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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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V457E mutation abolishes con action. (A) Bar shows BD of Construct 8. Sequence alignment of the region from helix αA through strand β2 is shown for point mutants, along with their respective mean g(30 mM cGMP)/g(3 mM cGMP) ratios. Dots in alignment indicate residues conserved between fCNGA2 and rCNGA4. Amino acids mutated in each construct are circled. (B) Dose–response relation of X-fA2 V457E activated with cGMP (down-triangles; n ≥ 3 for all points). Conductances were converted to Po using the methods of Fig. 3, fixing Po = 0.359 at 3 mM cGMP. Black curve is a Hill equation fit. Fitted parameters (±SE) are: K1/2 = 350 ± 97 µM, h = 0.78 ± 0.17, and Pmax = 0.45 ± 0.03. For comparison, gray curve shows cGMP dose–response data for X-fA2 (Young et al., 2001) as in Fig. 4 B.
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fig5: V457E mutation abolishes con action. (A) Bar shows BD of Construct 8. Sequence alignment of the region from helix αA through strand β2 is shown for point mutants, along with their respective mean g(30 mM cGMP)/g(3 mM cGMP) ratios. Dots in alignment indicate residues conserved between fCNGA2 and rCNGA4. Amino acids mutated in each construct are circled. (B) Dose–response relation of X-fA2 V457E activated with cGMP (down-triangles; n ≥ 3 for all points). Conductances were converted to Po using the methods of Fig. 3, fixing Po = 0.359 at 3 mM cGMP. Black curve is a Hill equation fit. Fitted parameters (±SE) are: K1/2 = 350 ± 97 µM, h = 0.78 ± 0.17, and Pmax = 0.45 ± 0.03. For comparison, gray curve shows cGMP dose–response data for X-fA2 (Young et al., 2001) as in Fig. 4 B.

Mentions: For selected constructs, the Po for the 3-mM cGMP normalization condition (after run-up) was estimated as the conductance ratio g(3 mM cGMP)/g(30 mM cAMP+Ni), where the denominator is the conductance in the presence of 30 mM cAMP and 10 µM Ni2+ ion (Varnum et al., 1995; Young et al., 2001). Ratio values recorded for constructs in this study were as follows: Construct 2, 0.180 ± 0.050 (n = 3); Construct 3, 0.73 ± 0.15 (n = 3); Construct 8, 0.337 ± 0.092 (n = 4); X-fA2 V457E, 0.359 ± 0.079 (n = 4). This method assumes Po ≈ 1 in the nickel-potentiated fully liganded channel and that unitary open-channel conductance is unaffected by either cyclic nucleotide or Ni2+. These two assumptions were validated for previously studied X-chimeras (Young et al., 2001); all X-chimera sequences are identical in the transmembrane domain (including the pore region determining unitary conductance; Goulding et al., 1993) and the C-linker (including the Ni2+-binding histidine residue; Gordon and Zagotta, 1995). Dose–response relations in Figs. 4 and 5 were fitted with the Hill equation, Po = Pmax/(1 + K1/2/[cNMP])h.


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)

V457E mutation abolishes con action. (A) Bar shows BD of Construct 8. Sequence alignment of the region from helix αA through strand β2 is shown for point mutants, along with their respective mean g(30 mM cGMP)/g(3 mM cGMP) ratios. Dots in alignment indicate residues conserved between fCNGA2 and rCNGA4. Amino acids mutated in each construct are circled. (B) Dose–response relation of X-fA2 V457E activated with cGMP (down-triangles; n ≥ 3 for all points). Conductances were converted to Po using the methods of Fig. 3, fixing Po = 0.359 at 3 mM cGMP. Black curve is a Hill equation fit. Fitted parameters (±SE) are: K1/2 = 350 ± 97 µM, h = 0.78 ± 0.17, and Pmax = 0.45 ± 0.03. For comparison, gray curve shows cGMP dose–response data for X-fA2 (Young et al., 2001) as in Fig. 4 B.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig5: V457E mutation abolishes con action. (A) Bar shows BD of Construct 8. Sequence alignment of the region from helix αA through strand β2 is shown for point mutants, along with their respective mean g(30 mM cGMP)/g(3 mM cGMP) ratios. Dots in alignment indicate residues conserved between fCNGA2 and rCNGA4. Amino acids mutated in each construct are circled. (B) Dose–response relation of X-fA2 V457E activated with cGMP (down-triangles; n ≥ 3 for all points). Conductances were converted to Po using the methods of Fig. 3, fixing Po = 0.359 at 3 mM cGMP. Black curve is a Hill equation fit. Fitted parameters (±SE) are: K1/2 = 350 ± 97 µM, h = 0.78 ± 0.17, and Pmax = 0.45 ± 0.03. For comparison, gray curve shows cGMP dose–response data for X-fA2 (Young et al., 2001) as in Fig. 4 B.
Mentions: For selected constructs, the Po for the 3-mM cGMP normalization condition (after run-up) was estimated as the conductance ratio g(3 mM cGMP)/g(30 mM cAMP+Ni), where the denominator is the conductance in the presence of 30 mM cAMP and 10 µM Ni2+ ion (Varnum et al., 1995; Young et al., 2001). Ratio values recorded for constructs in this study were as follows: Construct 2, 0.180 ± 0.050 (n = 3); Construct 3, 0.73 ± 0.15 (n = 3); Construct 8, 0.337 ± 0.092 (n = 4); X-fA2 V457E, 0.359 ± 0.079 (n = 4). This method assumes Po ≈ 1 in the nickel-potentiated fully liganded channel and that unitary open-channel conductance is unaffected by either cyclic nucleotide or Ni2+. These two assumptions were validated for previously studied X-chimeras (Young et al., 2001); all X-chimera sequences are identical in the transmembrane domain (including the pore region determining unitary conductance; Goulding et al., 1993) and the C-linker (including the Ni2+-binding histidine residue; Gordon and Zagotta, 1995). Dose–response relations in Figs. 4 and 5 were fitted with the Hill equation, Po = Pmax/(1 + K1/2/[cNMP])h.

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