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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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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.
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fig1: 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.

Mentions: Recently, a new form of negative agonism has been described for cGMP acting on CNG channels. CNG channels are nonselective cation channels activated by direct binding of cGMP and cAMP; the channels consist of homotetramers or heterotetramers of homologous subunits. Each subunit has a conserved architecture of six transmembrane helices and a cytoplasmic C-terminal region that contains the ligand-binding domain (BD). Cells such as photoreceptors and olfactory sensory neurons express various types of CNG subunits (i.e., paralogues) in different combinations (Bradley et al., 2001; Kaupp and Seifert, 2002; Matulef and Zagotta, 2003). Negative agonism is observed in a particular subtype of CNG channels—the CNGA2 subtype from catfish olfactory neurons (fCNGA2) (Goulding et al., 1992)—and in recombinant CNG channels derived from this subtype (Young et al., 2001; Chan and Young, 2009). Notably, the dose–response relation of activity versus cGMP concentration shows a rising phase for low concentrations and a falling phase for higher concentrations. This contrasts with the normal (monotonically rising) dose–response curve of the positive agonist cAMP. Single-channel recordings have shown (Young et al., 2001) that the falling phase of the cGMP dose–response reflects a decrease in steady-state open probability (Po) induced by a new cGMP molecule binding to a partially liganded channel (see reaction scheme in Fig. 1). That is, it does not involve a reduction of open-channel unitary conductance as typically found with agonist pore block (Ogden and Colquhoun, 1985; Karashima et al., 2007), or a spontaneous gate-closing conformational change as found with traditional desensitization in ligand-gated channels (Cachelin and Colquhoun, 1989; Robert and Howe, 2003) or inactivation in voltage-gated channels (Hoshi et al., 1991). We have termed this unusual phenomenon bimodal agonism because cGMP exhibits two opposite modes of action: “pro action” that enhances receptor activity by a positive agonism mechanism, and “con action” that suppresses activity by a negative agonism mechanism. Our distinct terminology “con action” is used to denote negative agonism in this special context where more than one agonism mode is possible.


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)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: 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.
Mentions: Recently, a new form of negative agonism has been described for cGMP acting on CNG channels. CNG channels are nonselective cation channels activated by direct binding of cGMP and cAMP; the channels consist of homotetramers or heterotetramers of homologous subunits. Each subunit has a conserved architecture of six transmembrane helices and a cytoplasmic C-terminal region that contains the ligand-binding domain (BD). Cells such as photoreceptors and olfactory sensory neurons express various types of CNG subunits (i.e., paralogues) in different combinations (Bradley et al., 2001; Kaupp and Seifert, 2002; Matulef and Zagotta, 2003). Negative agonism is observed in a particular subtype of CNG channels—the CNGA2 subtype from catfish olfactory neurons (fCNGA2) (Goulding et al., 1992)—and in recombinant CNG channels derived from this subtype (Young et al., 2001; Chan and Young, 2009). Notably, the dose–response relation of activity versus cGMP concentration shows a rising phase for low concentrations and a falling phase for higher concentrations. This contrasts with the normal (monotonically rising) dose–response curve of the positive agonist cAMP. Single-channel recordings have shown (Young et al., 2001) that the falling phase of the cGMP dose–response reflects a decrease in steady-state open probability (Po) induced by a new cGMP molecule binding to a partially liganded channel (see reaction scheme in Fig. 1). That is, it does not involve a reduction of open-channel unitary conductance as typically found with agonist pore block (Ogden and Colquhoun, 1985; Karashima et al., 2007), or a spontaneous gate-closing conformational change as found with traditional desensitization in ligand-gated channels (Cachelin and Colquhoun, 1989; Robert and Howe, 2003) or inactivation in voltage-gated channels (Hoshi et al., 1991). We have termed this unusual phenomenon bimodal agonism because cGMP exhibits two opposite modes of action: “pro action” that enhances receptor activity by a positive agonism mechanism, and “con action” that suppresses activity by a negative agonism mechanism. Our distinct terminology “con action” is used to denote negative agonism in this special context where more than one agonism mode is possible.

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
Related in: MedlinePlus