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Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (beta) subunit of the olfactory cyclic nucleotide-gated channel.

Young EC, Sciubba DM, Siegelbaum SA - J. Gen. Physiol. (2001)

Bottom Line: Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)).In particular, only channels containing the roll subdomain of the beta subunit had high efficacy.Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurobiology and Behavior, Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA. ecy4@columbia.edu

ABSTRACT
CNG channels in vivo are heteromers of homologous alpha and beta subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric alphabeta channels activate with greater sensitivity to ligand than do homomeric alpha channels; however, ligand-gating of channels containing only beta subunit BDs has never been studied because beta subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the beta subunit BD to ligand-gating, we constructed a chimeric subunit, X-beta, whose BD sequence was that of the beta subunit CNG5 from rat, but whose sequence outside the BD was derived from alpha subunits. For comparison, we constructed another chimera, X-alpha, whose sequence outside the BD was identical to that of X-beta, but whose BD sequence was that of the alpha subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-beta and X-alpha each formed functional homomeric channels activated by both cAMP and cGMP. This is the first demonstration that the beta subunit BD can couple ligand binding to activation in the absence of alpha subunit BD residues. Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (beta-roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-beta and X-alpha, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the beta subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.

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Analysis of cAMP activation data for chimeras using a thermodynamic linkage cycle. (A) Efficacies of cAMP activation for all single-channel patches in this study are plotted using the same axes as in Fig. 6 A; each solid point plots data obtained from an individual patch from a distinct oocyte. (B) BDs of four chimeras arranged at the corners of the linkage cycle. Wide arrows represent replacements of fCNG2 C-helix by rCNG5 C-helix; dashed arrows represent replacements of fCNG2 roll subdomain by rCNG5 subdomain. For each replacement, ΔΔGsat,cAMP (defined as the change in ΔGsat,cAMP that results from performing that replacement) is shown, in units of kJ/mol. The independence hypothesis fails because parallel sides of the cycle have different values of ΔΔGsat,cAMP.
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Figure 8: Analysis of cAMP activation data for chimeras using a thermodynamic linkage cycle. (A) Efficacies of cAMP activation for all single-channel patches in this study are plotted using the same axes as in Fig. 6 A; each solid point plots data obtained from an individual patch from a distinct oocyte. (B) BDs of four chimeras arranged at the corners of the linkage cycle. Wide arrows represent replacements of fCNG2 C-helix by rCNG5 C-helix; dashed arrows represent replacements of fCNG2 roll subdomain by rCNG5 subdomain. For each replacement, ΔΔGsat,cAMP (defined as the change in ΔGsat,cAMP that results from performing that replacement) is shown, in units of kJ/mol. The independence hypothesis fails because parallel sides of the cycle have different values of ΔΔGsat,cAMP.

Mentions: The above results show that differences in activation properties of X-α and X-β are derived from elements in both the C-helix and roll subdomains. Do these two regions act independently or do they interact during activation of the channel? If these subdomains act independently of one another, then their energetic contributions to channel activation will be invariant when quantified separately and will be strictly additive. This independence hypothesis can be tested by thermodynamic linkage analysis of the four chimeras we have studied here (Fig. 8; see materials and methods), which form corners of a linkage cycle (Wyman 1964; Weber 1975); the chimeras represent all combinations of the roll and C-helix subdomains from fCNG2 and rCNG5. Thus, if the C-helix acts independently of the roll subdomain, we would predict that the effects on activation of exchanging the α subunit C-helix for the β subunit C-helix should be the same whether we measure the difference in activation between X-α and X-αR/βC or whether we measure the difference in activation between X-βR/αC and X-β (see Fig. 8 B, double arrows). (The same principles have been applied to point mutations in “double mutant cycle” analysis [Carter et al. 1984; Scott and Tanaka 1998].)


Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (beta) subunit of the olfactory cyclic nucleotide-gated channel.

Young EC, Sciubba DM, Siegelbaum SA - J. Gen. Physiol. (2001)

Analysis of cAMP activation data for chimeras using a thermodynamic linkage cycle. (A) Efficacies of cAMP activation for all single-channel patches in this study are plotted using the same axes as in Fig. 6 A; each solid point plots data obtained from an individual patch from a distinct oocyte. (B) BDs of four chimeras arranged at the corners of the linkage cycle. Wide arrows represent replacements of fCNG2 C-helix by rCNG5 C-helix; dashed arrows represent replacements of fCNG2 roll subdomain by rCNG5 subdomain. For each replacement, ΔΔGsat,cAMP (defined as the change in ΔGsat,cAMP that results from performing that replacement) is shown, in units of kJ/mol. The independence hypothesis fails because parallel sides of the cycle have different values of ΔΔGsat,cAMP.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233835&req=5

Figure 8: Analysis of cAMP activation data for chimeras using a thermodynamic linkage cycle. (A) Efficacies of cAMP activation for all single-channel patches in this study are plotted using the same axes as in Fig. 6 A; each solid point plots data obtained from an individual patch from a distinct oocyte. (B) BDs of four chimeras arranged at the corners of the linkage cycle. Wide arrows represent replacements of fCNG2 C-helix by rCNG5 C-helix; dashed arrows represent replacements of fCNG2 roll subdomain by rCNG5 subdomain. For each replacement, ΔΔGsat,cAMP (defined as the change in ΔGsat,cAMP that results from performing that replacement) is shown, in units of kJ/mol. The independence hypothesis fails because parallel sides of the cycle have different values of ΔΔGsat,cAMP.
Mentions: The above results show that differences in activation properties of X-α and X-β are derived from elements in both the C-helix and roll subdomains. Do these two regions act independently or do they interact during activation of the channel? If these subdomains act independently of one another, then their energetic contributions to channel activation will be invariant when quantified separately and will be strictly additive. This independence hypothesis can be tested by thermodynamic linkage analysis of the four chimeras we have studied here (Fig. 8; see materials and methods), which form corners of a linkage cycle (Wyman 1964; Weber 1975); the chimeras represent all combinations of the roll and C-helix subdomains from fCNG2 and rCNG5. Thus, if the C-helix acts independently of the roll subdomain, we would predict that the effects on activation of exchanging the α subunit C-helix for the β subunit C-helix should be the same whether we measure the difference in activation between X-α and X-αR/βC or whether we measure the difference in activation between X-βR/αC and X-β (see Fig. 8 B, double arrows). (The same principles have been applied to point mutations in “double mutant cycle” analysis [Carter et al. 1984; Scott and Tanaka 1998].)

Bottom Line: Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)).In particular, only channels containing the roll subdomain of the beta subunit had high efficacy.Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurobiology and Behavior, Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA. ecy4@columbia.edu

ABSTRACT
CNG channels in vivo are heteromers of homologous alpha and beta subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric alphabeta channels activate with greater sensitivity to ligand than do homomeric alpha channels; however, ligand-gating of channels containing only beta subunit BDs has never been studied because beta subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the beta subunit BD to ligand-gating, we constructed a chimeric subunit, X-beta, whose BD sequence was that of the beta subunit CNG5 from rat, but whose sequence outside the BD was derived from alpha subunits. For comparison, we constructed another chimera, X-alpha, whose sequence outside the BD was identical to that of X-beta, but whose BD sequence was that of the alpha subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-beta and X-alpha each formed functional homomeric channels activated by both cAMP and cGMP. This is the first demonstration that the beta subunit BD can couple ligand binding to activation in the absence of alpha subunit BD residues. Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (beta-roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-beta and X-alpha, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the beta subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.

Show MeSH
Related in: MedlinePlus