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

Matthew R Fleming,Maile R Brown,Jack Kronengold,Yalan Zhang,David P Jenkins,Gulia Barcia,Rima Nabbout,Anne E Bausch,Peter Ruth,Robert Lukowski,Dhasakumar S Navaratnam,Leonard K Kaczmarek

doi:10.1016/j.celrep.2016.07.024

Abstract

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.

Highlights

The Na+-activated K+ channel Slack (KNa1.1, KCNT1, SLO2.2) is widely distributed throughout the nervous system (Kaczmarek, 2013)
Mutations in Slack have been described in malignant migrating partial seizures of infancy (MMPSI), a condition that produces infantile seizures coupled with very severe intellectual disability (Barcia et al, 2012; Kim et al, 2014)
Activation of Slack Decreases Mass at the Plasma Membrane To monitor the interactions of Slack channels with its potential cytoplasmic partners in real-time within living cells, we used resonance-wavelength grating (RWG) optical biosensors, a technique that has been used to monitor the activation of G protein coupled receptors (Fang et al, 2007; Fleming and Kaczmarek, 2009; Lee, 2009)

Summary

Introduction

The Na+-activated K+ channel Slack (KNa1.1, KCNT1, SLO2.2) is widely distributed throughout the nervous system (Kaczmarek, 2013). Mutations in Slack have been described in malignant migrating partial seizures of infancy (MMPSI), a condition that produces infantile seizures coupled with very severe intellectual disability (Barcia et al, 2012; Kim et al, 2014). The majority of these mutations produce single amino acid substitutions within the cytoplasmic C-terminal domain of Slack. These gain-of-function mutations increase Slack current amplitude (Kim et al, 2014)

Results

Discussion

Conclusion