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 (KNa) channel Kcnt1 (Slack) is abundantly expressed in nociceptor (pain sending) neurons of the dorsal root ganglion (DRG), where they transmit the large outward conductance IKNa 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 IKNa expression underlies DRG sensitization. An important component of the inflammatory milieu, Protein Kinase A (PKA) internalizes Slack channels from the DRG membrane, reduces IKNa and produces DRG 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 (CME). We provide mass spectrometric and biochemical evidence of an association of native neuronal AP-2 adaptor proteins with Slack channels, facilitated by a di-leucine 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 hyperexcitability after PKA activation. Together, these findings uncover AP-2 and clathrin as players in Slack channel regulation. Given Slack's significant role in nociceptive neuronal excitability, the AP-2 CME trafficking mechanism may enable targeting of peripheral and possibly, central neuronal sensitization.