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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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PB cassette substitution masks con action by raising gating efficacy: refutation of necessity hypothesis. (A) Dose–response relations of Construct 3 activated with cGMP (down-triangles) or cAMP (up-triangles). Points show means and error bars show SD (n ≥ 3 for each point); points are joined by straight lines without fitting. To plot Po values, conductances were normalized to the steady-state conductance measured at 3 mM cGMP (solid down-triangle) in the same patch. Then, Po at 3 mM cGMP was fixed as 0.73 based on Ni2+ potentiation (see Materials and methods). For comparison, gray curves show dose–response relations for Construct 2 activated by cGMP (solid) or cAMP (dashed); these were previously reported on a relative scale without conversion to Po values (Chan and Young, 2009), and new Ni2+ potentiation experiments in this study fix the Po at 3 mM cGMP as 0.18. All measures for both constructs in this plot were collected after completion of spontaneous run-up. (B) Example macroscopic current trace from one patch of Construct 3 before and after completion of run-up, tracking progressive increase in pro-action efficacy and masking of con action. Po scale at right is based on fixing Po = 0.73 for 3 mM cGMP after run-up. Arrows and asterisks, respectively, mark steady-state and spike currents as in Fig. 1. For this example, Psteady/Pspike = 0.80 before run-up and 0.95 after run-up.
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fig3: PB cassette substitution masks con action by raising gating efficacy: refutation of necessity hypothesis. (A) Dose–response relations of Construct 3 activated with cGMP (down-triangles) or cAMP (up-triangles). Points show means and error bars show SD (n ≥ 3 for each point); points are joined by straight lines without fitting. To plot Po values, conductances were normalized to the steady-state conductance measured at 3 mM cGMP (solid down-triangle) in the same patch. Then, Po at 3 mM cGMP was fixed as 0.73 based on Ni2+ potentiation (see Materials and methods). For comparison, gray curves show dose–response relations for Construct 2 activated by cGMP (solid) or cAMP (dashed); these were previously reported on a relative scale without conversion to Po values (Chan and Young, 2009), and new Ni2+ potentiation experiments in this study fix the Po at 3 mM cGMP as 0.18. All measures for both constructs in this plot were collected after completion of spontaneous run-up. (B) Example macroscopic current trace from one patch of Construct 3 before and after completion of run-up, tracking progressive increase in pro-action efficacy and masking of con action. Po scale at right is based on fixing Po = 0.73 for 3 mM cGMP after run-up. Arrows and asterisks, respectively, mark steady-state and spike currents as in Fig. 1. For this example, Psteady/Pspike = 0.80 before run-up and 0.95 after run-up.

Mentions: Patches were held at −40 mV, and steady-state currents in the presence of cyclic nucleotide were corrected by subtraction of leak currents in the absence of cyclic nucleotide. Spontaneous activity changes (run-up and run-down) of CNG channels in excised patches were typically complete after 10–20 min, somewhat longer than previously noted for bCNGA1 (Molokanova et al., 1997). With the exception of specific experiments examining this run-up (Fig. 3 B), all reported measurements were collected after run-up was completed, as shown by a difference of <10% among multiple conductance measurements in 3 mM cGMP. Steady-state conductance in 3 mM cGMP after completion of run-up was used in each patch to normalize conductances in other conditions, including those before the completion of run-up (Fig. 3 B). Unless otherwise noted, means are reported ± SD with n the sample size, and conductance ratios were compared with unity using t test.


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)

PB cassette substitution masks con action by raising gating efficacy: refutation of necessity hypothesis. (A) Dose–response relations of Construct 3 activated with cGMP (down-triangles) or cAMP (up-triangles). Points show means and error bars show SD (n ≥ 3 for each point); points are joined by straight lines without fitting. To plot Po values, conductances were normalized to the steady-state conductance measured at 3 mM cGMP (solid down-triangle) in the same patch. Then, Po at 3 mM cGMP was fixed as 0.73 based on Ni2+ potentiation (see Materials and methods). For comparison, gray curves show dose–response relations for Construct 2 activated by cGMP (solid) or cAMP (dashed); these were previously reported on a relative scale without conversion to Po values (Chan and Young, 2009), and new Ni2+ potentiation experiments in this study fix the Po at 3 mM cGMP as 0.18. All measures for both constructs in this plot were collected after completion of spontaneous run-up. (B) Example macroscopic current trace from one patch of Construct 3 before and after completion of run-up, tracking progressive increase in pro-action efficacy and masking of con action. Po scale at right is based on fixing Po = 0.73 for 3 mM cGMP after run-up. Arrows and asterisks, respectively, mark steady-state and spike currents as in Fig. 1. For this example, Psteady/Pspike = 0.80 before run-up and 0.95 after run-up.
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Related In: Results  -  Collection

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fig3: PB cassette substitution masks con action by raising gating efficacy: refutation of necessity hypothesis. (A) Dose–response relations of Construct 3 activated with cGMP (down-triangles) or cAMP (up-triangles). Points show means and error bars show SD (n ≥ 3 for each point); points are joined by straight lines without fitting. To plot Po values, conductances were normalized to the steady-state conductance measured at 3 mM cGMP (solid down-triangle) in the same patch. Then, Po at 3 mM cGMP was fixed as 0.73 based on Ni2+ potentiation (see Materials and methods). For comparison, gray curves show dose–response relations for Construct 2 activated by cGMP (solid) or cAMP (dashed); these were previously reported on a relative scale without conversion to Po values (Chan and Young, 2009), and new Ni2+ potentiation experiments in this study fix the Po at 3 mM cGMP as 0.18. All measures for both constructs in this plot were collected after completion of spontaneous run-up. (B) Example macroscopic current trace from one patch of Construct 3 before and after completion of run-up, tracking progressive increase in pro-action efficacy and masking of con action. Po scale at right is based on fixing Po = 0.73 for 3 mM cGMP after run-up. Arrows and asterisks, respectively, mark steady-state and spike currents as in Fig. 1. For this example, Psteady/Pspike = 0.80 before run-up and 0.95 after run-up.
Mentions: Patches were held at −40 mV, and steady-state currents in the presence of cyclic nucleotide were corrected by subtraction of leak currents in the absence of cyclic nucleotide. Spontaneous activity changes (run-up and run-down) of CNG channels in excised patches were typically complete after 10–20 min, somewhat longer than previously noted for bCNGA1 (Molokanova et al., 1997). With the exception of specific experiments examining this run-up (Fig. 3 B), all reported measurements were collected after run-up was completed, as shown by a difference of <10% among multiple conductance measurements in 3 mM cGMP. Steady-state conductance in 3 mM cGMP after completion of run-up was used in each patch to normalize conductances in other conditions, including those before the completion of run-up (Fig. 3 B). Unless otherwise noted, means are reported ± SD with n the sample size, and conductance ratios were compared with unity using t test.

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