From Bench to Biomolecular Simulation: Phospholipid Modulation of Potassium Channels

Figures

• 

  Figure 1. Topological representation and atomic structures of K+ channels. (A) Voltage-gated potassium (Kv) channel topology. (B) A structural example for of a Kv channel: hKCNQ1 (PDB ID: 6V01).56(C) Two-pore potassium (K2P) channel topology. (D) Example structure of the K2P channel, mTREK-1 (PDB ID: 6V37). (E) Ca2+ gated potassium (KCa) channel topology. (F) Structure of the KCa channel hSlo1 (PDB ID: 6V38).143(G) Inwardly rectified potassium (Kir) channel topology. (H) Structure of the cKir2.2 channel (PDB ID: 3SPI).144 Topology of potassium channels where transmembrane helices are represented as cylinders. The structures are shown in cartoon. The voltage sensor domain (VSD) is coloured green. The pore domains are all represented in blue. The cytoplasmic domain of Kv, KCa and Kir are shown in yellow, red and lilac respectively. The cap domain of K2P channel is shown in orange. N and C represent each termini of the protein. The PIP2 cartoon represents a putative binding site based on experimental and/or structural data.

• 

  Figure 2. PIP2 binding site on cKir2.2 and hKCNQ1 channel. (A) Atomic structure of the cKir2.2 channel in Apo (PDB ID: 3JYC)145 and PIP2-bound state (PDB ID: 3SPI).87 The pore domain is shown in blue and the cytoplasmic domain is shown in pink. (B) Atomic structure model of KCNQ1 channel in KCNE3-CaM bound (PDB ID: 6V00) and KCNE3-CaM-PIP2-bound state (PDB ID: 6V01).56 The pore domain is shown in blue, voltage sensor domain is coloured in green, S6 helices in the cytoplasm are shown in yellow, calmodulin proteins are shown in pink and KCNE3 subunits are shown in orange. In both panels, carbon atoms on PIP2 are represented in purple and others are coloured based on CPK convention. All PIP2 bound residues are shown as sticks.

• 

  Figure 3. Computational methods associated with K+ Channel lipid interactions. (A) Potential of mean force (PMF) calculation. PIP2 lipids (acyl tail - green, glycerol - yellow, phosphates - orange, inositol ring - cyan) are sampled with a harmonic potential along the collective variable (CV) parallel to the bilayer. Each PIP2 represents a sampling position. The protein is displayed only in coarse-grained backbone where the transmembrane region is shown in blue and cytoplasmic region is shown in pink. PIP2 binding free energy to the protein is then calculated using weighted histogram analysis method (WHAM) based on the energies it samples along the CV (right). (B) The thermodynamic scheme underpinning free energy perturbation (FEP) calculation. Transformation of PIP2 to PC in both bound and free configurations are calculated in ΔG1 and ΔG2 (blue). This allows ΔG3 and ΔG4 to be obtained (green) using the equation shown (bottom).

• 

  Figure 4. Experimental methods associated with K+ channel lipid interactions. (A) Reconstituted flux Assay. Schematic diagram showing the fluorescence properties of the liposome based on.72 The liposome contains K+ and fluorescence sensitive dye (green). Upon an addition of CCCP - uncoupler (grey circle) and the opening of the channel (blue), K+ ions (pink) move out of the liposome, counteracting with the movement of proton (red) into the liposome. The protons then quench the fluorescence signal (green to grey). The degree of quenching is depending upon the concentration of the channel activator. (B) PIP2 sensitivity. The FVPP labels its application to prevent rundown. The channel is then allowed to rundown almost to 0 through the wash-out of FVPP. PIP2 at different concentrations (low - dark blue, high - yellow) are applied to the patch to observe an increase in channel current. The y-axis is labelled as the fraction of the maximal current. Neomycin sensitivity. (C) Neomycin at different concentrations (low - dark blue, high - yellow) are applied to the patch to observe decrease in channel current as PIP2 is quenched from the membrane. (D) Fluorescence binding assay. Decrease in…