). Error bars represent SEM. We also tested the inhibitory response to baclofen (50 m) Cast of VGCC activity recorded from 0.05, Student’s test) in surface expression of receptors in = 8; data not shown). Open in a separate window Figure 2. Confocal laser-scanning microscopy and flow cytometry to detect cell-surface receptors in DRG neurons. cIAP1 Ligand-Linker Conjugates 14 = 5) and 44 8% (= 5) of the initial peak inhibitions in = 3) and 45 7% (= 4) of the initial peak inhibitions in = 7) (Fig. 5= 5) of Ca2+ currents recorded from = 7) and 10 4% (= 4) in = 14) of the amplitude of currents recorded in the absence of a prepulse (?PP) (Fig. 6 0.05) larger increase (106 3%; = 9) in current amplitude in experiments performed on 0.05, ANOVA, Tukey’s test) and presence (** 0.01) of GTP–S. Error bars represent SEM. We further explored the possibility of disrupted inhibitory G-protein coupling to VGCCs caused by the absence of -arrestin2 by comparing the effect on VGCCs of GTP–S (300 m) applied through the recording electrode to the inside of cIAP1 Ligand-Linker Conjugates 14 0.01) greater in = 10 of control) compared with = 13) (Fig. 6= 6) in the presence and absence of naltrexone (1 m), respectively (data not shown). GTP–S is nonhydrolysable and therefore interacts irreversibly with activated G-protein subunits. Thus, it is not possible to reverse such an interaction should it have taken place before administration of the inverse agonist naltrexone. There are several mechanisms that may facilitate the exchange of GDP, bound to G subunits, by intracellular GTP–S: (1) endogenous agonist-mediated GPCR activity, (2) constitutive GPCR activity, and (3) intrinsic G subunit activity. Therefore, although it is possible that naltrexone slows the exchange process by inhibiting constitutive receptor activity in 0.01) inhibition of the 0.01, ANOVA, Tukey’s test). Error bars represent SEM. Constitutive recycling of receptors is impaired in 0.05, Student’s test; = 6). In contrast, monensin had no effect on receptor levels in = 10), indicating that constitutive recycling of the receptor is -arrestin2 dependent. These data suggest that receptors are constitutively recycled in wild-type DRG neurons in a monensin-sensitive manner. Such GPCR cIAP1 Ligand-Linker Conjugates 14 recycling typically occurs within 200 nm of the plasma membrane and is therefore undetectable by traditional CLSM. This.