Cholesterol enantiospecifically binds prokaryotic and eukaryotic Kir channels.

<p>(<b>A</b>) Representative time course of ⁸⁶Rb⁺ uptake into 9∶1 POPE∶POPG liposomes (+1% PI(4,5)P₂ for Kir2.1) containing 0% cholesterol (▪), 5% cholesterol (•) or 5% <i>ent-</i>cholesterol (▴) reconstituted with KirBac1.1 (left), KirBac3.1 (middle), or Kir2.1 (right) protein, with 450 mM internal and 0 mM external [K⁺]. (<b>B</b>) KirBac1.1 (blue), KirBac3.1 (green) and human Kir2.1 (red) channel activity-cholesterol relationship obtained from ⁸⁶Rb⁺ uptake counts. The data are renormalized to uptake in liposomes containing 0% cholesterol. (n = 6 for each protein, * P<0.05 assessed by ANOVA).</p

Cholesterol inhibits activity of reconstituted KirBac1.1, KirBac3.1 and human Kir2.1.

<p>(<b>A</b>) Representative time course of ⁸⁶Rb⁺ uptake into 9∶1 POPE∶POPG liposomes (+1% PI(4,5)P₂ for Kir2.1) containing increasing amounts of cholesterol reconstituted (0% = ▪; 1% = •; 5% = ▴; 10% = ▾) with KirBac1.1 (left), KirBac3.1 (middle), or Kir2.1 (right) protein, incubated with 450 mM internal and 0 mM external [K⁺]. Uptake was normalized to valinomycin-induced uptake in the same liposomes. (<b>B</b>) Channel activity-cholesterol relationship obtained from ⁸⁶Rb⁺ uptake at 240 s for KirBac1.1 (blue; n = 7±s.e.m), at 90 mins for KirBac3.1 (green; n = 6±s.e.m) and at 60 mins for human Kir2.1 (red; n = 6±s.e.m) for each cholesterol concentration. The data are renormalized to uptake in liposomes containing 0% cholesterol. (<b>C</b>) ⁸⁶Rb⁺ uptake from KirBac1.1 (blue) and Kir2.1 (red) channels reconstituted into liposomes containing 25% POPG (+1% PI(4,5)P₂ for Kir2.1) ±5% cholesterol on a POPE background (n = 8 for each). The data were normalized to valinomycin-induced uptake in the same liposomes and re-normalized to uptake in liposomes containing 0% cholesterol. (* P<0.05 assessed by ANOVA).</p

Calculated secondary structure of the NavSp channel in liposomes of different compositions based on SRCD measurements.

<p>The percentage values listed are the average secondary structures calculated from several methods of analyses applied to the same data sets (the +/− values indicate the variations between algorithms). All liposomes include PE; the “guest” lipid is the second lipid present in the liposome sample.</p

Lipid dependence of purified NavSp channels.

<p>²²Na⁺ uptake counts of full-length (<b>A</b>) or pore-only (<b>B</b>) constructs of NavSp channels in liposomes composed of POPE and the indicated lipid. (<b>C</b>) Relative ²²Na⁺ uptake of NavSp at 120 mins normalized to the uptake into liposomes that do not contain NavSp protein (ie. “Blank”) (n = 3 and 6, respectively, +/− s.e.m.).</p

Model structures of the pore-only constructs of NavSp and NavMs.

<p>Comparison of the electrostatic surface potentials (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061216#pone-0061216-g006" target="_blank">Figure 6</a>) of the pore-only constructs of NavMs and NavSp. The NavMs is the crystal structure of the NavMs pore (PDBID: 4F4L) and the NavSp is a homology model based on that crystal structure.</p

Effect of PI(4,5)P₂ on bacterial voltage-gated sodium channels.

<p>(<b>A–C</b>) Time-dependent uptake of ²²Na⁺ into proteoliposomes containing 3∶1 POPE∶POPG (or PI in the case of NavSp) plus 1% PI(4,5)P₂ and full-length NaChBac, NavMs, or NavSp proteins, respectively. (<b>D</b>) Relative ²²Na⁺ uptake at 10 mins and 60 mins from the time course measurements in parts A–C indicate that NaChBac channels are unaffected by the presence of 1% PI(4,5)P₂, NavSp channels are approximately 50% inhibited by 1% PI(4,5)P₂ and NavMs channels have complex regulation by PI(4,5)P₂ (* P<0.05).</p

Model structures of the three homologues.

<p>Comparison of the electrostatic potentials at +/−5 KT/e plotted on the solvent-accessible surfaces of (<b>A</b>) NavSp, (<b>B</b>) NavMs and (<b>C</b>) NaChBac, based on homology models constructed from NavAb in the activated closed state (PDBID:3RVY), viewed along the extracellular surface (left panel), the membrane normal (middle panel) and from the cytoplasmic surface (right panel). The black spheres indicate the positions of ordered lipids in the original NavAb (PDBID: 3RVY) crystal structure used to produce the models.</p

Lipid dependence of purified NaChBac channels.

<p>(<b>A</b>) ²²Na⁺ uptake counts of NaChBac channels in liposomes composed of POPE and the indicated lipid. (<b>B</b>) Relative ²²Na⁺ uptake of NaChBac at 120 mins normalized to uptake into liposomes that do not contain NaChBac protein (n = 6, +/− s.e.m.).</p

Sequence alignment of bacterial voltage-gated sodium channels.

<p>The black arrows indicate the region visible in the NavAb crystal structure (PDBID 3RVY) which was used to create homology models of each channel. Positively charged residues are shown in blue, negatively charged residues in red. The pink upward arrows indicate residues within 5 Å of the phosphocholine headgroups observed in the NavAb crystal structures (PDBID 4EKW or 3RVY). The green downward arrow indicates the start of the pore-only constructs and the orange downward arrow indicates the site of the C-terminal truncation of the NavMs constructs.</p

Purification of NaChBac, NavSp and NavMs channels.

<p>(<b>A</b>) SDS-PAGE of purified proteins in Cymal-5. The positions of the predicted monomeric (M), dimeric (D), trimeric (Tri) and tetrameric (Tet) forms are indicated. (<b>B</b>) SDS-PAGE of purified pore-only proteins in DM. The position of the predicted monomeric (M), dimeric (D), trimeric (Tri) and tetrameric (Tet) forms are indicated. (<b>C</b>) Gel filtration chromatography profiles for NaChBac (black solid), NavSp (grey dashed) and NavMs (grey solid). The predominant peak corresponds to the molecular mass of tetrameric voltage-gated sodium channel plus Cymal-5 micelle. (<b>D</b>) Gel filtration chromatography profiles for NavSp pore (grey dashed) and NavMs pore (grey solid). The predominant peak corresponds to the molecular mass of tetrameric voltage-gated sodium channel plus DM micelle.</p