Protein kinase A–induced internalization of Slack channels from the neuronal membrane occurs by adaptor protein-2/clathrin–mediated endocytosis

Abstract

The sodium-activated potassium (K) channel Kcnt1 (Slack) is abundantly expressed in nociceptor (pain-sensing) neurons of the dorsal root ganglion (DRG), where they transmit the large outward conductance and arbitrate membrane excitability. Slack channel expression at the DRG membrane is necessary for their characteristic firing accommodation during maintained stimulation, and reduced membrane channel density causes hyperexcitability. We have previously shown that in a pro-inflammatory state, a decrease in membrane channel expression leading to reduced Slack-mediated expression underlies DRG neuronal sensitization. An important component of the inflammatory milieu, PKA internalizes Slack channels from the DRG membrane, reduces , and produces DRG neuronal hyperexcitability when activated in cultured primary DRG neurons. Here, we show that this PKA-induced retrograde trafficking of Slack channels also occurs in intact spinal cord slices and that it is carried out by adaptor protein-2 (AP-2) via clathrin-mediated endocytosis. We provide mass spectrometric and biochemical evidence of an association of native neuronal AP-2 adaptor proteins with Slack channels, facilitated by a dileucine motif housed in the cytoplasmic Slack C terminus that binds AP-2. By creating a competitive peptide blocker of AP-2-Slack binding, we demonstrated that this interaction is essential for clathrin recruitment to the DRG membrane, Slack channel endocytosis, and DRG neuronal hyperexcitability after PKA activation. Together, these findings uncover AP-2 and clathrin as players in Slack channel regulation. Given the significant role of Slack in nociceptive neuronal excitability, the AP-2 clathrin-mediated endocytosis trafficking mechanism may enable targeting of peripheral and possibly, central neuronal sensitization.

Magi‐1 scaffolds Na v 1‐8 and Slack K Na channels in dorsal root ganglion neurons regulating excitability and pain

“…S3A). We expected the decrease in surface Slack K Na channels to result in repetitive firing (14,15); however, the observed severely stunted APs suggested that Magi-1 deficiency was also affecting sodium channel functioning.…”

“…For electrophysiological experiments, DRG neurons were cotransfected with the GFP-containing plasmid pTRACER and siRNA duplexes for positive indication of transfection. Imunofluorescence procedures on cultured neurons were performed as previously described (15). The investigator was blinded to the transfection condition.…”

“…Peptides were initially dissolved in DMSO and diluted to a final working concentration in saline. A peptide concentration of 10 mM was used as previously described (15), and the final DMSO concentration was 0.05%. Intraplantar dosing was chosen based on a prior study by Weng et al (39) demonstrating the analgesic effects of hind paw intradermal injections of a different myristoylated peptide.…”

“…For example, PKA-mediated internalization of the Slack K Na channel in dorsal root ganglion (DRG) neurons resulted in a loss of firing accommodation and hyperexcitability (14). This channel internalization process was shown to be dependent upon adaptor protein 2clathrin-mediated endocytosis (AP2-CME) (15). Inhibiting AP2-CME with a cell-penetrating peptide prevented PKAinduced repetitive firing of DRG neurons (15).…”

“…This channel internalization process was shown to be dependent upon adaptor protein 2clathrin-mediated endocytosis (AP2-CME) (15). Inhibiting AP2-CME with a cell-penetrating peptide prevented PKAinduced repetitive firing of DRG neurons (15). Importantly, immunohistochemistry studies found overlapping Slack K Na immunolabeling with Na V 1.8 channels in DRG neurons (16), and electrophysiological studies suggested that K Na channels reside in close proximity and possibly are functionally coupled to Na V channels (17).…”