1. Volume 95, Issue 6, Pages 2750-2758 (September 2008)
Thermodynamics of Activation Gating in Olfactory-Type Cyclic Nucleotide-Gated (CNGA2) Channels 
Vasilica Nache, Jana Kusch, Christoph Biskup, Eckhard Schulz, Thomas Zimmer, Volker Hagen, Klaus Benndorf 
Biophysical Journal 
Volume 95, Issue 6, Pages (September 2008)
DOI: /biophysj
Copyright © 2008 The Biophysical Society Terms and Conditions

2. Figure 1
Effect of temperature on CNGA2 currents. (A) Effect of temperature on CNGA2 currents activated by 3μM cGMP. Solid symbols indicate the currents that were measured in a representative patch at a cGMP concentration of 3μM during a temperature ramp from 20.0 to 10.0°C. Current amplitudes that are corrected for the effects of the temperature on the single-channel current amplitude and the recording system (see Methods) are indicated by shaded symbols. (B) Effect of temperature on CNGA2 currents at saturating concentration. CNGA2 currents were measured at a cGMP concentration of 100μM during a temperature ramp from ϑ=20°C to 10.0°C and normalized with respect to the current amplitude at 20.0°C. Each data point represents the average of 11 patches. The ratio f(ϑ)=I(100μM, ϑ) / I(100μM, 20°C) is used to correct current amplitudes for the effects of the temperature on the single-channel current and the recordings system (see Methods). (C) Effect of temperature on CNGA2 currents in a representative patch activated by 0.35μM cGMP. Measured and corrected currents are represented by solid and shaded symbols, respectively. (D) Corrected and normalized CNGA2 currents as function of temperature. The current amplitudes I(c, ϑ) were corrected for the effects of the temperature on the single-channel current amplitude and the recordings system and normalized with respect to the current measured at 20.0°C, I(c,20°C) (see Methods). The plot shows that the effect of temperature is more pronounced at lower cGMP concentrations.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

3. Figure 2
Thermodynamic analysis of CNGA2 channels under steady-state conditions. (A) Concentration-response relationship of CNGA2 currents at four temperatures. The current amplitudes I(c, ϑ) were normalized with respect to the current amplitude at the saturating concentration of 100μM cGMP, I(100μM, ϑ). The data points were fitted with the Hill equation (Eq. 1), yielding as parameters at 20.0°C: KA=0.994μM−n, n=1.86; at 18.0°C: KA=0.902μM−n, n=1.88; at 14.0°C: KA=0.681μM−n, n=1.91; and at 10.0°C: KA=0.503μM−n, n=2.09. Cooling shifts the relationships to lower cGMP concentrations. (B) van’ t Hoff plot. lnKA was plotted as a function of 1/T, yielding ΔH=−11.2kcal M−1 and ΔS=−38.1cal M−1K−1.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

4. Figure 3
Activation time courses of CNGA2 currents elicited by cGMP jumps. (A) Activation time courses at 20.3°C. (B) Activation time courses at 10.3°C. Activation time courses were obtained from different patches at the indicated cGMP concentrations. The traces were fitted (red curves) with the sum of two exponentials (Eq. 3) yielding the indicated time constants τfast and τslow. For comparison, the traces were selected such that the cGMP concentrations of two traces side by side are similar.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

5. Figure 4
Temperature dependence of the speed of activation on the cGMP concentration. (A) Plot of the activation time constants τfast and τslow as function of the cGMP concentration at 20.3 and 10.3°C. Each data point was obtained from 5 to 20 patches. Here and in the following diagrams, the cGMP concentration was normalized to the EC50 value, which was 1.64μM at 20.3°C and 1.12μM at 10.3°C, respectively. The speed of activation was fast at high cGMP and slow at low cGMP but did not depend on the cGMP concentration in a simple monotonous fashion. (B) Plot of the temperature coefficients Q10,fast=τfast10.3∘Cτfast20.3∘C and Q10,slow=τslow10.3∘C/τslow20.3∘C as function of [cGMP]/EC50. At cGMP concentrations where time constants were only available for one temperature, the time constants for the other temperature were obtained by linear interpolation. Both time constants generate a maximum Q10 at [cGMP]/EC50 near 0.1. (C) Mean activation time constant τmean as function of [cGMP]/EC50 at 20.3 and 10.3°C. The value τmean was calculated according to Eq. 4. The profile is similar to that of the individual time constants. (D) Temperature coefficient Q10 for τmean as function of [cGMP]/EC50. Similar to Q10 for the individual time constants, also Q10,mean exhibits a maximum at [cGMP]/EC50 near 0.1.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

6. Figure 5
Arrhenius plots of the activation rate constants of CNGA2 currents over a wide range of cGMP concentrations. The activation rate, k, was obtained from the reciprocal mean activation time constant, τmean, at the temperatures 20.3, 15.3, and 10.3°C. At each cGMP concentration lnk is plotted as function of the reciprocal temperature 1/T and the three data points were fitted by a straight line, yielding the activation energy Ea. Error bars (see Fig. 4 C) were omitted for clarity.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

7. Figure 6
Thermodynamics of cGMP-jump induced activation of CNGA2 channels as function of the cGMP concentration. (A) Plot of the activation energy Ea, as function of [cGMP]/EC50. Ea was obtained from the slope in the Arrhenius plots (Fig. 5). (B) Plot of the free activation enthalpy ΔG‡ as function of [cGMP]/EC50. ΔG‡ was calculated by Eq. 7. (C) Plot of the activation enthalpy ΔH‡ as function of [cGMP]/EC50. ΔH‡ was calculated by Eq. 9. ΔΔH‡ is the difference between ΔH‡ at the highest and lowest cGMP concentration. (D) Plot of the activation entropy ΔS‡ as function of [cGMP]/EC50. ΔS‡ was calculated by Eq. 10. The entropy is only positive at very low cGMP. ΔΔS‡ is the difference between ΔS‡ at the highest and lowest cGMP concentration.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

8. Figure 7
Degree of liganding as function of the cGMP concentration according to the C4L model proposed by Biskup et al. (19). (A) Scheme of the C4L model. The model consists of five closed states (C0….C4) and five open states (O0…..O4). The equilibrium association constants are indicated in M−1 (horizontal reactions); the closed-open isomerization constants are dimensionless (vertical reactions). (B) Plot of the degree of liganding with respect to the sum of all degrees of liganding as a function of [cGMP]/EC50. The degrees of liganding add to unity at each cGMP concentration. At [cGMP]/EC50<0.1, only the single-liganded state (C1+O1) contributes to channel opening.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions

9. Scheme 1
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2008 The Biophysical Society Terms and Conditions