Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites

José A Poveda, A Marcela Giudici, M Lourdes Renart, Oscar Millet, Andrés Morales, José M González-Ros, Victoria Oakes, Simone Furini, Carmen Domene

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Abstract

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

Original languageEnglish
Article number183029
JournalBiochimica Et Biophysica Acta-Biomembranes
Volume1861
Issue number10
Early online date24 Jul 2019
DOIs
Publication statusPublished - 1 Oct 2019

Keywords

  • Ion channel inactivation
  • Ion channel kinetics
  • Membrane lipid-protein interactions
  • Molecular dynamics simulations
  • Patch-clamp recordings

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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