Mutations stabilizing KCNQ1 towards the inactivated state.

<p>(A) and (B) Representative current traces of WT and L233W and Q244W, respectively, recorded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001943#pone-0001943-g002" target="_blank">Figure 2 A</a>. (C) Normalized conductance of L233W (n = 13) (black squares), compared to WT (open squares). (D) Percent of macroscopic inactivation of WT, Q244W and L233W (n = 7–20) as measured by the ratio between the sustained and the peak current amplitudes.</p

Impact of KCNE1 expression on WT Kv7.1 and mutant R228C.

<p>(A) Representative trace of WT Kv7.1 coexpressed with WT KCNE1. (B) Effects of external Cu-Phen on mutant R228C. Oocytes were bathed in ND96 in the absence and presence of 100 µM Cu-Phen. Shown are representative traces and current-voltage relations were determined as indicated. (C) Shown are representative traces and current-voltage relations of R228C+WT KCNE1 channels, when oocytes were bathed with ND96 in the absence of presence of 100 µM Cu-Phen. Also shown, is the reversal by DTT of the current decrease produced by Cu-Phen. (D) Representative traces of R228C+WT KCNE1 channels, when oocytes were bathed with ND96 containing 100 µM Cu-Phen. Currents were evoked by a train of step depolarization to +30 mV. Similar results have been obtained in 5 other cells.</p

Gating parameters of WT and mutant Kv7.1 channels expressed in the presence of WT KCNE1.

<p>V₅₀ (half activation voltage) and z (equivalent gating charge) were derived from fitting single Boltzmann function; I₆₀ corresponds to the current density measured at +60 mV in pA/pF. ΔG₀ and ΔΔG₀^c were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001943#s4" target="_blank">methods</a>. Data are expressed as mean ± SEM and in parentheses are indicated the number of cells.^*, p<0.05 compared to WT (two-tailed, Student's unpaired t test). ND, not determined; NA, not applicable as R231W mutant is a constitutively open K⁺ leak channel.</p

Effect of KCNE1 co-expression with mutant R231W and I235W.

<p>Representative current traces of mutant R231W expressed without (A) or with KCNE1 (B). Conductance-voltage relations (C) and current-voltage relations (D) of WT Kv7.1 and mutant R231W co-expressed with KCNE1. Representative current traces of mutant I235W expressed without (E) or with KCNE1 (F). Conductance-voltage relations (G) and current-voltage relations (H) of WT Kv7.1 and mutant I235W co-expressed with KCNE1.</p

Effect of KCNE1 co-expression with mutant R237W and R243W.

<p>Representative current traces of mutant R237W expressed without (A) or with KCNE1 (B). Conductance-voltage relations (C) and current-voltage relations (D) of WT Kv7.1 and mutant R237W co-expressed with KCNE1. Representative current traces of mutant R243W expressed without (E) or with KCNE1 (F). Conductance-voltage relations (G) and current-voltage relations (H) of WT Kv7.1 and mutant R243W co-expressed with KCNE1.</p

Gating parameters of WT and mutant Kv7.1 channels.

<p>V₅₀ (half activation voltage) and z (equivalent gating charge) were derived from fitting single Boltzmann function; I₆₀ corresponds to the current density measured at +60 mV in pA/pF. ΔG₀ and ΔΔG₀^c were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001943#s4" target="_blank">methods</a>. Data are expressed as mean ± SEM and in parentheses are indicated the number of cells.^*, p<0.05 compared to WT (two-tailed, Student's unpaired t test). NA, not applicable as R231W mutant is a constitutively open K⁺ leak channel.</p

Mutations stabilizing Kv7.1 to the open state.

<p>(A) and (B) Representative current traces of WT and A226W, respectively. From a holding potential of −90 mV, the membrane was stepped for 3 s from −70 mV to +60 mV in 10 mV increments and then repolarized for 1.5 s to −60 mV to generate the tail currents. (C) and (D) Normalized conductance was plotted as a function of step voltages, for the mutants (black squares) A226W (n = 6) and V241W (n = 11), respectively, and compared to WT (n = 20) (open squares). The activation curves were fitted using one Boltzmann function. (E) Representative current traces of R231W. Membrane was stepped for 3 s from −140 mV to +60 mV in 20 mV increments and then repolarized for 1.5 s to −60 mV. (F) Current-voltage relations of R231W (n = 8) (black squares) and WT (open squares). Current density (pA/pF) was plotted as a function of step voltages.</p

Summary of the tryptophan scan of Kv7.1 S4.

<p>(A) Sequence alignment of the S4 segment of human Kv7.1 with various Kv channels. (B) Impact of the perturbations projected onto a helical wheel diagram. The cut-off for significance was ∥ΔΔG₀^c∥≥1.5 kcal.mol⁻¹. The red, blue and green circled mutated residues shift the gating equilibrium in favor of the closed, open and inactivated state, respectively. (C) Impact of the perturbations expressed as a bar graph along the S4 sequence. The color coding is as in B. The black bars correspond to residues whose perturbation is not significant (∥ΔΔG₀^c∥<1.5 kcal.mol⁻¹).</p

Summary of the tryptophan scan of Kv7.1 S4 in the presence of KCNE1.

<p>The cut-off for significance was ∥ΔΔG₀^c∥≥1.5 kcal.mol⁻¹. The red and blue bars of the mutated residues shift the gating equilibrium in favor of the closed and open state, respectively. The black bars correspond to residues whose perturbation is not significant (∥ΔΔG₀^c∥<1.5 kcal.mol⁻¹).</p