KCNE1 Constrains the Voltage Sensor of Kv7.1 K+ Channels

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K+ channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K+ channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions

S1 Constrains S4 in the Voltage Sensor Domain of Kv7.1 K+ Channels

Voltage-gated K+ channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (∼190°) and outward translation of S4 (∼12 Å) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1–S4 interaction to an inter-VSD S1–S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating

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

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

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

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

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

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

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