Action potential simulation for Kv11.1-A and Kv11.1-3.1.

<p>Typical current responses for the 1^st, 5^th and 61^st pulse for A. Kv11.1-1A and B. Kv11.1-3.1 during a pulse protocol where cells were repetitively depolarized to +40 mV for 5 ms, from a holding potential of <b>−</b>70 mV with interpulse interval of 15 ms (voltage protocol shown at top of panel). Currents were measured at 1 ms (red dashed line) and 3 ms (blue dashed line) and the values normalized to the peak tail current recorded for each cell at <b>−</b>120 mV after a 1 s step to +40 mV to fully activate the channels (data not shown). C. Normalized currents measured at 1 ms (red symbols) plotted against time for Kv11.1-1A (○), Kv11.1-1A/Kv11.1-3.1 (*) and Kv11.1-3.1 (•) with the 1^st, 5^th and 61^st pulse highlighted by the black arrows. D. Normalized currents measured at 3 ms (blue symbols) plotted against time for Kv11.1-1A (○) and Kv11.1-3.1 (•). 1^st, 5^th and 61^th pulse highlighted with black arrows. The data for the Kv11.1-1A/Kv11.1-3.1 channels has been left out of panel D for purposes of clarity (due to overlapping error bars). Panel C and D only show every second data point for purposes of clarity.</p

Rates of activation for Kv11.1 channels at 0 mV.

<p>A. Typical examples of Kv11.1-3.1 currents recorded at 37°C during an envelope-of-tails voltage clamp protocol to measure rates of activation at 0 mV. The voltage protocol is shown at the top of the panel. The dashed line highlights the peak tail current for each current trace. B. Normalized peak tail current plotted against duration of the test pulse for Kv11.1-1A (○) and Kv11.1-3.1 (•). The inset shows the mean ± SEM for time constants of activation (n = 4−5). τ_{act, 0 mV} for Kv11.1-1A (58±5 ms, n = 5) was not significantly larger than that for Kv11.1-3.1 (57±5 ms, n = 4).</p

Rates of deactivation for Kv11.1-1Aand Kv11.1-3.1A.

<p>A. Typical currents recorded at 37°C during a protocol to measure rates of deactivation (voltage protocol show at top of panel, dashed box indicates the part of the voltage protocol for which current traces are shown) for Kv11.1-1A (left) and Kv11.1-3.1 (right). B. Magnification of the first 100 ms of the −120 mV tail current for Kv11.1-1A (grey) and Kv11.1-3.1 (black) show the characteristic hooked appearance reflecting recovery from inactivation followed by deactivation. C. Summary of τ_deact (mean ± SEM) over the voltage range of −130 mV to −60 mV for Kv11.1-1A (○) and Kv11.1-3.1 (•). ***: p<0.001.</p

Inactivation properties of Kv11.1-1A and Kv11.1-3.1.

<p>A. Typical family of Kv11.1-1A (left) and Kv11.1-3.1 (right) current traces recorded at 37°C during a protocol to measure rates of inactivation (voltage protocol shown at top of panel A, dashed box highlights the part of the traces shown). B. Magnification of the first 8 ms of the +60 mV step for Kv11.1-1A (grey) and Kv11.1-3.1 (black) indicating slower inactivation for Kv11.1-3.1 compared to Kv11.1-1A. C. Summary of rates of recovery from inactivation for Kv11.1-1A (□) and Kv11.1-3.1 (▪) (τ_recov, measured from protocol shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045624#pone-0045624-g003" target="_blank">Fig. 3</a>) over the voltage range −130 mV to −40 mV and rates of inactivation (τ_inact) for Kv11.1-1A (○) and Kv11.1-3.1 (•) over the voltage range <b>−</b>30 mV to +60 mV. Solid lines are the best fits of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045624#pone.0045624.e002" target="_blank">equation 2</a> to the data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045624#s2" target="_blank">materials and methods</a>). D. Midpoint of stead-state inactivation for Kv11.1-1A (○) and Kv11.1-3.1 (•) measured as the voltage at which τ_inact = τ_recov (see methods for details). ***: p<0.001.</p

Voltage dependence of steady-state activation for Kv11.1-1A and Kv11.1-3.1.

<p>A. Typical families of current traces recorded at 37°C from Kv11.1-1A (left) and Kv11.1-3.1 (right) showing the last 100–150 ms of the activating step and 500 ms of the tail current recorded at −60 mV. Arrow indicates position where peak tail current was recorded. Inset at top of panel shows voltage protocol used to measure steady-state activation. B. Normalized peak tail currents plotted against voltages of the preceding test pulse for Kv11.1-1A (○) and Kv11.1-3.1 (•). Solid lines are fits of the Boltzmann function (see Eq. 1) giving V_0.5 for steady-state activation of −31.4±1 mV for Kv11.1-1A and −35±1 mV for Kv11.1-3.1 (P<0.05).</p

Summary data for individual β9-strand mutants.

<p>(A) Scatter plot of the V_0.5 values for the 3 s isochronal activation (open symbols) and 3 s isochronal deactivation (closed symbols) for WT (black), AAA (grey), F860A (red), N861A (magenta), L862A (blue), F860L (orange), F860Y (green) and F860R (cyan). (B) Scatter plot of the V_0.5 values for the steady-state inactivation (open symbols) for WT, AAA, F860A, N861A, L862A, F860L, F860Y and F860R (same colour scheme as in panel A). In all panels, the mean and SEM are indicated by horizontal bars and asterisks indicate values that are statistically significantly different to WT (<i>P</i><0.05, ANOVA). The dashed horizontal lines indicate mean values for WT. The values for all mutants are summarized in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077032#pone.0077032.s001" target="_blank">File S1</a>.</p

Topology of Kv11.1 channels and sequence analysis of cNBH domains.

<p>(A) Topology of Kv11.1 channel showing the intracellular N-terminal PAS domain (blue), transmembrane voltage sensing domain (green), pore domain (yellow) and intracellular C-terminal C-linker and cNBH domains (orange). Inset shows the homology model of the cNBH domain of Kv11.1 generated based on the mEAG crystal structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077032#pone.0077032-MarquesCarvalho1" target="_blank">[13]</a>. (B) Sequence alignment of mHCN2, zELK, mEAG and human Kv11.1 extracted from a Clustalw alignment of the entire family of KCNHx/HCNx/CNGx ion channels. Sequences shown correspond to the dotted box region shown in panel A. Sequence similarity to the Kv11.1 are marked by white text/red box (identical) and black text/yellow box (similar). Non-conserved sequences are in grey. Clear rods and arrows represent the consensus α-helices and β-strands while filled rods and arrows indicate the differences with orange, green and blue representing mHCN2, zELK and mEAG, respectively. The hydrogen bond between asparagine (arrow) and tyrosine (asterisk) in zELK is not observed in the others.</p

Trafficking assay of LQT2 mutants located within β9-strand.

<p>(A) Typical western blot of WT, N861I and N861H mutant channels. WT shows two bands at ∼155 kDa and ∼135 kDa. The ∼155 kDa band disappears following digestion of surface proteins with proteinase K. The N861H mutant shows only a single ∼135 kDa band. N861I contains both ∼155 kDa and ∼135 kDa bands. Arrow indicates degradation band after proteinase K digestion. (B) Normalized expression levels of N861H and N861I relative to WT for the fully glycosylated (∼155 kDa band) and core-glycosylated (∼135 kDa band) proteins. (C) The partially trafficking defective N861I can be rescued by incubation with cisparide whereas N861H was not rescued by cisapride. (D) Co-imunpreciptation of HA-tagged mutant subunits with Flag-tagged WT subunits. (E) Top panel: Summary of 3 s isochronal activation V_0.5 (open symbols) and 3 s isochronal deactivation V_0.5 (closed symbols) for WT (black), N861H (magenta) and N861I (blue). Asterisks indicate <i>P</i><0.05 (ANOVA) compared to WT. Bottom panel: Summary of the V_0.5 of steady-state inactivation for WT, N861H and N861I (same colours as in top panel). Mean data for all mutants are summarized in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077032#pone.0077032.s001" target="_blank">File S1</a>.</p

Sequence alignment and structure of hERG S4–S5 linker.

<p>A. Sequence alignment of hERG and Kv1.2 for the distal S4, S4–S5 linker and proximal S5 domains. The leucine residues (red) of Kv1.2 S4–S5 linker correspond to tyrosine and valine residues in hERG. Glycine residue (green) in both channels is also conserved. B. Chemical shift index (CSI) plot for NMR structure of hERG S4–S5. CSI values less than −0.1 ppm are indicative of α-helical structure. C. 20 lowest energy structures for hERG S4–S5 with side chains colour coded according to physiochemical properties (basic: blue, acidic: red, polar: green, aromatic: yellow, hydrophobic: grey).</p

Secondary structure prediction and MD simulations of cNBH domain.

<p>Sequence prediction of the cNBH domain around the β9-strand for (A) WT (i) and AAA mutant (ii). (B) RMSD (i) and RMSF (ii) of WT (red) and AAA mutant (black) from the 60 ns of MD simulations. The blue box highlights the most significant difference between WT and AAA mutant in the backbone fluctuation. (C) The structures that have the lowest structural fluctuation to the centroid structure in the most populated cluster from the last 10 ns for WT (i) and AAA mutant (ii). Residues involved in hydrophobic interactions, defined by being within 4 Å of residues 860, 861 and 862 (cyan), are highlighted in magenta for WT (i) and AAA mutant (ii). There are reduced hydrophobic interactions in the AAA mutant. (D) Summary of residues that participate in hydrogen bonds with residues 860, 861 and 862 in WT (i) and the AAA mutant (ii) that are present for more than 5% of the 60 ns of MD simulation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077032#pone-0077032-t001" target="_blank">Table 1</a> for details).</p