Nache V et al. (2013), Hysteresis of ligand binding in CNGA2 ion chann...

XB-ART-49864

Nat Commun 2013 Jan 01;4:2866. doi: 10.1038/ncomms3866.

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Hysteresis of ligand binding in CNGA2 ion channels.

Nache V , Eick T , Schulz E , Schmauder R , Benndorf K .

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Tetrameric cyclic nucleotide-gated (CNG) channels mediate receptor potentials in olfaction and vision. The channels are activated by the binding of cyclic nucleotides to a binding domain embedded in the C terminus of each subunit. Here using a fluorescent cGMP derivative (fcGMP), we show for homotetrameric CNGA2 channels that ligand unbinding is ~50 times faster at saturating than at subsaturating fcGMP. Analysis with complex Markovian models reveals two pathways for ligand unbinding; the partially liganded open channel unbinds its ligands from closed states only, whereas the fully liganded channel reaches a different open state from which it unbinds all four ligands rapidly. Consequently, the transition pathways for ligand binding and activation of a fully liganded CNGA2 channel differ from that of ligand unbinding and deactivation, resulting in pronounced hysteresis of the gating mechanism. This concentration-dependent gating mechanism allows the channels to respond to changes in the cyclic nucleotide concentration with different kinetics.

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Species referenced: Xenopus laevis
Genes referenced: cnga2

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	Figure 1. Experimental data of fcGMP binding and gating in CNGA2 channels.(a) Concentration–binding relationship and concentration–activation relationship under steady-state conditions over a wide concentration range between 30 nM and 29.1 μM. Each data point is the mean of measurements obtained from 6 to 15 patches. Error bars denote s.e.m. The arrows indicate the concentrations used for the analyses of the time courses as shown in b. (b) Time courses of activation and deactivation as well as fcGMP binding and unbinding following a concentration pulse to either 0.5 μM (13.0 s), 2.0 μM (7.5 s), or 12.0 μM (9.0 s) fcGMP and back to zero. The traces are averages of 7, 7, 10 currents, respectively, arising from different patches. For better comparison the traces were graphically cut after reaching a steady state. All traces were normalized with respect to the late current (I/Imax) or late fluorescence intensity (F/Fmax). (c) Superimposition of the time courses of ligand unbinding. The colours correspond to b. The orange curves indicate monoexponential fits yielding the time constants for the ligand unbinding starting from different fcGMP concentrations. The red arrow indicates the unbinding time course at 12.0 μM fcGMP, which is ~50 times faster than the unbinding at the two lower concentrations. The obtained time constants are: 20.5 ms from 12 μM fcGMP, 943.2 ms from 2.0 μM fcGMP and 796.3 ms from 0.5 μM fcGMP. (d) Superimposition of the deactivation time courses. The colors correspond to b.
	Figure 2. Fit of ligand binding and activation with the C4L model.(a) Scheme of the C4L model. Cx and Ox (n=0…4) denote closed and open states, respectively. L is a ligand. The numbers are the values of the rate constants (s−1 or s−1 M−1) determined by the fit. Rate constants set equal in the fit are indicated by using the same colours. The errors for each rate constant are given in Supplementary Table S1. (b) Time courses of binding and unbinding (green) as well as activation and deactivation (black) for three fcGMP concentrations. The time interval between the concentration jump from 0 to the respective fcGMP concentration and back to 0 is indicated for each trace by the black bar at the top. (c) Steady-state relationships for activation (black symbols) and ligand binding (green symbols). Each data point is the mean of measurements obtained from 6 to 15 patches. Error bars denote s.e.m. The red curves in b and c are the result of the best global fit of the time courses of ligand binding and activation as well as the steady-state relationships with the parameters shown in a. The reduced χr2 for the activation/binding and steady-state was 5.0.
	Figure 3. Fit with the C4L-O4L model including ligand unbinding and deactivation.(a) Scheme of the C4L-O4L model. Cx and Ox (n=0…4) denote closed and open states, respectively. Ox denotes states of an extra pathway used only in deactivation. L is a ligand. Again, the numbers provide the values of the rate constants (s−1 or s−1M−1) determined by the fit. Rate constants set equal in the fit are indicated by using the same colours. The errors for each rate constant are given in Supplementary Table S2. (b) The time courses of ligand binding and unbinding as well as activation and deactivation are the same as those in Fig. 2b. (c) The steady-state relationships are the same as those in Fig. 2c. Each data point is the mean of measurements obtained from 6 to 15 patches. Error bars denote s.e.m. The red curves in b and c are the result of the best global fit with the C4L-O4L model. The reduced χr2 was 6.0.
	Figure 4. Time-dependent state population in the C4L-O4L model.Using the parameters given in Fig. 3a, the population of all states in the C4L-O4L model was computed for three fcGMP concentrations. (a) Upper time courses: open probability, Po (red curve, same curves as in Fig. 3b) and probability of being either in Ox, PO,x (x=0….3) or Ox, PO,x (x=0….4; coloured curves). The PO,x and PO,x values sum up to Po. Lower time courses: part of the respective upper time courses at an expanded timescale. (b) Probability of being in the closed state x, PC,x (x=0….4). The PO,x, PO,x, and PC,x, values sum up to unity. (c) Ligand binding to the total channel (red curve) to the individual open (x/4 × PO,x; x/4 × P*O,x; coloured continuous) and closed states (x/4 × PC,x; coloured dashed), given as fraction of unity (left ordinates). x/4 weighs the contribution of each liganded state to the total liganding. NL indicates the number of ligands bound to the channel and to the individual states under the assumption that maximally four ligands can bind to the channel (right ordinate).
	Figure 5. Predicted and measured time course of ligand unbinding at 4.5 μM fcGMP.The approximated time courses of ligand unbinding at 0.5 μM, 2.0 μM, 4.5 μM and 12 μM fcGMP are obtained by the global fit with the C4L-O4L model (c.f. Fig. 3). The time course at 4.5 μM (blue curve), simulated with C4L-O4L model, approximately matches the experimentally determined time course at this concentration (green trace). The experimentally determined time course is the average from five individual time courses.
	Figure 6. Transition pathways in the C4L-O4L model.The net probability fluxes along specified transition pathways between state X and Y, Fp,XY, were computed by integrating the net probability flux densities and employing the principles of the transition pathway theory (see Supplementary Methods). The weight of the pathway net probability fluxes is semiquantitatively encoded by the thickness of the arrows (fluxes below 2 × 10−4 are indicated by dotted lines. The numbers besides the coloured graphs indicate the exact weights as fraction of unity. (a) 12 μM fcGMP. Pathway net probability fluxes along the pathways C0…O4, C0…O3, and C0…O2 after applying fcGMP (red) and after its removal (blue). (b,c) Respective pathway net probability fluxes at 2.0 μM and 0.5 μM fcGMP, respectively.

References [+] :

Benndorf, Probability fluxes and transition paths in a Markovian model describing complex subunit cooperativity in HCN2 channels. 2012, Pubmed

	Figure 1. Experimental data of fcGMP binding and gating in CNGA2 channels.(a) Concentration–binding relationship and concentration–activation relationship under steady-state conditions over a wide concentration range between 30 nM and 29.1 μM. Each data point is the mean of measurements obtained from 6 to 15 patches. Error bars denote s.e.m. The arrows indicate the concentrations used for the analyses of the time courses as shown in b. (b) Time courses of activation and deactivation as well as fcGMP binding and unbinding following a concentration pulse to either 0.5 μM (13.0 s), 2.0 μM (7.5 s), or 12.0 μM (9.0 s) fcGMP and back to zero. The traces are averages of 7, 7, 10 currents, respectively, arising from different patches. For better comparison the traces were graphically cut after reaching a steady state. All traces were normalized with respect to the late current (I/Imax) or late fluorescence intensity (F/Fmax). (c) Superimposition of the time courses of ligand unbinding. The colours correspond to b. The orange curves indicate monoexponential fits yielding the time constants for the ligand unbinding starting from different fcGMP concentrations. The red arrow indicates the unbinding time course at 12.0 μM fcGMP, which is ~50 times faster than the unbinding at the two lower concentrations. The obtained time constants are: 20.5 ms from 12 μM fcGMP, 943.2 ms from 2.0 μM fcGMP and 796.3 ms from 0.5 μM fcGMP. (d) Superimposition of the deactivation time courses. The colors correspond to b.
	Figure 2. Fit of ligand binding and activation with the C4L model.(a) Scheme of the C4L model. Cx and Ox (n=0…4) denote closed and open states, respectively. L is a ligand. The numbers are the values of the rate constants (s−1 or s−1 M−1) determined by the fit. Rate constants set equal in the fit are indicated by using the same colours. The errors for each rate constant are given in Supplementary Table S1. (b) Time courses of binding and unbinding (green) as well as activation and deactivation (black) for three fcGMP concentrations. The time interval between the concentration jump from 0 to the respective fcGMP concentration and back to 0 is indicated for each trace by the black bar at the top. (c) Steady-state relationships for activation (black symbols) and ligand binding (green symbols). Each data point is the mean of measurements obtained from 6 to 15 patches. Error bars denote s.e.m. The red curves in b and c are the result of the best global fit of the time courses of ligand binding and activation as well as the steady-state relationships with the parameters shown in a. The reduced χr2 for the activation/binding and steady-state was 5.0.
	Figure 3. Fit with the C4L-O4L model including ligand unbinding and deactivation.(a) Scheme of the C4L-O4L model. Cx and Ox (n=0…4) denote closed and open states, respectively. Ox denotes states of an extra pathway used only in deactivation. L is a ligand. Again, the numbers provide the values of the rate constants (s−1 or s−1M−1) determined by the fit. Rate constants set equal in the fit are indicated by using the same colours. The errors for each rate constant are given in Supplementary Table S2. (b) The time courses of ligand binding and unbinding as well as activation and deactivation are the same as those in Fig. 2b. (c) The steady-state relationships are the same as those in Fig. 2c. Each data point is the mean of measurements obtained from 6 to 15 patches. Error bars denote s.e.m. The red curves in b and c are the result of the best global fit with the C4L-O4L model. The reduced χr2 was 6.0.
	Figure 4. Time-dependent state population in the C4L-O4L model.Using the parameters given in Fig. 3a, the population of all states in the C4L-O4L model was computed for three fcGMP concentrations. (a) Upper time courses: open probability, Po (red curve, same curves as in Fig. 3b) and probability of being either in Ox, PO,x (x=0….3) or Ox, PO,x (x=0….4; coloured curves). The PO,x and PO,x values sum up to Po. Lower time courses: part of the respective upper time courses at an expanded timescale. (b) Probability of being in the closed state x, PC,x (x=0….4). The PO,x, PO,x, and PC,x, values sum up to unity. (c) Ligand binding to the total channel (red curve) to the individual open (x/4 × PO,x; x/4 × P*O,x; coloured continuous) and closed states (x/4 × PC,x; coloured dashed), given as fraction of unity (left ordinates). x/4 weighs the contribution of each liganded state to the total liganding. NL indicates the number of ligands bound to the channel and to the individual states under the assumption that maximally four ligands can bind to the channel (right ordinate).
	Figure 5. Predicted and measured time course of ligand unbinding at 4.5 μM fcGMP.The approximated time courses of ligand unbinding at 0.5 μM, 2.0 μM, 4.5 μM and 12 μM fcGMP are obtained by the global fit with the C4L-O4L model (c.f. Fig. 3). The time course at 4.5 μM (blue curve), simulated with C4L-O4L model, approximately matches the experimentally determined time course at this concentration (green trace). The experimentally determined time course is the average from five individual time courses.
	Figure 6. Transition pathways in the C4L-O4L model.The net probability fluxes along specified transition pathways between state X and Y, Fp,XY, were computed by integrating the net probability flux densities and employing the principles of the transition pathway theory (see Supplementary Methods). The weight of the pathway net probability fluxes is semiquantitatively encoded by the thickness of the arrows (fluxes below 2 × 10−4 are indicated by dotted lines. The numbers besides the coloured graphs indicate the exact weights as fraction of unity. (a) 12 μM fcGMP. Pathway net probability fluxes along the pathways C0…O4, C0…O3, and C0…O2 after applying fcGMP (red) and after its removal (blue). (b,c) Respective pathway net probability fluxes at 2.0 μM and 0.5 μM fcGMP, respectively.