Benndorf K et al. (1999), Gating by cyclic GMP and voltage in the alpha s...

XB-ART-12288

J Gen Physiol 1999 Oct 01;1144:477-90.

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Gating by cyclic GMP and voltage in the alpha subunit of the cyclic GMP-gated channel from rod photoreceptors.

Benndorf K , Koopmann R , Eismann E , Kaupp UB .

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	Scheme S1.
	Scheme S2.
	Figure 2. Voltage-dependent activity of cGMP-gated channels at 7 and 700 μM cGMP. The final filter frequency was 2 kHz. (A) 7 μM cGMP. The voltage was stepped repetitively between −50 and +50 mV every 3 s. In each 3-s interval, three traces were recorded for 500 ms. The patch contained at least three channels. The corresponding ensemble averaged currents (top, calculated from 12 individual traces each) show outward rectification. (B) 700 μM cGMP. The patch contained only one channel. Although the Po at −50 mV is also smaller than that at +50 mV (for values see text) the degree of outward rectification is lower than at 7 μM cGMP.
	Figure 1. Single-channel currents and corresponding amplitude histograms in a patch containing at least three CNG channels at 70 (left) and 7 (right) μM cGMP. The patch was held at +50 mV. The individual traces of 500-ms duration were recorded in intervals of 1 s. The final cut-off frequency was 1 kHz. The amplitude histograms were formed with the variance-mean technique with a window width of 280 μs (seven sampling points). The distributions were fitted with sums of either four or two Gaussian functions. The respective single-channel currents i and standard deviations σ are indicated. At 70 μM cGMP, the amplitude histograms were formed from consecutive traces, whereas at 7 μM cGMP only nonempty traces were used. Significant sublevel openings were not present.
	Figure 5. Voltage dependence of macroscopic currents through cGMP-gated channels at different [cGMP]. Recordings at 20 and 70 μM cGMP were obtained from one patch and at 700 μM and 7 mM cGMP from another patch. The membrane voltage Vm was stepped from −100 mV to another voltage between −80 and +100 mV in 20-mV increments. The duration and frequency of pulses were either 50 ms and 2 Hz (20 and 70 μM cGMP) or 4 ms and 10 Hz (700 μM and 7 mM cGMP), respectively. Traces represent the average of 10–20 consecutive current recordings. Each trace was corrected for capacitive and small leakage currents by subtracting a current that was averaged from 5–10 control traces recorded in the absence of cGMP.
	Figure 3. Open-time histograms at 7 and 700 μM cGMP and +50 and −50 mV each. The data were obtained from a multichannel patch at 7 μM cGMP and a single-channel patch at 700 μM cGMP. The distributions were fitted with sums of two exponentials yielding the indicated contributions An and open-time constants τn. The combination of high [cGMP] and positive voltage caused a dramatic prolongation of both time constants. Filter, 2 kHz.
	Figure 4. Dependence of open times on [cGMP]. All data were filtered at 2 kHz. (A) Fast open time τo1 and slow open time τo2 of single cGMP-gated channels as function of [cGMP]. Kinetic constants were measured at 7, 20, 70, and 700 μM cGMP and at −50 and +50 mV. Parentheses indicate values at 7 and 70 μM, respectively. 16–63 traces of 500-ms duration were analyzed. When switching from −50 to +50 mV, both τo1 and τo2 increased at 70 μM cGMP and even more so at 700 μM cGMP, whereas at 7 and 20 μM cGMP, no statistically significant increase of either τo1 and τo2 was observed. (B) Voltage-dependent incidence of the relative contribution of fast and slow exponential in the open-time histograms as function of [cGMP]. Plotted is the ratio An (+50 mV)/An (−50 mV). At low [cGMP], A2 (+50 mV)/A2 (−50 mV) is larger than A1 (+50 mV)/A1 (−50 mV), suggesting that the contribution of the slow exponential is more influenced by voltage than that of the fast exponential.
	Figure 6. Voltage and [cGMP] dependence of macroscopic current. (A) Dose–response relationships for the channel activation by cGMP at +100 and −100 mV. The error bars indicate SD; for several conditions, SD is smaller than the size of the symbols. Three different types of measurements were used to determine the dose–response relationships (see text). Each data point was calculated from 4–10 individual experiments. The data points at the two voltages were simultaneously fitted with the allosteric model (Fig. 1; see text for parameters). (B) Po/Vm relationships at 20, 70, and 700 μM, and 7 mM [cGMP]. In the diagram, the data points at −100 and +100 mV were taken from A. The data points for all other voltages were obtained from the instantaneous tail currents at −100 mV after test pulses to the indicated voltages at the abscissa (compare Fig. 5). The error bars indicate SD that was computed according to the error propagation law. The curves provide the best simultaneous fit to all data with the allosteric model (see text for parameters).
	Figure 7. Time course of the activation at saturating [cGMP] (7 mM). The activation time course was fitted with a single exponential yielding the indicated time constants τ. The measured current at +40 mV was scaled to the current at +100 mV.
	Figure 8. Comparison of computed with measured currents at three [cGMP] and voltage steps of 50-ms duration from −100 to +100 mV. (A) Measured currents. The currents at 70 and 700 μM cGMP were recorded from the same patch. The current at 20 μM [cGMP] was recorded from another patch and it was scaled with the ratio of currents at 700 μM [cGMP] in the two patches. (B) Computed currents. Po was calculated with the allosteric model (Fig. 1) and then scaled with the mean single channel current (dotted lines; −1.6 pA at −100 mV; 2.6 pA at +100 mV). The parameters were: k1 = 3 × 107 M−1 s−1, k2 = 1.5 × 103 s−1, k3,0 = 1.39 × 103 s−1, k4,0 = 1.67 × 102 s−1, z = 0.23.

References [+] :

Bader, A voltage-clamp study of the light response in solitary rods of the tiger salamander. 1979, Pubmed