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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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Extensive BD sequence substitutions preserve con action. (A) Topology of the CNG channel subunit, highlighting the BD region substituted in the X-chimera series of constructs. Alignment compares the substituted BD sequences (bounded by vertical dotted lines) originating from rCNGA4 (in X-rA4) and fCNGA2 (in X-fA2). Invariant non-BD sequences (Young et al., 2001) include bCNGA1 sequence in the C-linker and the extreme C-terminal region, respectively, before and after the BD. Dots in the sequence alignment indicate fCNGA2 residues conserved with rCNGA4; secondary structure elements are marked (α for helices, β for strands) as predicted by comparative modeling (Fig. 4). (B) X-chimera testing substitutions in the C-terminal portion of the BD. Bar represents BD sequence; below the bars, secondary structure elements are marked (letters for helices, numbers for strands, and PB for the PB cassette). Gray background color in bar represents amino acids conserved between fCNGA2 and rCNGA4; black ticks mark unconserved amino acids found in fCNGA2 but not in rCNGA4. Mean g(10 mM cGMP)/g(3 mM cGMP) values (±SD, number of patches in parentheses) include those previously reported for Construct 1 (Young et al., 2001) and for Construct 2 and X-rA4 (Chan and Young, 2009). (C) Examples of current traces for selected constructs during 10-mM cGMP pulses, with g(10 mM cGMP)/g(3 mM cGMP) as marked (based on applications of 3 mM cGMP; not depicted). Horizontal bar, 10 s; vertical bar, 1 nA.
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fig2: Extensive BD sequence substitutions preserve con action. (A) Topology of the CNG channel subunit, highlighting the BD region substituted in the X-chimera series of constructs. Alignment compares the substituted BD sequences (bounded by vertical dotted lines) originating from rCNGA4 (in X-rA4) and fCNGA2 (in X-fA2). Invariant non-BD sequences (Young et al., 2001) include bCNGA1 sequence in the C-linker and the extreme C-terminal region, respectively, before and after the BD. Dots in the sequence alignment indicate fCNGA2 residues conserved with rCNGA4; secondary structure elements are marked (α for helices, β for strands) as predicted by comparative modeling (Fig. 4). (B) X-chimera testing substitutions in the C-terminal portion of the BD. Bar represents BD sequence; below the bars, secondary structure elements are marked (letters for helices, numbers for strands, and PB for the PB cassette). Gray background color in bar represents amino acids conserved between fCNGA2 and rCNGA4; black ticks mark unconserved amino acids found in fCNGA2 but not in rCNGA4. Mean g(10 mM cGMP)/g(3 mM cGMP) values (±SD, number of patches in parentheses) include those previously reported for Construct 1 (Young et al., 2001) and for Construct 2 and X-rA4 (Chan and Young, 2009). (C) Examples of current traces for selected constructs during 10-mM cGMP pulses, with g(10 mM cGMP)/g(3 mM cGMP) as marked (based on applications of 3 mM cGMP; not depicted). Horizontal bar, 10 s; vertical bar, 1 nA.

Mentions: Simple allosteric reaction scheme for bimodal agonism. Reaction scheme (Chan and Young, 2009) is illustrated by a cell-free inside-out macroscopic patch recording of bimodal CNG channels with cGMP washed into and out from the bath (solid and dashed black bars, respectively). Applying cGMP elicits inward channel current with no desensitization up to 3 mM cGMP, but with 10 mM cGMP, the steady-state macroscopic conductance (white arrow) is smaller than for 3 mM. During wash-in and wash-out of the 10-mM cGMP solution, there are brief intervals with bath cGMP concentration near 3 mM giving rise to “spikes” of maximal conductance. Details of reaction scheme notation: Closed- (C) and open- (O) channel states are equilibrating rapidly compared with agonist wash-in and wash-out. Sizes of black reaction arrows indicate the favored direction of equilibrium. Po increases with cGMP binding of up to m molecules of cGMP (pro action). Binding of the (m+1)th cGMP molecule decreases the steady-state Po (con action) to below the value obtained with m ligands. Experimental details of current trace: The previously characterized channel, X-fA2, incorporates the BD from fCNGA2 channel in a chimera with sequence from other CNG channel types (see Young et al., 2001, and Fig. 2). X-fA2 was expressed as homomers in Xenopus oocytes; patches were held at −40 mV. White arrows mark times where steady-state conductance, g, was measured with cGMP concentrations fixed at their nominal concentrations; for this example, g(10 mM cGMP)/g(3 mM cGMP) = 0.63.


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)

Extensive BD sequence substitutions preserve con action. (A) Topology of the CNG channel subunit, highlighting the BD region substituted in the X-chimera series of constructs. Alignment compares the substituted BD sequences (bounded by vertical dotted lines) originating from rCNGA4 (in X-rA4) and fCNGA2 (in X-fA2). Invariant non-BD sequences (Young et al., 2001) include bCNGA1 sequence in the C-linker and the extreme C-terminal region, respectively, before and after the BD. Dots in the sequence alignment indicate fCNGA2 residues conserved with rCNGA4; secondary structure elements are marked (α for helices, β for strands) as predicted by comparative modeling (Fig. 4). (B) X-chimera testing substitutions in the C-terminal portion of the BD. Bar represents BD sequence; below the bars, secondary structure elements are marked (letters for helices, numbers for strands, and PB for the PB cassette). Gray background color in bar represents amino acids conserved between fCNGA2 and rCNGA4; black ticks mark unconserved amino acids found in fCNGA2 but not in rCNGA4. Mean g(10 mM cGMP)/g(3 mM cGMP) values (±SD, number of patches in parentheses) include those previously reported for Construct 1 (Young et al., 2001) and for Construct 2 and X-rA4 (Chan and Young, 2009). (C) Examples of current traces for selected constructs during 10-mM cGMP pulses, with g(10 mM cGMP)/g(3 mM cGMP) as marked (based on applications of 3 mM cGMP; not depicted). Horizontal bar, 10 s; vertical bar, 1 nA.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig2: Extensive BD sequence substitutions preserve con action. (A) Topology of the CNG channel subunit, highlighting the BD region substituted in the X-chimera series of constructs. Alignment compares the substituted BD sequences (bounded by vertical dotted lines) originating from rCNGA4 (in X-rA4) and fCNGA2 (in X-fA2). Invariant non-BD sequences (Young et al., 2001) include bCNGA1 sequence in the C-linker and the extreme C-terminal region, respectively, before and after the BD. Dots in the sequence alignment indicate fCNGA2 residues conserved with rCNGA4; secondary structure elements are marked (α for helices, β for strands) as predicted by comparative modeling (Fig. 4). (B) X-chimera testing substitutions in the C-terminal portion of the BD. Bar represents BD sequence; below the bars, secondary structure elements are marked (letters for helices, numbers for strands, and PB for the PB cassette). Gray background color in bar represents amino acids conserved between fCNGA2 and rCNGA4; black ticks mark unconserved amino acids found in fCNGA2 but not in rCNGA4. Mean g(10 mM cGMP)/g(3 mM cGMP) values (±SD, number of patches in parentheses) include those previously reported for Construct 1 (Young et al., 2001) and for Construct 2 and X-rA4 (Chan and Young, 2009). (C) Examples of current traces for selected constructs during 10-mM cGMP pulses, with g(10 mM cGMP)/g(3 mM cGMP) as marked (based on applications of 3 mM cGMP; not depicted). Horizontal bar, 10 s; vertical bar, 1 nA.
Mentions: Simple allosteric reaction scheme for bimodal agonism. Reaction scheme (Chan and Young, 2009) is illustrated by a cell-free inside-out macroscopic patch recording of bimodal CNG channels with cGMP washed into and out from the bath (solid and dashed black bars, respectively). Applying cGMP elicits inward channel current with no desensitization up to 3 mM cGMP, but with 10 mM cGMP, the steady-state macroscopic conductance (white arrow) is smaller than for 3 mM. During wash-in and wash-out of the 10-mM cGMP solution, there are brief intervals with bath cGMP concentration near 3 mM giving rise to “spikes” of maximal conductance. Details of reaction scheme notation: Closed- (C) and open- (O) channel states are equilibrating rapidly compared with agonist wash-in and wash-out. Sizes of black reaction arrows indicate the favored direction of equilibrium. Po increases with cGMP binding of up to m molecules of cGMP (pro action). Binding of the (m+1)th cGMP molecule decreases the steady-state Po (con action) to below the value obtained with m ligands. Experimental details of current trace: The previously characterized channel, X-fA2, incorporates the BD from fCNGA2 channel in a chimera with sequence from other CNG channel types (see Young et al., 2001, and Fig. 2). X-fA2 was expressed as homomers in Xenopus oocytes; patches were held at −40 mV. White arrows mark times where steady-state conductance, g, was measured with cGMP concentrations fixed at their nominal concentrations; for this example, g(10 mM cGMP)/g(3 mM cGMP) = 0.63.

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