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Figure 1. . Structure of HCN2 C-linker and CNBD. Structure of the HCN2 COOH-terminal region (Zagotta et al., 2003), viewed from the side (left) and from the membrane (right). The structure is positioned below the membrane-spanning portion of the channel, as it is thought to be in vivo. The structure contains four subunits, two in dark gray and two in light gray, with the C-linkers making up the top half of the structure and the CNBDs the bottom half. cAMP (yellow) is bound in the CNBD of each subunit. Residues of the putative salt bridges are shown in CPK format: K472 (blue), E502 and D542 (both in red). Enlargement of the region with these salt bridges (inset). Only two subunits are shown here, with the A'–D' helices and β roll labeled, and the residues are now colored according to their elements (carbon, gray; nitrogen, blue; and oxygen, red). Plus sign within a square is K472, minus sign within a square is D542 from the same subunit, and minus sign within a circle is E502 from the neighboring subunit.
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Figure 2. . Behavior of wild-type HCN2 and CNGA1cys-free channels. (A) Behavior of wild-type HCN2 channels. Currents (left) and conductance–voltage relations (right) are shown in the absence (black) and presence of saturating cAMP (red). The currents were recorded in response to voltage pulses from a holding potential of 0 mV to test potentials between −70 and −150 mV, returning to a tail potential of −40 mV, and the conductance–voltage relations were obtained from normalized tail currents. (B) Behavior of CNGA1cys-free channels. Leak-subtracted currents (left) in response to voltage pulses from a holding potential of 0 mV to test potentials from −100 to +100 mV are shown in the absence (black, not leak subtracted) and presence of saturating cAMP (red), cIMP (blue), and cGMP (green). Box plot (right) of the ΔG for the opening transition with bound cGMP, cIMP, and cAMP. Center line is the median of the data, box is the 25th to 75th percentile of the data, and whiskers are the 5th and 95th percentile of the data. Outliers are shown as symbols.
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Figure 3. . Sequence alignment of COOH-terminal regions from HCN, CNG, and related channels. Sequence alignment of the COOH-terminal region includes all members of the CNG channel family (bovine CNGA1, rat CNGA2, bovine CNGA3, rat CNGA4, bovine CNGB1, murine CNGB3) and HCN channel family (murine HCN2, mouse HCN1, rat HCN3, rat HCN4), as well as SPIH (the sea urchin sperm homologue of HCN), and the human ether-a-go-go related gene (HERG) and Drosophila Eag K+ channels. Above the sequence alignment, the tertiary structure elements are shown: α-helices (red rectangles), β-sheets (blue arrow), and uncoiled regions (line). Residues highlighted in blue are similar. Putative salt bridge residues studied are boxed and labeled.
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Figure 4. . Mutations of intersubunit salt bridge. (A–D) Behavior of wild-type HCN2 (A), K472E (B), E502K (C), and K472E + E502K (D) channels. Currents in response to voltage pulses to −130 mV are shown in the absence (black) and presence of saturating cAMP (red). Diagrams to the left show attractive electrostatic interactions by solid lines and repulsive electrostatic interactions by dotted lines with wild-type residues as open symbols and mutant residues as shaded symbols. (E–H) Behavior of CNGA1cys-free (E), R431E (F), E462R (G), and R431E + E462R (H) channels. For R431E (F), the current values for cIMP are so similar to cGMP that the two traces almost overlay. Current in response to voltage pulses to +100 mV are shown in the presence of saturating cAMP (red), cIMP (blue), and cGMP (green).
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Figure 5. . Mutations of intrasubunit salt bridge. (A–D) Behavior of wild-type HCN2 (A), K472E (B), D542K (C), and K472E + D542K (D) channels. Currents in response to voltage pulses to −130 mV are shown in the absence (black) and presence of saturating cAMP (red). Diagrams to the left show attractive electrostatic interactions by solid lines and repulsive electrostatic interactions by dotted lines with wild-type residues as open symbols and mutant residues as shaded symbols. (E–H) Behavior of CNGA1cys-free (E), R431D (F), D502R (G), and R431D + D502R (H) channels. Current in response to voltage pulses to +100 mV are shown in the presence of saturating cAMP (red), cIMP (blue), and cGMP (green).
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Figure 6. . Conductance–voltage relations for HCN2 channels. (A–F) Conductance versus voltage plots for wild-type HCN2 (A), K472E (B), E502K (C), D542K (D), K472E+E502K (E), and K472E+D542K (F). Each plot indicates GV curves, obtained from normalized tail currents at −40 mV, in the absence (black) and presence of saturating cAMP (red). The data are fit with a Boltzmann relation. Diagrams on the left and the right show attractive electrostatic interactions by solid lines and repulsive electrostatic interactions by a dotted line with wild-type residues as open symbols and mutant residues as shaded symbols.
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Figure 7. . Thermodynamic mutant cycle analysis for CNGA1 channels. (A) Box plot of ΔGcGMP values for CNGA1cys-free and mutant CNGA1 channels. The dashed line indicates the median ΔGcGMP value for CNGA1cys-free. (B and C) Thermodynamic mutant cycles for the intersubunit salt bridge (B) and intrasubunit salt bridge (C). The letters are the amino acid abbreviations for the residues, the number indicates the residue number, and the charge of the residue is also shown. The lines indicate when a salt bridge is possible between residues with opposite charges. ΔΔΔG values were calculated from ΔG values (see materials and methods) and are shown as mean ΔΔΔG ± SEM of ΔΔΔG.
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Figure 8. . Double negative-to-positive HCN2 mutant has same phenotype as B' helix site positive-to-negative mutant. Behavior of K472E (A) and E502K+D542K (B) HCN2 channels. Currents in response to voltage pulses to −130 mV, as well as conductance–voltage relations of the normalized conductance from tail currents at −40 mV, are shown in the absence (black) and presence of saturating cAMP (red). Diagrams at the top of each column show attractive electrostatic interactions by solid lines and repulsive electrostatic interactions by dotted lines with wild-type residues as open symbols and mutant residues as shaded symbols. The GV curves are fit with a Boltzmann relation.
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Figure 9. . Model of modular gating scheme for HCN and CNG channels. (A) HCN and CNG gating model with three modules: pore, C-linker, and CNBD. Each module is represented by a boxed equilibrium, indicating that each module can be in one of two configurations. Pore can be closed (C) or open (O), C-linker can be resting (R) or activated (A), and CNBD can be unbound (U) or bound (B) with ligand. Equilibrium constants are shown for each module: L for pore, M for C-linker, and K for CNBD. The pore and the C-linker influence each other through allosteric factor C, and the C-linker and CNBD through allosteric factor F. (B) Simulated conductance–voltage (GV) relations for wild-type (top) and K472E (bottom) HCN2 channels in the absence (black) and presence of saturating cAMP (red). A voltage-dependent module that was coupled to the pore was added for these calculations. The values of the parameters used are shown in the materials and methods. (C) Simulated current traces in response to voltages for CNGA1cys-free (top) and R431E (bottom) channels in the presence of saturating cAMP (red), cIMP (blue), and cGMP (green). For the R431E simulations, the current values for cIMP are so similar to cGMP that the two traces overlay. Diagrams to the left show attractive electrostatic interactions by solid lines and repulsive electrostatic interactions by dotted lines with wild-type residues as open symbols and mutant residues as shaded symbols.
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Figure 10. . Model for COOH-terminal region in the resting and activating configurations. Cartoons of HCN2 COOH-terminal region from the side view, both in the resting configuration (left) and the active configuration (right). COOH-terminal regions of two subunits (dark and light gray) are shown below the membrane-spanning portion of the channel. α helices are shown with narrow cylinders, and the β rolls of the CNBDs are shown with wide cylinders. Salt bridge residues are shown: the positive B' helix residue (plus sign within a square) and the two negative residues on the D' helix and the β roll (minus signs within a circle or square, respectively).
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(SCHEME 1).
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