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. 2023 Jan-Dec:19:17448069231222406.
doi: 10.1177/17448069231222406.

A-kinase anchoring protein 79/150 coordinates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor sensitization in sensory neurons

Yan Zhang  1 , Nathaniel A Jeske  1   2   3
Affiliations

A-kinase anchoring protein 79/150 coordinates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor sensitization in sensory neurons

Yan Zhang et al. Mol Pain. 2023 Jan-Dec.

Abstract

Changes in sensory afferent activity contribute to the transition from acute to chronic pain. However, it is unlikely that a single sensory receptor is entirely responsible for persistent pain. It is more probable that extended changes to multiple receptor proteins expressed by afferent neurons support persistent pain. A-Kinase Anchoring Protein 79/150 (AKAP) is an intracellular scaffolding protein expressed in sensory neurons that spatially and temporally coordinates signaling events. Since AKAP scaffolds biochemical modifications of multiple TRP receptors linked to pain phenotypes, we probed for other ionotropic receptors that may be mediated by AKAP and contribute to persistent pain. Here, we identify a role for AKAP modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor (AMPA-R) functionality in sensory neurons. Pharmacological manipulation of distinct AMPA-R subunits significantly reduces persistent mechanical hypersensitivity observed during hyperalgesic priming. Stimulation of both protein kinases C and A (PKC, PKA, respectively) modulate AMPA-R subunit GluR1 and GluR2 phosphorylation and surface expression in an AKAP-dependent manner in primary cultures of DRG neurons. Furthermore, AKAP knock out reduces sensitized AMPA-R responsivity in DRG neurons. Collectively, these data indicate that AKAP scaffolds AMPA-R subunit organization in DRG neurons that may contribute to the transition from acute-to-chronic pain.

Keywords: AKAP; AMPA; glutamate; pain.

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Conflict of interest statement

Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Pharmacological Manipulation of AMPA Subunits Reduce Mechanical Hyperalgesic Priming. Male rats were injected i.pl. with vehicle or Complete Freund’s Adjuvant (CFA, 50% in 50 μL saline). Over the next 4 days, rats were injected daily with vehicle or NBQX (AMPA GluR1 antagonist, 200 nmol/50 μL, i.pl., (a and b), or control peptide (Con) or TAT-GluA2-3g (TAT, GluR2 internalization inhibitor, 50 nmol/50 μL, i.pl., (c and d). Rats were tested for paw withdrawal threshold (PWT, g) on Day 5, then injected with PGE2 (100 ng in 50 μL saline, i.pl.), and again tested for PWT 0.25, 0.5, 2, and 4 h. n = 24 rats/treatment, triplicate readings per time point; compared to Veh/PGE2 (a, *p = .0167, ****p < .0001, c, **p = .0028, ****p < .0001), two-way ANOVA with Sidak’s multiple Comparisons test.
Figure 2.
Figure 2.
AMPA GluR Subunit PM Expression in DRG tissue following Hind Paw Incision treatment. Right hind paw injection with Complete Freund’s Adjuvant (CFA, 50% in 50 μL saline, i.pl.) compared to left sham (sham). Respective side DRG were homogenized and fractionated for plasma membrane fractions. Aliquots resolved by SDS-PAGE and probed for GluR1, GluR2, and AKAP. Representative WB results, n = 4 trials. Quantitative analysis of densitometry normalized to AKAP, *p = .0195, ***p = .0005, unpaired t test, n = 4.
Figure 3.
Figure 3.
SAP97 knockdown reduces AMPA GluR/AKAP Association. Sensory neurons were transfected in mock fashion or with SAP97 siRNA. Equal aliquots of crude plasma membrane preps were immunoprecipitated with AKAP antibody and probed for GluR1, GluR2, and AKAP immunoreactivities. Densitometries normalized to mock. Whole cell lysates were probed for SAP97 and β-arrestin to demonstrate siRNA specificity and efficacy. Representative WB results, n = 3 trials, means ± SEM shown, GluR1 **p = .040, GluR2 *p = .031, unpaired t test.
Figure 4.
Figure 4.
PKC/PKA phosphorylation of AMPA Requires AKAP. Sensory neurons cultured, transfected in mock fashion or with AKAP150 siRNA, serum-starved, then treated with PMA (1 μM, 15 min, PKC activator) or 8-Br-cAMP (10 μM, 15 min, PKA activator), and harvested for WB analysis. (a) Representative WB results, n = 4 trials. (b) Quantitative analysis of immunoreactive densitometry, means ± SEM, p-S831 PMA versus Veh ***p = .0003, p-S845 PMA versus Veh ***p = .0001, p-S880 8-Br-cAMP versus Veh **p = .009, NS = no significance, unpaired t test.
Figure 5.
Figure 5.
AKAP Mediates AMPA GluR1/2 Surface Expression. Sensory neurons cultured, transfected in mock fashion or with AKAP150 siRNA, serum-starved, then treated with vehicle, 8-Br-cAMP (10 μM, 10 min, PKA activator) or PMA (1 μM, 10 min, PKC activator), surface biotinylated, streptavidin precipitated, and harvested for WB analysis. (a) Representative WB results, n = 4 trials, means ± SEM shown. Densitometric quantification of AMPA GluR1 biotinylation (b), ***p = .0008, GluR2 biotinylation (c) ***p = .0007, NS = no significance, unpaired t test.
Figure 6.
Figure 6.
PKA/PKC Activation Increase AMPA Responsive Neurons from AKAP WT Only. DRG neurons cultured from male AKAP WT or KO male mice, pre-treated for 15 min with vehicle, PMA (1 μM), 8-Br-cAMP (8-Br, 10 μM), or PMA and 8-Br-cAMP (15 min) before AMPA (30 μM). Responder threshold set at 0.05 ΔF340/380 above baseline, from n = 37–90 KCl (+) cells/treatment, with each point representing the percentage of AMPA-responsive neurons/total KCl-responsive neurons/coverslip, *p = .0335, **p = .0012, ****p < .0001, NS = no significance, one-way ANOVA with Tukey post-hoc.
Figure 7.
Figure 7.
Mechanism of AMPA Modulation in Persistent Pain. Inflammation drives AKAP association with AMAP GluR1/2 heteromer. AKAP Scaffolds PKC phosphorylation of GluR2 (causing internalization of the subunit) and PKA phosphorylation of GluR1 (causing subunit tetramerization and glutamate-dependent Ca+2 influx.
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