TRESK background potassium channel is not gated at the helix bundle crossing near the cytoplasmic end of the pore

PLoS One. 2018 May 15;13(5):e0197622. doi: 10.1371/journal.pone.0197622. eCollection 2018.

Abstract

Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Barium / metabolism
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cytoplasm / metabolism
  • Female
  • HEK293 Cells
  • Humans
  • Ion Channel Gating
  • Kinetics
  • Kv1.3 Potassium Channel / antagonists & inhibitors
  • Kv1.3 Potassium Channel / metabolism
  • Mice
  • Oocytes / metabolism
  • Patch-Clamp Techniques
  • Phosphorylation
  • Point Mutation
  • Potassium Channels / chemistry
  • Potassium Channels / genetics
  • Potassium Channels / metabolism*
  • Potassium Channels, Tandem Pore Domain / antagonists & inhibitors
  • Potassium Channels, Tandem Pore Domain / chemistry
  • Potassium Channels, Tandem Pore Domain / genetics
  • Potassium Channels, Tandem Pore Domain / metabolism*
  • Protein Conformation
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Xenopus laevis

Substances

  • Cell Cycle Proteins
  • Kv1.3 Potassium Channel
  • Potassium Channels
  • Potassium Channels, Tandem Pore Domain
  • Recombinant Proteins
  • Trik protein, mouse
  • potassium channel protein TREK-1
  • Barium
  • Mark2 protein, mouse
  • Protein-Serine-Threonine Kinases
  • Cyclic AMP-Dependent Protein Kinases

Grant support

This work was supported by the Hungarian National Research Fund (OTKA K108496) (PE). M.L. was supported by the New National Excellence Program of the Ministry of Human Capacities (ÚNKP-17-3-I-SE-7). The work was supported by the Ministry of Human Capacities in the frame of Institutional Excellence Program for Higher Education.