Garneau L , Klein H , Lavoie MF , Brochiero E , Parent L , Sauvé R .

Canadian Institutes of Health Research

	Figure 1. Ribbon representation of the KCa3.1 pore region, including the S4–S5 cytosolic linker, the S5 and S6 transmembrane helices, the pore helix (gray), and the selectivity filter regions (stick representation). (A) Model structure generated by MODELLER9.11 using as template the crystal structure solved for a bacterial cyclic nucleotide–regulated channel MlotiK1 (Protein Data Bank accession no. 3BEH). Also shown is a space-filling representation of residues L243, F248, and V256, with the L243 side chain projecting opposite to the S5 and S6 transmembrane segments toward V256 of the selectivity filter. In contrast, F248 is predicted to be oriented with its side chain making contact with both the S5 and S6 helices. The residues documented to be facing the channel pore are colored in blue (Klein et al., 2009). (B) Model structure generated by MODELLER9.11 using as template the crystal structure solved for the mammalian Kv1.2 channel (Protein Data Bank accession no. 2A79). As in A, residue F248 is seen as projecting between the transmembrane helices S5 and S6. Only two subunits are presented for clarity. (C) Inside-out patch recordings supporting the formation of a disulfide bond between Cys engineered at 256 and 243. The reducing agent BMS caused channel activation when applied onto the L243C–V256C double mutation channel but failed to affect channel activity when applied to the V256C (single-channel) and L243C (multi-channel) single mutation channels. These observations support the orientation of the pore helix relative to the selectivity filter proposed in A and B. Channels were expressed in Xenopus oocytes, and current records were obtained at −60 mV in symmetrical 200-mM K2SO4 conditions at an internal Ca2+ concentration of 25 µM. The label “c” refers to the zero current level. Illustration by Discovery Studio Visualizer (Accelrys).
	Figure 2. Characterization of the interface formed by the pore helix with the S5 and S6 transmembrane segments based on the difference in SASA (ΔSASA) for the KCa3.1 model derived from the 3BEH (A and C) or 2A79 (B and D) templates. ΔSASA values were computed from MD 64-ns trajectories using the Charmm37 force field. The contribution of individual pore helix residue to the interface formed by the pore helix and S5 transmembrane segment or pore helix and S6 transmembrane helix (A and B) was estimated as described in the Results. The results in A and B show that the average contact area with either the S5 or S6 transmembrane segment is maximal for residue F248 independently of the template used. In contrast, residues T240, L241, and I244 contributed more to the interface between the pore helix and the S5 than the S6 segment, whereas residues W242 and L249 appeared to form an interface with S6 exclusively. Notably, residues L243 and I246 failed to contribute to the interface formed by the pore helix with the S5 and S6 transmembrane segments, in accordance with these residues being oriented toward the channel selectivity filter. A similar procedure applied to the interface formed by the pore helix and the S6 transmembrane segment (C and D) revealed important contributions coming from T278, C277, V275, G274, V272, G271, T270, and L268, with the V275 and L268 contribution coming essentially from intersubunit interactions. It is concluded that the interface between the pore helix and the S5 plus S6 segments of each subunit essentially involves F248 at the C-terminal end of the pore helix.
	Figure 3. Detailed representations of the KCa3.1 channel pore helix region (gray) derived either from the 3BEH (bacterial nucleotide–activated potassium channel) or 2A79 (Kv1.2) templates. A and B illustrate that the interface between F248 and the S5 transmembrane helix is template dependent, with F248 predicted to be interacting with W216 and T212 of S5 for the 3BEH-derived model but with L211 for the model obtained using the Kv1.2 structure (2A79) as template. C and D show in contrast a structural organization of the interface between F248 and the S6 transmembrane segment in which F248 is in both cases within proximity of the Gly hinge at 274 on S6 added to potential contacts with C277, M273, and T270. Illustrations by Discovery Studio Visualizer (Accelrys).
	Figure 4. Electrostatic and van der Waals interaction energy between F248 and residues L268 to L280 of S6 (A and B) or residues G210 to T218 of S5 (C and D). Energy calculations were based on 64-ns MD trajectories obtained for the KCa3.1 model derived either from the 3BEH (nucleotide-activated) or 2A79 (Kv1.2) templates. The bar graphs in A and B indicate that F248 of the pore helix strongly interacts with the Gly hinge at 274 for both models and to a lesser extent with T270, M273, C277, and T278. The interaction energy pattern remained overall template independent. In contrast, strong interaction energies were estimated between F248 and W216 for the KCa3.1 model derived from the 3BEH template, whereas the maximum interaction energy for the KCa3.1 2A79-derived model involves residues F248 and L211. This analysis thus indicates that the interaction energy pattern between F248 and residues in S5 varies according to the template used.
	Figure 5. Alanine scan of the KCa3.1 pore helix region. (A) Inside-out current recordings obtained in symmetrical 200-mM K2SO4 conditions at a pipette potential of 100 mV (T240A, I244A, and F248A) or 150 mV (T250A) from KCa3.1 mutants expressed in Xenopus oocytes. The label “c” refers to zero current level. The internal Ca2+ concentration was 25 µM throughout. The mutation F248A caused a highly significant increase of the channel open probability at saturating internal Ca2+ concentration (Pomax) relative to wild type (P < 0.0005) compared with T240A (P < 0.01). The substitution I244A caused a nonsignificant variation in Pomax relative to wild type. Because of its strategic position at the N-terminal end of the selectivity filter, the mutation of T250 to Ala resulted in an important decrease in channel conductance (9 pS compared with 30 pS), with Pomax being not significantly different from wild type. These results are summarized in B with Pomax values of 0.42 ± 0.08 (n = 4), 0.10 ± 0.04, 0.75 ± 0.01 (n = 3), and 0.16 ± 0.01 (n = 2) for the T240A, I244A, F248A, and T250A channels, respectively. Our results point toward the interactions involving F248 with the S5 and S6 helices as being determinant in setting Pomax. Also shown are all-point histograms computed from the entire recording.
	Figure 6. Examples of inside-out current recordings (A) and calculated Pomax (B) for KCa3.1 channels after the substitution of F248 by residues differing in volume size and/or hydrophobicity. Recordings were performed in symmetrical 200-mM K2SO4 conditions in saturating internal Ca2+ (25 µM) at a pipette potential of 100 mV. Low Pomax values compared with wild type were obtained with the F248T mutant only with a mean value of 0.19 ± 0.05 (n = 3). The substitution of F248 by either a smaller (F248A) or larger (F248W) residue led systematically to a Pomax increase. Also shown is an example of inside-out current trace obtained in conditions where F248 was replaced by the unnatural amino acid pMpa, a tyrosine analogue obtained by substituting the hydroxyl moiety of Tyr by an O-CH3 group. This substitution led to a drastic increase in Pomax with a mean value of 0.94 ± 0.03 (n = 4), compared with 0.22 ± 0.07 (n = 8) for wild type. (B) Bar graph summarizing the effect of substituting F248 by nonpolar residues differing in volume size and/or hydrophobicity. The resulting Pomax ranking reads: T ≈ F < H < Y < S < A < V < L ≈ W < pMpa, confirming the absence of correlation between Pomax and the volume size and/or hydrophobicity (A < S < T < H < V < Y < L < F < W) (Wimley et al., 1996) of the substituting residue. Also shown are all-point histograms computed from the entire recording. Bars, 2 pA and 0.5 s.
	Figure 7. (A) Effect on Pomax of substituting F248 by either nonaromatic or aromatic residues. Scatter plot of Pomax as a function of the residue volume. This analysis reveals two distinct sets of data. Whereas there was a modest but significant increase in Pomax as a function of volume size for Ala (A), Val (V), and Leu (L), drastic changes were observed with the aromatic (Phe [F], Tyr [Y], His [H], Trp [W], and pMpa) residues. Importantly, aromatic residues such as Phe (F) and Tyr (Y) showed significant lower Pomax values compared with nonaromatic amino acids of similar sizes, such as Val (V) or Leu (L), suggesting specific effects related to the presence of an aromatic residue at 248. (B and C) Ln(Keq_mutant/Keq_wild_type), where Keq = Pomax/(1 − Pomax) plotted as a function of the energy perturbation ΔΔEoc coming from mutating residue 248 for the F248T, F248V, F248A, and F248W mutants. ΔΔEoc was calculated as ΔEoc_mutant − ΔEoc_wild_type, where ΔEoc is the difference in nonbonded energy for residue 248 between the open and closed state. Nonbonded energies were computed from >32–64-ns MD trajectories by averaging the van der Waals plus electrostatic interactions between the residue at 248 and the surrounding atoms within a 10-Å radius (excluding lipids and water) (B), or between the residue at 248 and the S5 and S6 helices (C). The resulting analysis is in agreement with the Pomax ranking F248T ≈ F248 < F248A ≈ F248V < F248W observed experimentally, while establishing an energy equivalence (ΔΔEoc ≈ 0) between a Thr (T) and a Phe (F) at 248. The latter result argues for π–π interactions involving F248 and the S5 transmembrane segment in the closed state as being determinant in setting Pomax for the KCa3.1 wild-type channel. These simulations also predict an increase in Pomax relative to wild type with the F248W mutant despite the possibility of π–π interactions with S5, an effect attributable to the mutation F248W stabilizing to a greater extent the channel open than closed configurations. In contrast, the Pomax increase seen with the F248A and F248V mutants appeared to be related to a stronger destabilization for the channel closed than open state.
	Figure 8. Evidence indicating potential aromatic–aromatic interactions involving residue 248 of the pore helix and W216 of S5. (A) Inside-out current recordings demonstrating that the absence of aromatic–aromatic interactions resulting from the substitution of W216 by the nonaromatic Ala or Leu residue caused a strong increase in channel activity, with Pomax values of 0.68 ± 0.04 (n = 3) and 0.95 ± 0.01 (n = 3), respectively, compared with 0.22 ± 0.07 (n = 8) for the wild-type channel (W216). However, such behavior was not observed with the L215A mutant nor the T212A (not depicted). Substituting W216 by the smaller Phe aromatic residue (W216F) resulted in a single-channel fluctuation pattern characterized by a period of high activity (Pomax = 0.70 ± 0.2; n = 3), ending within <3 min in 50% of the recordings (n = 10) by a transition to stable closed state. Such behavior is compatible with strong aromatic–aromatic F248–W216F interactions maintaining the channel in the closed configuration. These results are summarized in the bar graph presented in C. These observations agree with a model whereby aromatic–aromatic interactions between F248 of the pore helix and W216 of S5 tend to stabilize the channel closed configuration, as suggested by the 3BEH-based model illustrated in B. Recordings were performed in symmetrical 200-mM K2SO4 conditions in saturating internal Ca2+ (25 µM) at a pipette potential of 100 mV. All-point histograms were computed from the entire recording. Illustration by Discovery Studio Visualizer (Accelrys).
	Figure 9. Effects of mutating residue L211 in S5 on KCa3.1 Pomax. (A) Inside-out current recordings demonstrating that increasing the volume size of the substituting residue at position 211 in S5 has no significant impact on Pomax relative to wild type. A significant increase was, however, observed with the L211F, suggesting possible aromatic–aromatic interactions between L211F and F248. The results of this analysis are presented in the scatter plot illustrated in B, which shows a Pearson correlation coefficient of 0.75 ± 0.15 between the volume size of the residue engineered at 211 and Pomax. (C) A better correlation was obtained between Pomax and the solvation energy of the residue at 211 with a Pearson correlation coefficient of 0.84 ± 0.07. This analysis confirms that L211 can come in close proximity of F248 during gating, as revealed by the Pomax increase seen with the L211F mutant. Recordings were performed in symmetrical 200-mM K2SO4 conditions in saturating internal Ca2+ (25 µM) at a pipette potential of 100 mV. All-point histograms were computed from the entire recording.
	Figure 10. Mutant cycle analyses demonstrating functional coupling between F248W of the pore helix and C277W or M273W of the S6 transmembrane segment. Energies are expressed in kilocalorie/mole. (A) Example of inside-out current recordings illustrating the effect of the M273W mutation on the change in Pomax resulting from the substitution at 248 of a Phe by a Trp. These observations are summarized in the cycle diagram illustrated in B, which shows that the mutation M273W resulted in a drastic Pomax increase (0.95 ± 0.005; n = 3), an effect that was partly impaired with a Trp at 248 (Pomax of 0.63 ± 0.05; n = 4). (C) Examples of inside-out current recordings illustrating the coupling between Trp at 248 and 277, respectively. The cycle diagram in D indicates in this regard that the mutation C277W totally prevented the Pomax increase normally resulting from the substitution F248W, while being ineffective in modifying Pomax with F248. These observations would be compatible with Trp–Trp interactions between F248W at the pore helix and C277W on S6 stabilizing the closed state (Fig. 11). On the basis of this analysis, it is concluded that both C277 and M273 are in proximity of F248. Recordings were performed in symmetrical 200-mM K2SO4 conditions in saturating internal Ca2+ (25 µM) at a pipette potential of 100 mV. All-point histograms were computed from the entire recording.
	Figure 11. Structure representations of single and double mutants supporting close proximity between C277 and M273 of S6 and F248 of the pore helix. Representation of the channel in the closed state (3BEH template). (A) The relative orientation of the Thr (W) for the F248W–C277W mutant suggests possible π–π interactions that would stabilize the pore helix in the closed configuration, thus accounting for the mutation C277W preventing the Pomax increase coming from the F248W mutation. (B) Also illustrated is the tight packing coming from the mutation M273W, which confirms the proximity of F248 and M273, as demonstrated through the double mutant cycle analysis in Fig. 10. Illustrations by Discovery Studio Visualizer (Accelrys).

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