Model for the Ca²⁺-dependent CaM/Q2AB interaction.

<p><i>(A)</i> Sequence alignment of segments A and B in which L339, R353 and S511 are underlined. The predicted secondary structure of Kv7.2 according to the GORV algorithm is indicated above the sequence (<a href="http://gor.bb.iastate.edu/" target="_blank">http://gor.bb.iastate.edu/</a>, h = alpha helix, e = extended, c = coiled). The circle beneath a residue indicates that it contacts the N-lobe, while those in contact with the C-lobe are indicated with a square, both of which are color coded according to the CaM surface contact. The contact surface area has been estimated using the Sobolev <i>et al.</i> algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Sobolev1" target="_blank">[52]</a>. <i>(B)</i> Interaction model. The Q2AB helices are depicted as rectangles, the CaM lobes as ovals. Binding to CaM is transient <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Mruk1" target="_blank">[23]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-GomezPosada1" target="_blank">[24]</a>, and the interaction with helix A is critical for function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo1" target="_blank">[25]</a>. Given the greater affinity for helix B <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Xu1" target="_blank">[22]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo1" target="_blank">[25]</a>, it is more likely that CaM docks initially to this helix <i>via</i> the N-lobe, facilitating the interactions between the C-lobe and helix A. Subsequently, a dynamic equilibrium is established: 1.- In the absence of Ca²⁺ the N-lobe dominates the interaction and initially binds to helix B. 2.- Subsequently, the C-lobe engages, establishing an equilibrium between binding to helix A and helix B. 3.- In the presence of Ca²⁺ the C-lobe binds to the IQ site of helix A. 4.- The holo-N-lobe alternates between helix A and helix B. Upon calcification, the interaction between helix B and the N lobe is weakened and the binding between helix A and the C-lobe becomes more significant. Concomitantly, the global affinity in the presence of Ca²⁺ is reduced.</p

The N-lobe binds preferentially to helix B in the absence of Ca²⁺, whereas the C-lobe binds preferentially to helix A in the presence of Ca²⁺.

<p>Competition curves with isolated CaM lobes (N or C) obtained using the individual CaM lobes and performed in the absence (filled symbols) or in the presence of Ca²⁺ (open symbols). D-CaM (12.5 nM) was mixed with helix A (hA, red symbols) or helix B (hB, black symbols) at a concentration corresponding to its calculated EC₅₀ for the increase in D-CaM fluorescence emission (46.4 and 65.6 nM in absence or presence of Ca²⁺ for helix A respectively, and 20.1 and 42.6 nM in absence or presence of Ca²⁺ for helix B respectively) and then each lobe was added incrementally at the concentrations indicated. The data represent the means ± standard error from three or more independent experiments, where some error bars were smaller than the symbols. The result of fitting a Hill equation to the competition curves is compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-t003" target="_blank">Table 3</a>.</p

Summary of the binding parameters obtained after fitting a one site Hill equation to the data in Fig. 4.

<p>See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-g004" target="_blank">Fig. 4</a> for the experimental conditions used. h: Hill coefficient; IC₅₀: concentration producing 50% inhibition. Kd: affinity constant derived assuming a 1∶1 binding.</p

Summary of the binding parameters obtained after fitting a two sites Hill equation to the Q2AB data in Fig. 3.

<p>See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-g003" target="_blank">Fig. 3</a> for the experimental conditions used. h: Hill coefficient; IC₅₀: concentration producing 50% inhibition.</p

Q2AB weakens the Ca²⁺-CaM interaction.

<p><i>(A)</i> Relative increase in D-CaM fluorescence emission (12.5 nM) in response to increased Ca²⁺ concentrations in the presence (open circles) or absence (filled circles) of a molar excess of Q2AB (200 nM) or the indicated segment A mutants. We have previously shown that maximal D-CaM fluorescence is attained at this concentration for WT, L339R and R353G <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo2" target="_blank">[26]</a>. The lines are the result of fitting a Hill equation to the data. The data represent the means ± standard error from three or more independent experiments. Some error bars were smaller than the symbols. The EC₅₀ values obtained are (in µM): CaM = 0.72±0.02, CaM/Q2AB WT = 3.64±0.26, CaM/Q2AB R353G = 1.26±0.16, CaM/Q2AB L339R = 0.75±0.02. <i>(B)</i> Plot of the apparent binding affinity derived from the data in A. ***, significance at P≤0.001, *P≤0.05, unpaired Student’s t test.</p

The competition assay defines lobe specific interactions with SK2.

<p>Competition curves with isolated CaM lobes (N or C), with an equimolar mixture of N- and C-lobes (N&C) or with intact CaM (N–C). D-CaM (12.5 nM) was mixed with SK2 at a concentration corresponding to its calculated EC₅₀ for the increase D-CaM fluorescence emission (see Fig. 1 <i>C</i>, 9.2 and 13.7 nM in the presence or absence of Ca²⁺, respectively) and the competing peptides were added incrementally at the concentrations indicated. The data represent the means ± standard error from three or more independent experiments. The error bars are smaller than the symbols. The result of fitting a Hill equation to the competition curves is compiled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone-0086711-t001" target="_blank">Table 1</a>. <i>(A)</i> The effect of incremental addition of the lobes indicated obtained in the absence of Ca²⁺ (Left, 10 mM EGTA added) and in the presence of 100 µM free Ca²⁺ (right). <i>(B)</i> The effect of incremental addition of CaM WT (N−C) and of an equimolar mixture of the lobes (N&C) obtained in the absence of Ca²⁺ (left, 10 mM EGTA added) and in the presence of 100 µM free Ca²⁺ (right). (<i>C</i>) Comparison of the arithmetic addition of the curves obtained for each individual lobe (N+C) with the effect of an equimolar mixture (N&C) and with CaM (N−C) at concentrations under 200 nM in absence (left) or in the presence of 3.9 µM Ca²⁺ (that were indistinguishable from the results obtained in the presence of 100 µM Ca²⁺). <i>(D)</i> Plot of the reduction in fluorescence at the indicated concentration of competing lobe(s). ***, significance at P≤0.001, **P≤0.01, unpaired Student’s t test. <i>(E)</i> A model for the Ca²⁺-dependent CaM/SK2 interaction that can be derived from this set of experiments. 1. Both lobes cooperate in binding and the C-lobe, but not the N-lobe, is bound to SK2 in absence of Ca²⁺. 2. Ca²⁺ does not affect the interaction with the C-lobe. As the Ca²⁺ concentration increases the N-lobe becomes calcified. 3. The calcified N-lobe binds to SK2, leading to the observed increase in affinity in the presence of Ca²⁺. The data do not allow the oligomerization state to be established and therefore the dimerization of the CaM/SK2 complex that takes place upon Ca²⁺ binding is based on the resolved structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Schumacher1" target="_blank">[9]</a>.</p

Dose-response enhancement of 12.5 nM D-CaM fluorescence emission by the CaMBD.

<p><i>(A)</i> Effect of an incremental addition of the Q2AB (left column) or SK2 CaM binding domains (right column) in the emission spectra of 12.5 nM D-CaM both in the absence of free Ca²⁺ (top panels, 10 mM EGTA added) and in the presence of 3.9 µM free Ca²⁺ (bottom panels). The color of the traces changes from red to blue as the ligand concentration increases. <i>(B)</i> Relative concentration-dependent enhancement of 12.5 nM D-CaM fluorescence emission by SK2 in the presence (open circles) or absence (filled circles) of 3.9 µM Ca²⁺. The parameters used to fit a Hill equation to the data (continuous and dashed lines) were: Max = 122±4.3, EC₅₀ = 13.7±1.6 nM, h = 1.6±0.3 in absence of Ca²⁺, and Max = 123±1.4, EC₅₀ = 9.2±0.4 nM, h = 1.3±0.1 in the presence of Ca²⁺. The data represent the means ± standard error from three or more independent experiments. The error bars are smaller than the symbols. For comparison, the result of the fit of a Hill equation to the data for the effect of Q2AB of D-CaM fluorescent emission taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086711#pone.0086711-Alaimo2" target="_blank">[26]</a> is plotted in grey in the absence (continuous grey line) or presence of Ca²⁺ (dotted grey line). <i>(C)</i> Plot of the apparent binding affinity derived from the data in B obtained in absence (black column) or in presence of Ca²⁺ (white columns) for the proteins indicated. ***, significance at P≤0.001, *P≤0.05, unpaired Student’s t test.</p

Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

<div><p>Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A – helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.</p> </div

Surface expression of WT and Del6 Kv7.2 subunits in human HEK293T cells.

<p>Analysis of confocal images of non-permeabilized cells expressing the indicated constructs. The subunits have a mCFP tag at the N-terminus (rendered in green) and an extracellular 2×HA tag, allowing simultaneously monitoring total (green) and surface expression (red). The proportion of the cells with surface staining in confocal images was determined in >40 mCFP positive cells for each construct in three independent experiments. <b><i>A.</i></b><i>-</i> Grey bars represent mean ± SEM of the percentage of cells expressing the channel at the surface. *** <i>P</i>≤0.001; unpaired Student’s <i>t</i> test. <b><i>B.</i></b><i>-</i> Representative images of cells expressing the indicated subunit. <b><i>C.</i></b>- Ratio of surface/total expression (red fluorescence/cyan fluorescence) from wide field epifluorescence images of cells expressing the indicated Kv7.2 subunits. *** <i>P</i>≤0.001; unpaired Student’s <i>t</i> test. <b><i>D.</i></b>- Plot of the cyan fluorescence <i>vs</i> red fluorescence intensity from wide field epifluorescence images of cells expressing the subunits indicated. There was no correlation between total and surface expression (>90 cells from more than 5 independent experiments).</p

Removal of the A-B linker resulted in functional Kv7.2 channels. <i>A.-</i>

<p>Representative currents recording from HEK293T cells transfected with WT-, Del6- or Del2-Kv7.2, activated from a holding potential (V_h) = −30 mV after 1,500 ms steps to the indicated voltages. <b><i>B.-</i></b> Current density-voltage relationship from tail currents of WT (<i>n</i> = 13) or Del6 (<i>n</i> = 15) channels. Each point represents the mean ± SEM. A Boltzmann equation D = Dmax/(1+e^((V-V_1/2^)/S)) was fitted to the data. The averaged Boltzmann parameters were: WT: V_1/2 =  −34.8±1.9 mV, Slope = 11.6±1.7, Dmax = 30.7±0.9 pA/pF; Del6: V_1/2 =  −30.6±5.1 mV, Slope = 11.2±4.5, Dmax = 34.4±2.6 pA/pF; Del2: V_1/2 =  −29.6±5.5 mV, Slope = 14.2±4.5, Dmax = 34.1±2.6 pA/pF.</p