Slack sodium-activated potassium channel membrane expression requires p38 mitogen-activated protein kinase phosphorylation

Abstract

p38 MAPK has long been understood as an inducible kinase under conditions of cellular stress, but there is now increasing evidence to support its role in the regulation of neuronal function. Several phosphorylation targets have been identified, an appreciable number of which are ion channels, implicating the possible involvement of p38 MAPK in neuronal excitability. The KNa channel Slack is an important protein to be studied as it is highly and ubiquitously expressed in DRG neurons and is important in the maintenance of their firing accommodation. We sought to examine if the Slack channel could be a substrate of p38 MAPK activity. First, we found that the Slack C-terminus contains two putative p38 MAPK phosphorylation sites that are highly conserved across species. Second, we show via electrophysiology experiments that KNa currents and further, Slack currents, are subject to tonic modulation by p38 MAPK. Third, biochemical approaches revealed that Slack channel regulation by p38 MAPK occurs through direct phosphorylation at the two putative sites of interaction, and mutating both sites prevented surface expression of Slack channels. Based on these results, we conclude that p38 MAPK is an obligate regulator of Slack channel function via the trafficking of channels into the membrane. The present study identifies Slack KNa channels as p38 MAPK substrates.

The Phe932Ile mutation in KCNT1 channels associated with severe epilepsy, delayed myelination and leukoencephalopathy produces a loss-of-function channel phenotype

“…The F932(911)I mutation resides in the C-terminal, a region containing various regulatory sites. In a recent study, Gururaj et al (2016) illustrate the importance of the C-terminal in membrane trafficking by showing that p38 phosphorylation is required for Slack channel membrane expression.…”

“…In addition, the Slack monoclonal antibody used in this study has been validated by multiple labs for biochemical analyses. (Bansal & Fisher, 2016; Gururaj et al, 2016; Lu et al, 2013; Martinez-Espinosa et al, 2015; Rizzi et al, 2016). This antibody only weakly reacts with the human channel due to amino acid differences in the epitope, necessitating the use of the rat variant.…”

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

“…For example, association with the ion channel TMEM16C was shown to increase membrane expression of Slack channels in DRG neurons such that neuronal excitability was alleviated (34). p38 MAPK phosphorylation-induced insertion of Slack channels into the DRG membrane is critical to tonic maintenance of firing accommodation (9). The present work demonstrates PKA-induced trafficking of channels from the DRG membrane to result in loss of firing accommodation, which, when prevented, restores it.…”

“…Among the cytoplasmic proteins identified heretofore as having a regulatory influence on Slack channels, intracellular protein kinases comprise a functionally significant fraction. The majority of these interactions can be attributed to the extensive intracellular C termini unique to K Na channel subunits, which house several consensus sites for phosphorylation by kinases (7)(8)(9) in the neighborhood of tandem regulators of K ϩ conductance (RCK) domains (10,11). For example, PKC phosphorylation of Ser 407 in the hinge region between the S6 transmembrane domain and the first RCK domain produces a constitutively active phenotype in Slack channels that mimics known KCNT1 mutations associated with malignant migrating partial seizures of infancy (12).…”

“…For example, PKC phosphorylation of Ser 407 in the hinge region between the S6 transmembrane domain and the first RCK domain produces a constitutively active phenotype in Slack channels that mimics known KCNT1 mutations associated with malignant migrating partial seizures of infancy (12). In the distal C terminus, phosphorylation by p38 MAPK is required for tonic conductance and surface expression of neuronal Slack channels, prevention of which causes significant reduction in total neuronal I K (9). Finally, the abovementioned indirect regulatory effect of PKA on Slack channels resulting in DRG neuronal hyperexcitability is bolstered by the fact that four of the five known PKA consensus sites are located outside the RCK domains (7).…”