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Ligand-binding domain subregions contributing to bimodal agonism in cyclic nucleotide-gated channels.

Wong WF, Chan KS, Michaleski MS, Haesler A, Young EC - J. Gen. Physiol. (2011)

Bottom Line: However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM.To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action.Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

ABSTRACT
Cyclic nucleotide-gated (CNG) channels bind cGMP or cAMP in a cytoplasmic ligand-binding domain (BD), and this binding typically increases channel open probability (P(o)) without inducing desensitization. However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM. We sought to clarify if low pro-action efficacy was either necessary or sufficient for con action to operate. To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action. Constructs were expressed in frog oocytes and tested by patch clamp of cell-free membranes. For nearly all BD elements, we found at least one construct where replacing that element preserved robust con action, with a ratio of steady-state conductances, g((10 mM cGMP))/g((3 mM cGMP)) < 0.75. When all of the BD sequence C terminal of strand β6 was replaced, g((10 mM cGMP))/g((3 mM cGMP)) was increased to 0.95 ± 0.05 (n = 7). However, this apparent attenuation of con action could be explained by an increase in the efficacy of pro action for all agonists, controlled by a conserved "phosphate-binding cassette" motif that contacts ligand; this produces high P(o) values that are less sensitive to shifts in gating equilibrium. In contrast, substituting a single valine in the N-terminal helix αA abolished con action (g((30 mM cGMP))/g((3 mM cGMP)) increased to 1.26 ± 0.24; n = 7) without large increases in pro-action efficacy. Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.

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Neighbor network around position 457 proposed by comparative modeling. All residues in this structural schematic are numbered according to fCNGA2 sequence position. Glutamate is shown in position 457, with black dashed lines indicating its interactions with first neighbors (see Discussion). Parentheses indicate residues that are not identified as first neighbors when valine occupies position 457. Gray dashed lines indicate interactions between first neighbors (black type) and second neighbors (gray type). E458 is capable of forming hydrogen bonds or polar contact with both E457 and K462 (thick dashed lines); other neighbor interactions shown are not polar.
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fig6: Neighbor network around position 457 proposed by comparative modeling. All residues in this structural schematic are numbered according to fCNGA2 sequence position. Glutamate is shown in position 457, with black dashed lines indicating its interactions with first neighbors (see Discussion). Parentheses indicate residues that are not identified as first neighbors when valine occupies position 457. Gray dashed lines indicate interactions between first neighbors (black type) and second neighbors (gray type). E458 is capable of forming hydrogen bonds or polar contact with both E457 and K462 (thick dashed lines); other neighbor interactions shown are not polar.

Mentions: Could V457 then make contact with cGMP, forming counterproductive interactions during con action? This would constitute a noncanonical cGMP-binding site that is structurally distinct from known sites, based on analysis of our comparative models as follows. We enumerated “first neighbor” residues located within 5 Å of the residue 457 side chain in our comparative models (Fig. 6, black dashed lines), and then identified “second neighbor” residues within 5 Å of each of the first neighbors’ side chains (Fig. 6, gray dashed lines). The residues of the “neighbor network” are limited to three noncontiguous helices: helix αA itself, plus helix αD′ of the C-linker, and helix αB at the C-terminal end of the BD (second neighbors only). All network residues other than V457 were conserved in all constructs of this study. Based on corresponding residues in the HCN2 template structure, none of the network residues are proximal to ligand bound at the canonical site. Moreover, none of them correspond to residues forming the previously reported noncanonical cAMP-binding site in Escherichia coli catabolite activator protein (Passner and Steitz, 1997), which relies on the β4–β5 loop along with the DNA BD that has no counterpart in CNG channels.


Ligand-binding domain subregions contributing to bimodal agonism in cyclic nucleotide-gated channels.

Wong WF, Chan KS, Michaleski MS, Haesler A, Young EC - J. Gen. Physiol. (2011)

Neighbor network around position 457 proposed by comparative modeling. All residues in this structural schematic are numbered according to fCNGA2 sequence position. Glutamate is shown in position 457, with black dashed lines indicating its interactions with first neighbors (see Discussion). Parentheses indicate residues that are not identified as first neighbors when valine occupies position 457. Gray dashed lines indicate interactions between first neighbors (black type) and second neighbors (gray type). E458 is capable of forming hydrogen bonds or polar contact with both E457 and K462 (thick dashed lines); other neighbor interactions shown are not polar.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105518&req=5

fig6: Neighbor network around position 457 proposed by comparative modeling. All residues in this structural schematic are numbered according to fCNGA2 sequence position. Glutamate is shown in position 457, with black dashed lines indicating its interactions with first neighbors (see Discussion). Parentheses indicate residues that are not identified as first neighbors when valine occupies position 457. Gray dashed lines indicate interactions between first neighbors (black type) and second neighbors (gray type). E458 is capable of forming hydrogen bonds or polar contact with both E457 and K462 (thick dashed lines); other neighbor interactions shown are not polar.
Mentions: Could V457 then make contact with cGMP, forming counterproductive interactions during con action? This would constitute a noncanonical cGMP-binding site that is structurally distinct from known sites, based on analysis of our comparative models as follows. We enumerated “first neighbor” residues located within 5 Å of the residue 457 side chain in our comparative models (Fig. 6, black dashed lines), and then identified “second neighbor” residues within 5 Å of each of the first neighbors’ side chains (Fig. 6, gray dashed lines). The residues of the “neighbor network” are limited to three noncontiguous helices: helix αA itself, plus helix αD′ of the C-linker, and helix αB at the C-terminal end of the BD (second neighbors only). All network residues other than V457 were conserved in all constructs of this study. Based on corresponding residues in the HCN2 template structure, none of the network residues are proximal to ligand bound at the canonical site. Moreover, none of them correspond to residues forming the previously reported noncanonical cAMP-binding site in Escherichia coli catabolite activator protein (Passner and Steitz, 1997), which relies on the β4–β5 loop along with the DNA BD that has no counterpart in CNG channels.

Bottom Line: However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM.To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action.Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

ABSTRACT
Cyclic nucleotide-gated (CNG) channels bind cGMP or cAMP in a cytoplasmic ligand-binding domain (BD), and this binding typically increases channel open probability (P(o)) without inducing desensitization. However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM. We sought to clarify if low pro-action efficacy was either necessary or sufficient for con action to operate. To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action. Constructs were expressed in frog oocytes and tested by patch clamp of cell-free membranes. For nearly all BD elements, we found at least one construct where replacing that element preserved robust con action, with a ratio of steady-state conductances, g((10 mM cGMP))/g((3 mM cGMP)) < 0.75. When all of the BD sequence C terminal of strand β6 was replaced, g((10 mM cGMP))/g((3 mM cGMP)) was increased to 0.95 ± 0.05 (n = 7). However, this apparent attenuation of con action could be explained by an increase in the efficacy of pro action for all agonists, controlled by a conserved "phosphate-binding cassette" motif that contacts ligand; this produces high P(o) values that are less sensitive to shifts in gating equilibrium. In contrast, substituting a single valine in the N-terminal helix αA abolished con action (g((30 mM cGMP))/g((3 mM cGMP)) increased to 1.26 ± 0.24; n = 7) without large increases in pro-action efficacy. Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.

Show MeSH