Figure 1.
Homology models of the hHCN4 channel.
A: Homology models of the TM1/S5-P-TM2/S6 regions of the hHCN4 channel in the closed (left) and open form (right), respectively. Only two of the four subunits are shown for clarity. Residues lining the internal cavity are shown in stick mode and labeled. M508 is also indicated even if it does not point towards the cavity. B: sequence alignment of the TM1/S5-P-TM2/S6 regions of hHCN4 with the corresponding regions of mHCN1, mHCN2, Streptomyces lividans KcsA, and the mammalian K+ channel Kv1.2. The secondary structure elements, as defined in the crystal structure of KcsA, are indicated. P is pore, SF is selectivity filter. Residues of HCN channels identical and similar to those of KcsA and/or Kv1.2 are highlighted by green and yellow boxes, respectively. Residues that face the internal cavity of the hHCN4 channel in the open and closed forms and residues relevant to mHCN2 block by cilobradine [24] and to mHCN1/mHCN2 block by ZD7288 [2],[24],[25] are indicated in red bold font.
Figure 2.
Ivabradine-induced block of WT and mutant hHCN4 channels.
A,B: time-courses of current amplitudes (left panels) and sample current traces (right panels: a, control; b, steady-state block) recorded at −140 mV before, during, and after superfusion with 30 µM ivabradine (horizontal bars). Horizontal bars in right panels: 150 ms. C: Dose-response relations of ivabradine block of WT and mutant channels. Fitting with the Hill equation (curves) yielded half-block concentrations (IC50) of 2.1, 3.1, 3.6, 57.7, 3.7, 1.9, 44.0, 47.7 µM and Hill coefficients (nH) of 0.74, 0.84, 0.72, 1.02, 0.75, 0.77, 0.73, 0.83 for WT, C478A, A503V, Y506A, A507V, M508A, F509A, and I510A channels, respectively. Dose-response relations for Y506A, F509A, and I510A curves are significantly different from WT (P<0.05).
Figure 3.
Block of double (Y506A-I510A and Y506A-F509A) and triple (Y506A-F509A-I510A) mutant channels by ivabradine.
A, top, sample currents recorded at −140 mV from the two double mutant channels Y506A-I510A and Y506A-F509A in control (a) and after steady-state block by ivabradine 30 µM (b); A, bottom, time course of currents at −140 mV during ivabradine perfusion. B: dose-response relations; Hill fitting (full lines) yielded IC50, nH values of 2213.0 µM, 0.7, 42.5 µM, 0.8, and 1215.0 µM and 0.68 for Y506A-I510A, Y506A-F509A, and Y506A-F509A-I510A respectively. Hill fitting of WT and Y506A block from Fig. 2 also drawn for comparison (dotted lines). The two double-mutant curves are significantly different between themselves and from that of WT channels (P<0.05); the Y506A-I510A and the Y506A-F509A-I510A curves are not significantly different (P<0.05).
Figure 4.
Ivabradine block of WT and F509A channels in the open state.
A, representative traces showing the action of ivabradine (3 and 30 µM) on the current recorded from WT and F509A channels during a long (100 s) step to −100 mV. B, dose-response relations for ivabradine block measured as in A. Hill fitting resulted in IC50, nH values of 120.7 µM, 0.5 and 91.3 µM, 0.5 for WT (filled circles) and F509A channels (open circles), respectively (non-significantly different, P>0.05).
Figure 5.
Detailed stereo view of the ivabradine docking in the hHCN4 WT channel.
Stereo view of the interior of the WT hHCN4 channel in its closed (A) and open (B) form with the best-docked pose of ivabradine shown in magenta. The S5-P-S6 regions of the four hHCN4 subunits are shown as ribbon in green yellow, orange and pale blue, respectively. Side-chains of residues interacting with the ivabradine molecule are shown in stick representation in both panels. For the closed state only, the main-chain atoms of C478 are shown since the carbonyl oxygen atoms may form additional H-bonds with ivabradine. I510 is also shown in the channel closed form, though this residue does not interact directly with ivabradine. Black spheres indicate positions corresponding to K+ ion bound in pore of the KcsA crystal structure [12]. For clarity only residues of one subunit are labeled and L477 of the pale blue subunit is omitted.
Figure 6.
Detailed stereo view of the ivabradine docking to hHCN4 mutant channels.
A,B,C: stereo views of the interior of hHCN4 mutants Y506A (A), I510A (B), and Y506A-I510A (C) in the closed form. D, E: stereo views of the interior of the F509A mutant in the open (D) and closed (E) form. Side-chains of residues relevant to ivabradine binding are shown as ball-and-stick in all panels. In all mutant models the best pose of the docked ivabradine is shown in magenta, while the S5-P-S6 regions of the four hHCN4 subunits are shown as ribbon in grey, yellow, orange and pale blue, respectively. For clarity, residues have been labelled only in one hHCN4 subunit, with mutated residues indicated in bold characters.