Sunderman ER and Zagotta WN (1999), Sequence of events underlying the allosteric tr...

XB-ART-13086

J Gen Physiol 1999 May 01;1135:621-40.

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Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels.

Sunderman ER , Zagotta WN .

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Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.

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Genes referenced: camp

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	Figure 2. Current families for wild-type and mutant channels activated by cGMP. Current families are shown from inside-out patches excised from Xenopus laevis oocytes expressing BROD (A) and CHM15 (B) channels with mutations at position 604. Currents were elicited by 16 mM cGMP and voltage pulses from 0 mV to potentials between â80 and +80 mV in 20-mV steps. Leak currents in the absence of cyclic nucleotides were subtracted. The currents were normalized (using the +80-mV trace) to the maximum current obtained after Ni2+ potentiation (Gordon and Zagotta, 1995a) in the presence of a saturating concentration of the best agonist.
	Figure 3. Current families for wild-type and mutant channels activated by cIMP. Current families are shown from inside-out patches excised from Xenopus laevis oocytes expressing BROD (A) and CHM15 (B) channels with mutations at position 604. Currents were elicited by 16 mM cIMP and voltage pulses from 0 mV to potentials between â80 and +80 mV in 20-mV steps. Leak currents in the absence of cyclic nucleotides were subtracted. The currents were normalized (using the +80-mV trace) to the maximum current obtained after Ni2+ potentiation (Gordon and Zagotta, 1995a) in the presence of a saturating concentration of the best agonist.
	Figure 4. Current families for wild-type and mutant channels activated by cAMP. Current families are shown from inside-out patches excised from Xenopus laevis oocytes expressing BROD (A) and CHM15 (B) channels with mutations at position 604. Currents were elicited by 16 mM cAMP and voltage pulses from 0 mV to potentials between â80 and +80 mV in 20-mV steps. Leak currents in the absence of cyclic nucleotides were subtracted. The currents were normalized (using the +80-mV trace) to the maximum current obtained after Ni2+ potentiation (Gordon and Zagotta, 1995a) in the presence of a saturating concentration of the best agonist.
	Figure 5. Thermodynamic mutant cycles identify direct interactions between cyclic nucleotides and D604. (A) The effect of substituting the olfactory amino terminal region for the BROD amino terminal region was cyclic nucleotide independent. (B) The effect of mutating D604 to D604M was strongly cyclic nucleotide dependent, indicating that the amino acid at position 604 is critical for cGMP vs. cAMP discrimination by the channel.
	Figure 6. Representative single-channel traces at a saturating concentration of cGMP. Single-channel currents for all 10 constructs were recorded in the presence of 16 mM cGMP with the membrane voltage clamped at +80 mV. The upper and lower dotted lines indicated the open and closed levels, respectively, and are separated by 2.3 pA.
	Figure 8. Representative single-channel traces at a saturating concentration of cIMP. Single-channel currents for all 10 constructs were recorded in the presence of 16 mM cIMP with the membrane voltage clamped at +80 mV. The upper and lower dotted lines indicated the open and closed levels, respectively, and are separated by 2.3 pA.
	Figure 10. Representative single-channel traces at a saturating concentration of cAMP. Single-channel currents for all 10 constructs were recorded in the presence of 16 mM cAMP with the membrane voltage clamped at +80 mV. The upper and lower dotted lines indicated the open and closed levels, respectively, and are separated by 2.3 pA.
	Figure 7. Amplitude histograms for activation by a saturating concentration of cGMP. Amplitude histograms corresponding to the representative traces in Fig. 6 are shown. The histograms were fit to the sum of two Gaussians, and the peak of the closed level Gaussian was used to subtract off leak currents. The parameters for the amplitude histograms were as follows: for BROD, Ïclosed = 480 fA, Ïopen = 560 fA, Popen = 0.95; for CHM15, Ïclosed = 1.6 pA, Ïopen = 340 fA, Popen = 0.99; for BROD-D604E, Ïclosed = 490 fA, Ïopen = 430 fA, Popen = 0.71; for CHM15-D604E, Ïclosed = 850 fA, Ïopen = 240 fA, Popen = 0.97; for BROD-D604Q, Ïclosed = 380 fA, Ïopen = 890 fA, Popen = 0.06; for CHM15-D604Q, Ïclosed = 240 fA, Ïopen = 270 fA, Popen = 0.91; for BROD-D604N, Ïclosed = 270 fA, Ïopen = 560 fA, Popen = 0.07; for CHM15-D604N, Ïclosed = 260 fA, Ïopen = 290 fA, Popen = 0.92; for BROD-D604M, Ïclosed = 440 fA, Ïopen = 1.33 pA, Popen = 0.003; for CHM15-D604M, Ïclosed = 270 fA, Ïopen = 390 fA, Popen = 0.42.
	Figure 9. Amplitude histograms for activation by a saturating concentration of cIMP. Amplitude histograms corresponding to the representative traces in Fig. 8 are shown. The histograms were fit to the sum of two Gaussians, and the peak of the closed level Gaussian was used to subtract off leak currents. The parameters for the amplitude histograms were as follows: for BROD, Ïclosed = 240 fA, Ïopen = 360 fA, Popen = 0.74; for CHM15, Ïclosed = 530 fA, Ïopen = 270 fA, Popen = 0.96; for BROD-D604E, Ïclosed = 300 fA, Ïopen = 360 fA, Popen = 0.62; for CHM15-D604E, Ïclosed = 930 fA, Ïopen = 250 fA, Popen = 0.96; for BROD-D604Q, Ïclosed = 250 fA, Ïopen = 610 fA, Popen = 0.011; for CHM15-D604Q, Ïclosed = 320 fA, Ïopen = 360 fA, Popen = 0.81; for BROD-D604N, Ïclosed = 260 fA, Ïopen = 650 fA, Popen = 0.044; for CHM15-D604N, Ïclosed = 290 fA, Ïopen = 320 fA, Popen = 0.80; for BROD-D604M, Ïclosed = 320 fA, Ïopen = 610 fA, Popen = 0.009; for CHM15-D604M, Ïclosed = 210 fA, Ïopen = 340 fA, Popen = 0.59.
	Figure 11. Amplitude histograms for activation by a saturating concentration of cAMP. Amplitude histograms corresponding to the representative traces in Fig. 10 are shown. The histograms were fit to the sum of two Gaussians, and the peak of the closed level Gaussian was used to subtract off leak currents. The parameters for the amplitude histograms were as follows: for BROD, Ïclosed = 290 fA, Ïopen = 780 fA, Popen = 0.004; for CHM15, Ïclosed = 330 fA, Ïopen = 410 fA, Popen = 0.31; for BROD-D604E, Ïclosed = 230 fA, Ïopen = 630 fA, Popen = 0.05; for CHM15-D604E, Ïclosed = 300 fA, Ïopen = 390 fA, Popen = 0.61; for BROD-D604Q, Ïclosed = 360 fA, Ïopen = 580 fA, Popen = 0.009; for CHM15-D604Q, Ïclosed = 270 fA, Ïopen = 340 fA, Popen = 0.62; for BROD-D604N, Ïclosed = 250 fA, Ïopen = 660 fA, Popen = 0.015; for CHM15-D604N, Ïclosed = 300 fA, Ïopen = 390 fA, Popen = 0.71; for BROD-D604M, Ïclosed = 470 fA, Ïopen = 770 fA, Popen = 0.082; for CHM15-D604M, Ïclosed = 330 fA, Ïopen = 370 fA, Popen = 0.95.
	Figure 12. Box plot summaries for k01 and k10. Box plots of the k01 (A) and k10 (B) rate constants for the C0 â O1 â C2 scheme are shown for all 10 constructs for cGMP, cIMP, and cAMP. Values for the rate constants were determined by HMM analysis. The horizontal line within each box indicates the median of the data; boxes show the 25th and 75th percentiles of the data; whiskers show the 5th and 95th percentiles. Extreme data points are also indicated.
	Figure 13. Box plot summaries for k12 and k21. Box plots of the k12 (A) and k21 (B) rate constants for the C0 â O1 â C2 scheme are shown for all 10 constructs for cGMP, cIMP, and cAMP. Values for the rate constants were determined by HMM analysis.
	Figure 14. Box plot comparison between the values for k01/k10 and for L calculated from macroscopic experiments using Ni2+ potentiation. Box plots comparing the values for ÎG0 for the allosteric transition from singles and macroscopic current experiments are shown for the BROD (A) and CHM15 (B) channels.
	Figure 15. Free energy profile for the allosteric transition. The free energy profiles for the allosteric transition (C0 â O1) are illustrated based on the median values of the rate constants determined from HMM analysis of the single-channel currents. The rate constants were converted to energies using simple transition-state theory, with the highest energy intermediate postulated to break down to product at the vibrational frequency of a covalent bond. (A) Free energy profiles for cGMP, cIMP, and cAMP on BROD channels. (B) Free energy profiles for cAMP with and without Ni2+. (C) The free energy profiles of the allosteric transition for BROD and BROD-D604M channels activated by a saturating concentration of cGMP. (D) The free energy profiles of the allosteric transition for BROD and CHM15 channels activated by a saturating concentration of cIMP.
	Figure 16. Fraction of energetic effect occurring after the allosteric transition. The fraction of the energetic effect occurring after the allosteric transition was calculated as ÎÎGâ¡/ÎÎG0 = \|(ÎGâ¡1 â ÎGâ¡2)/(ÎG01 â ÎG02)\|, where ÎGâ¡ is the activation energy for the closing transition, ÎG0 is the standard free energy for the allosteric transition, and the subscript indicates the different conditions of allosteric modulation. ÎGs were the median values determined from an HMM analysis of the single-channel currents. The ÎÎGâ¡/ÎÎG0 for the interactions with different cyclic nucleotides was the average of the values for BROD channels between cGMP and cIMP, cGMP and cAMP, and cIMP and cAMP. The ÎÎGâ¡/ÎÎG0 for the interactions with D604 was the average of the values between BROD channels and D604M channels and between CHM15 channels and CHM15-D604M channels for all three cyclic nucleotides. The ÎÎGâ¡/ÎÎG0 for the interactions with Ni2+ was the average of the values between BROD channels with and without Ni2+ for cIMP and cAMP (Sunderman and Zagotta, 1999). The ÎÎGâ¡/ ÎÎG0 for the interactions with the amino terminal region was the average of the values between BROD channels and CHM15 channels for all three cyclic nucleotides.

References [+] :

Altenhofen, Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium. 1991, Pubmed, Xenbase