Pivoting between calmodulin lobes triggered by calcium in the Kv7.2/calmodulin complex

Abstract

Kv7.2 (KCNQ2) is the principal molecular component of the slow voltage gated M-channel, which strongly influences neuronal excitability. Calmodulin (CaM) binds to two intracellular C-terminal segments of Kv7.2 channels, helices A and B, and it is required for exit from the endoplasmic reticulum. However, the molecular mechanisms by which CaM controls channel trafficking are currently unknown. Here we used two complementary approaches to explore the molecular events underlying the association between CaM and Kv7.2 and their regulation by Ca(2+). First, we performed a fluorometric assay using dansylated calmodulin (D-CaM) to characterize the interaction of its individual lobes to the Kv7.2 CaM binding site (Q2AB). Second, we explored the association of Q2AB with CaM by NMR spectroscopy, using (15)N-labeled CaM as a reporter. The combined data highlight the interdependency of the N- and C-lobes of CaM in the interaction with Q2AB, suggesting that when CaM binds Ca(2+) the binding interface pivots between the N-lobe whose interactions are dominated by helix B and the C-lobe where the predominant interaction is with helix A. In addition, Ca(2+) makes CaM binding to Q2AB more difficult and, reciprocally, the channel weakens the association of CaM with Ca(2+).

Figure 1. Dose-response enhancement of 12.5 nM D-CaM fluorescence emission by the CaMBD.

(A) 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. (B) 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 is plotted in grey in the absence (continuous grey line) or presence of Ca²⁺ (dotted grey line). (C) 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.

Figure 2. The competition assay defines lobe specific interactions with SK2.

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 C, 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 Table 1. (A) 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). (B) 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). (C) 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⁺). (D) 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. (E) 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 .

Figure 3. Q2AB binds preferentially to the N-lobe in the absence of Ca²⁺ and to the C-lobe in the presence of Ca²⁺.

Relative concentration-dependent reduction in 12.5 nM D-CaM fluorescence emission when complexed with Q2AB. To achieve maximal sensitivity in the assay, concentrations of Q2AB that caused 50% of the maximal increase in D-CaM fluorescence emission were used (27 nM and 11 nM in the presence and absence of Ca²⁺, respectively). D-CaM was mixed with Q2AB and then each lobe (N or C), both lobes (N&C) or intact CaM (N−C) was added incrementally at the concentrations indicated. The data represent the means ± standard error from three or more independent experiments. Some error bars were smaller than the symbols. The result of fitting a two sites Hill equation to the competition curves is compiled in Table 2. (A) The effect of incremental addition of the indicated lobes obtained in the absence of Ca²⁺ (Left, 10 mM EGTA added) and in the presence of 100 µM free Ca²⁺ (right). (B) 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). (C) 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 of 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⁺). (D) Plot of the reduction on fluorescence at the indicated concentration of competing lobe(s). ***, significance at P≤0.001, **P≤0.01, *P≤0.05, unpaired Student’s t test.

Figure 4. 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⁺.

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 Table 3.

Figure 5. Monitoring of the interaction between CaM and Q2AB by (¹H, ¹⁵N)-HSQC spectroscopy.

(A) Details of ¹H,¹⁵N-HSQC spectra of CaM in the absence (black) and in the presence of 2.5 equivalents of Q2AB for apo-CaM (blue) and holo-CaM (red). (B) Structural mapping of the chemical shift perturbation (CSP) induced by Q2AB binding to apo-CaM (top, PDB entry 1CFC) and holo-CaM (bottom, PDB entry 2K0E). The lobes are indicated by the labels “N” and “C”, and the four EF hands are numbered 1–4. The CSPs are color coded as indicated by the gradient bar. Figure created using Pymol. (C) CSPs induced by Q2AB to apo-CaM (top) and holo-CaM (bottom) plotted in function of the CaM residue number.

Figure 6. Q2AB weakens the Ca²⁺-CaM interaction.

(A) 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 . 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. (B) 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.

Figure 7. Model for the Ca²⁺-dependent CaM/Q2AB interaction.

(A) 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 (http://gor.bb.iastate.edu/, 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 et al. algorithm . (B) Interaction model. The Q2AB helices are depicted as rectangles, the CaM lobes as ovals. Binding to CaM is transient ; , and the interaction with helix A is critical for function . Given the greater affinity for helix B ; , it is more likely that CaM docks initially to this helix via 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.