Salicylate Induced GABA(A)R Internalization by Dopamine D1-Like Receptors Involving Protein Kinase C (PKC) in Spiral Ganglion Neurons

BACKGROUND: Sodium salicylate (SS) induces excitotoxicity of spiral ganglion neurons (SGNs) by inhibiting the response of γ-aminobutyric acid type A receptors (GABA(A)Rs). Our previous studies have shown that SS can increase the internalization of GABA(A)Rs on SGNs, which involves dopamine D1-like receptors (D1Rs) and related signaling pathways. In this study, we aimed to explore the role of D1Rs and their downstream molecule protein kinase C (PKC) in the process of SS inhibiting GABA(A)Rs. MATERIAL/METHODS: The expression of D1Rs and GABARγ2 on rat cochlear SGNs cultured in vitro was tested by immunofluorescence. Then, the SGNs were exposed to SS, D1R agonist (SKF38393), D1R antagonist (SCH23390), clathrin/dynamin-mediated endocytosis inhibitor (dynasore), and PKC inhibitor (Bisindolylmaleimide I). Western blotting and whole-cell patch clamp technique were used to assess the changes of surface and total protein of GABARγ2 and GABA-activated currents. RESULTS: Immunofluorescence showed that D1 receptors (DRD1) were expressed on SGNs. Data from western blotting showed that SS promoted the internalization of cell surface GABA(A)Rs, and activating D1Rs had the same result. Inhibiting D1Rs and PKC decreased the internalization of GABA(A)Rs. Meanwhile, the phosphorylation level of GABA(A)Rγ2 S327 affected by PKC was positively correlated with the degree of internalization of GABA(A)Rs. Moreover, whole-cell patch clamp recording showed that inhibition of D1Rs or co-inhibition of D1Rs and PKC attenuated the inhibitory effect of SS on GABA-activated currents. CONCLUSIONS: D1Rs mediate the GABA(A)R internalization induced by SS via a PKC-dependent manner and participate in the excitotoxic process of SGNs.