Stimulation of Slack K+ Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex

Abstract

Summary Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways, and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.

Phactr1 regulates Slack (KCNT1) channels via protein phosphatase 1 (PP1)

“…11,42 For some of the disease-causing mutations, it has been shown that they behave as channels that are constitutively phosphorylated at this site. 4,44,45 Thus far, however, only for Phactr1 is there direct evidence that these interactions are altered in disease-causing Slack mutant channels. 5,43 In addition to Phactr1, a number of other proteins have been identified as Slack-interacting binding partners of Slack channels including FMRP, PSD95, CYFIP1, and TMEM16C.…”

“…11 One of the mechanisms that has been proposed to explain how mutations lead to neuronal dysfunction is an alteration in the interactions of Slack channels with its cytoplasmic binding partners. 4 This protein is a member of the phosphatase and actin regulator family of proteins, which are highly expressed in the central nervous system and at lower levels also in heart, lung, kidney, and testis. 4,44,45 Thus far, however, only for Phactr1 is there direct evidence that these interactions are altered in disease-causing Slack mutant channels.…”

“…4 Using primary cultures of cortical neurons, we first examined the localization of both proteins by immunocytochemistry ( Figure 1A-B). 4 Using primary cultures of cortical neurons, we first examined the localization of both proteins by immunocytochemistry ( Figure 1A-B).…”

“…11 Application of PKC activators such as TPA (12-O-Tetradecanoylphorbol-13-acetate) to cells expressing wild-type Slack channels leads to a 2-3 fold increase in current amplitude, but these activators have no effect on current amplitude in cells that express channels in which this serine has been mutated to an alanine. 4 Because Phactr1 is a phosphatase-binding protein, we tested whether its effects on Slack current amplitude could be mediated by phosphorylation at the S407 site. 4 Because Phactr1 is a phosphatase-binding protein, we tested whether its effects on Slack current amplitude could be mediated by phosphorylation at the S407 site.…”

“…31 Although Phactr1 has been shown to dissociate from Slack channels following their activation or phosphorylation, the functional consequences of the Slack-Phactr1 interaction are not known. 4 Recent data indicate that the Phactr1/PP1 complex is a PP1 holoenzyme. 32 Phosphorylation of Slack at a serine residue (S407) in the cytoplasmic C-terminal domain by PKC increases Slack currents.…”