Modulation of the IKS channel by PIP2 requires two binding sites per monomer

Abstract

The phosphatidyl-inositol-4,5-bisphosphate (PIP₂) lipid has been shown to be crucial for the coupling between the voltage sensor and the pore of the potassium voltage-gated K_V7 channel family, especially the K_V7.1 channel. Expressed in the myocardium membrane, K_V7.1 forms a complex with KCNE1 auxiliary subunits to generate the I_KS current. Here we present molecular models of the transmembrane region of this complex in its three known states, namely the Resting/Closed (RC), the Intermediate/Closed (IC), and the Activated/Open (AO), robustness of which is assessed by agreement with a range of biophysical data. Molecular Dynamics (MD) simulations of these models embedded in a lipid bilayer including phosphatidyl-inositol-4,5-bisphosphate (PIP₂) lipids show that in presence of KCNE1, two PIP₂ lipids are necessary to stabilize each state. The simulations also show that KCNE1 interacts with both PIP₂ binding sites, forming a tourniquet around the pore and preventing its opening. The present investigation provides therefore key molecular elements that govern the role of PIP₂ in KCNE1 modulation of I_KS channels, possibly a common mechanism by which auxiliary KCNE subunits might modulate a variety of other ion channels.

Graphical abstract

Fig. 1

I_KS MD systems. Representation of I_KS models in absence (left) and presence (right) of KCNE1’s (gray ribbons) PIP₂ binding site, each embedded in a POPC membrane (in yellow spheres) with PIP₂ lipids (in red spheres). Two K_V7.1 subunits are shown for clarity. The transmembrane segments are represented in ribbons. The VSD segments are colored in cyan, S4 in blue, S4-S5_LINKER in brown and PD segments in orange. The membrane-embedded models are surrounded by water (in transparent blue surface) as well as K⁺ (purple spheres) and Cl⁻ (green spheres) ions.

Fig. 2

Validation of I_KS models through pore radii calculations The graphs report the average pore radii (left) along the conduction pathway of K_V7.1 (black curves) I_KS 8PIP₂ (purple curves) and I_KS 4PIP₂ (beige curves) models in A. AO state B. Intermediate state and C. RC state. Averages radii at the levels of residues G345, S349 and L353 are depicted in yellow, orange and red curves, respectively. The right panels show cartoon representations of K_V7.1 (black) I_KS 8PIP₂ (purple) and I_KS 4PIP₂ (beige) models. Potassium ionic (1.33 Å) and hydrodynamic radii (3.6 Å) are represented by green and blue dashed lines in the graphs, respectively. Pore solvent accessible surfaces are colored as follows. Pore radii values inferior to K⁺ ionic radius, are colored in red. Pore radii values ranging between K⁺ ionic radius and K⁺ hydrodynamic radius, are colored in green. Pore radii values superior to K⁺ hydrodynamic radius, are colored in blue.

Fig. 3

Conserved basic residues in the of KCNE ancillary subunits. The picture shows a sequence alignment of the transmembrane domains (TMD) of KCNE subunits, highlighted in cyan. The conserved basic residues, located at the end of the TMD, near their CTERM domains, are highlighted in blue.

Fig. 4

Structural mapping of KCNE1 basic residues and PIP₂ lipids in the pore domain of I_KS models in 8PIP₂ systems Intracellular view (top panels) and side view (bottom panels) of K_V7.1 pore domain segments (in orange surface) along with S4-S5_LINKER (in brown ribbons), KCNE1 subunits (in gray ribbons) and PIP₂ phosphate groups (in red spheres) in A. RC model, B. IO model and C. AO model. KCNE1 basic residues R67, K69 and K70 are shown in blue spheres (See Tab S4 for their detailed interactions). S4-S5_LINKER residue R249 that interact with PIP₂ intra in a state independent fashion is shown in blue sticks. The successive rotational movements of KCNE1 subunits predicted to occur during RC-IC and IC-AO transitions are shown in gray circled arrows. For each state, a detailed intracellular view of KCNE1 basic residues interactions (framed in black) are depicted on the right side of the side view panels.