PKA/AKAP/VR-1 module: A common link of Gs-mediated signaling to thermal hyperalgesia

Abstract

Inflammatory mediators not only activate "pain-"sensing neurons, the nociceptors, to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. We demonstrate for the first time at the cellular level that heat-activated ionic currents were potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation was prevented in the presence of the selective PKA inhibitor PKI(14-22), suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits only translocated to the cell periphery when activated. The translocation and the current potentiation were abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation was greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia.

Fig. 1.

FSK-induced potentiation of native heat-activated ionic currents (I_heat). a, Example of a DRG neuron that was repetitively stimulated with noxious heat (linear temperature rise from room temperature to 46°C, 5 sec duration, 1 min intervals) before and after FSK (10⁻⁵m). b–d, Mean ± SEM responses before and immediately after FSK, and FSK with the selective PKA inhibitor peptide PKI_14–22 or the InCELLect AKAP St-Ht31 inhibitor peptide in the patch pipette. Peak stimulation temperatures were similar in the different samples (47.0 ± 1.5°C in controls; 46.7 ± 0.6°C for PKI; 47.2 ± 0.9°C for StHt31). ★p < 0.05 indicates significant differences.

Fig. 2.

Expression of VR-1, PKA subunits, and AKAPs in DRG-neurons. a, RT-PCR products for PKA-RI, PKA-RII, and PKA-C of total mRNA isolated from DRG (D) or HEK293t cells (H) and negative control without reverse transcriptase (∅RT).M, Marker. b, Indirect immunocytochemistry for VR-1 and PKA subunit, respectively. Cells were fixed and stained with antibodies against VR-1 and PKA-RI, RII, and PKA-C, respectively. c, Size distribution of neurons in culture double labeled for PKA-RI and VR-1 and PKA-C and VR-1, respectively. The cell size was determined off-line with the noncommercial image processing software IPB by Marc Nischik (Institute of Physiology, Erlangen). d, Immunostaining of HEK293t cells for different PKA subunits.

Fig. 3.

Translocation of PKA-C after FSK stimulation (35 sec, 10⁻⁵m) in DRG neurons.a, Examples of neurons stained without or after FSK stimulation. Confocal images indicate the position of the line scan profile used for calculations in b. b, Confocal line scan profile taken with the COMOS software.c, Columns show the average fluorescence magnitude in the peripheral and central regions of the cell diameter divided by the total average fluorescence of the cell.con, Control. d, e, Time course of the PKA-C translocation after FSK, depicted as the difference between peripheral (P) and central (C) fluorescence and effects of the selective inhibitors PKI_14–22, InCELLect AKAP inhibitor peptide plus inactive control peptide, and the inactive analog 1,9-dideoxy-FSK.

Fig. 4.

Sensory neurons and HEK293t cells express AKAPs.a, Expression of mRNA for dAKAP2, AKAP79, and AKAP220 but not AKAP-KL in DRGs and HEK293t cells and negative control without RT (øRT). The expected size of the fragments is between 400 and 510 bp. M, Marker.b, Immunostaining of DRG neurons in culture reveals expression of VR-1 together with AKAP79 or AKAP220.

Fig. 5.

FSK-induced potentiation of heat-activated currents in VR-1-transfected HEK293t cells. a, Whole-cell current responses evoked by repeated noxious thermal stimuli before and after a pretreatment period with FSK (10⁻⁵m) for 1 min. The interval between two thermal stimuli was 1 min. Heat response (mean ± SEM) before and after FSK pretreatment (b) and in the presence of the selective PKA inhibitor PKI_14–22(c), or the InCELLect AKAP St-Ht31 inhibitor peptide in the ICS (d,e).

Fig. 6.

Mean heat responses of wild-type and mutant VR-1 expressed in HEK293t cells before and after FSK pretreatment (n = 7–9; p < 0.05). * indicates statistically significant difference of current potentiation after FSK in mutants compared with wild-type. Heat stimuli H1 through H6 were applied at 1 min intervals.