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Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels.

Craven KB, Zagotta WN - J. Gen. Physiol. (2004)

Bottom Line: The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels.This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore.Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290, USA.

ABSTRACT
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

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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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fig9: 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.

Mentions: Thus far, we have considered the two negatively charged residues separately, focusing on how each interacts with the positively charged B' helix site within its own salt bridge. But is there coupling between mutations at the D' helix site and mutations at the β roll site? We were not able to perform thermodynamic mutant cycle analysis for these two residues in CNGA1, due to lack of functional expression of the double mutant channel. However, we were able to express this double mutation in the HCN2 channel. Although in HCN2 channels we cannot calculate the energetic effects of these mutants, we can compare the magnitude of the effects of each single negative-to-positive mutation at the D' helix site and the β roll site with the effects of mutating both negative residues to positive residues. The channel with both negative-to-positive mutations at the D' helix site and the β roll site shows a much greater (nonadditive) change from wild type than either of the single negative-to-positive mutant channels (compare Figs. 6, C and D, with Fig. 8 B). In fact, the behavior of the double negative-to-positive mutant channel (E502K+D542K) resembles the dramatic phenotype of the positive-to-negative mutation of the B' helix site (K472E) (Fig. 8). Both of these channels showed many of the same changes from wild-type behavior: a lack of cAMP-induced changes in activation kinetics and shift in voltage dependence, slight inhibition of steady-state currents instead of augmentation, and reduction of GV slope (Fig. 8; Table II). One difference between the behaviors of the two mutants was that E502K+D542K did not show the same shift in control Vhalf that was seen with K472E. Overall, it appears that in addition to an interaction between the residues within a salt bridge, there is also an interaction, exhibited by the nonadditive effect of the double negative-to-positive mutant, between the negatively charged residues in different salt bridges.


Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels.

Craven KB, Zagotta WN - J. Gen. Physiol. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234033&req=5

fig9: 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.
Mentions: Thus far, we have considered the two negatively charged residues separately, focusing on how each interacts with the positively charged B' helix site within its own salt bridge. But is there coupling between mutations at the D' helix site and mutations at the β roll site? We were not able to perform thermodynamic mutant cycle analysis for these two residues in CNGA1, due to lack of functional expression of the double mutant channel. However, we were able to express this double mutation in the HCN2 channel. Although in HCN2 channels we cannot calculate the energetic effects of these mutants, we can compare the magnitude of the effects of each single negative-to-positive mutation at the D' helix site and the β roll site with the effects of mutating both negative residues to positive residues. The channel with both negative-to-positive mutations at the D' helix site and the β roll site shows a much greater (nonadditive) change from wild type than either of the single negative-to-positive mutant channels (compare Figs. 6, C and D, with Fig. 8 B). In fact, the behavior of the double negative-to-positive mutant channel (E502K+D542K) resembles the dramatic phenotype of the positive-to-negative mutation of the B' helix site (K472E) (Fig. 8). Both of these channels showed many of the same changes from wild-type behavior: a lack of cAMP-induced changes in activation kinetics and shift in voltage dependence, slight inhibition of steady-state currents instead of augmentation, and reduction of GV slope (Fig. 8; Table II). One difference between the behaviors of the two mutants was that E502K+D542K did not show the same shift in control Vhalf that was seen with K472E. Overall, it appears that in addition to an interaction between the residues within a salt bridge, there is also an interaction, exhibited by the nonadditive effect of the double negative-to-positive mutant, between the negatively charged residues in different salt bridges.

Bottom Line: The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels.This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore.Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290, USA.

ABSTRACT
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

Show MeSH
Related in: MedlinePlus