Rettinger J , Schmalzing G .

	Figure 1. . Schematic drawing of the fast solution exchange device. (A) Cross-section (top) and top view (bottom) of the perfusion chamber. The oocyte is placed in a small cavity possessing an internal volume of 10 μl and superfused at a constant flow rate of ≈200 μl/s through a glass-capillary of 1.2-mm internal diameter connected with a manifold (not depicted) that is fixed by a micromanipulator. Bath solution exceeding the internal volume of the cavity is removed with a diaphragm pump through a glass-capillary mounted close to the chamber cavity. Two Ag/AgCl pellets are used for grounding the bath and as reference for the intracellular voltage electrode, respectively. (B) The onset of muscle type nAChR currents was used to assess the speed of solution exchange. Shown are seven superimposed current traces elicited repetitively in 1-min intervals by 5-s 30 μM acetylcholine pulses. A current rise time t10–90% of ∼150 ms was determined for a 10–90% increase of the current amplitude.
	Figure 2. . Desensitization of the P2X1 receptor by incubation at submicromolar ATP concentrations. (A) Shown are current traces elicited by saturating test pulses of 30 μM ATP after preincubation of individual oocytes at 18 nM ATP for the indicated time period. (B) Rate of entry into the desensitized receptor state by preincubation in ATP-containing Mg-ORI. Desensitization was induced using 3 (▪), 10 (•), 18 (▴), 30 (▾), 56 (♦), and 100 nM ATP (+). The lines represent fits of a monoexponential function to the data (6–18 determinations per data point). (C) ATP concentration dependence of steady-state desensitization. Steady-state desensitization levels represented by Iss values were derived from the exponential fits of (B). The curve represents a nonlinear fit of the Hill equation to the data (K1/2 = 3.2 ± 0.1 nM, n = 1.24 ± 0.06; 6–13 determinations per data point). The value at 1 nM ATP was obtained by preincubating the oocytes for 2 h at 1 nM ATP in a Petri dish before transferring them to the superfusion chamber, where the residual current response was elicited by the 30 μM ATP test pulse. (D) ATP concentration dependence of τod, the time-constant of onset of desensitization. τod values are derived from the nonlinear fits shown in (B).
	Figure 3. . Recovery from desensitization. Test responses were elicited by 200 nM ATP applied for a duration of 60 s in intervals ranging from 1 min to 60 min. Between ATP applications oocytes were washed in ATP-free Mg-ORI. One minute after each test response, a control response to 200 nM ATP was elicited which was used to normalize the preceding response of interest. The solid line represents the fit of a monoexponential function to the data yielding the time constant τ = 11.6 ± 1.0 min (N = 4–7).
	Figure 4. . Analysis of P2X1 receptor activation at submicromolar ATP concentrations. (A) ATP dose-response curve for 1 nM to 30 μM ATP (EC50 = 0.7 ± 0.06 μM, n = 1.13 ± 0.08; mean of 11–55 determinations per data point). (B) Scaled current traces elicited by 10–300 nM ATP. ATP was applied until the desensitization process was completed (10 min at 10 nM ATP; 10 s at 300 nM). The gray curves underlying the original current traces (in black) represent fits of the Bateman equation to the data. (C) ATP concentration dependence of activation (▪) and desensitization (▴) time-constants (21–70 determinations per data point). The solid line represents a fit of equation τ = τ0[ATP]−n to the data points (n = 1.75 ± 0.02). In addition, time constants of onset of desensitization (○) taken from Fig. 2 D were replotted to demonstrate that they follow a virtually identical ATP dependence. (D) Areas under current traces elicited by 18–300 nM ATP (11–51 determinations per column). The areas were calculated using the parameters obtained by fitting the Bateman function to the current traces in (A). Numerical integration of the receptor currents yielded virtually identical results (not depicted).
	Figure 5. . Analysis of simulated receptor currents. Receptor currents were simulated for the cyclic three-state model by using the Gepasi Software and analyzed in the same manner as the experimental data. (A) ATP dependence of simulated peak currents in dependence of assumed genuine KD values for receptor activation of 0.03 (▪), 3 (•), 30 (▴), 100 (▾), 300 (♦) and 1.000 nM (◂). A fit of the Hill equation to the simulated data yielded EC50 values of 0.72–1.3 μM for KD values ranging from 0.03 to 1,000 nM, respectively. (B) Relative deviations of the calculated EC50 taken from A from the experimentally determined EC50 value in dependence of the assumed genuine KD values. (C) Steady-state desensitization was calculated using the same set of simulated currents as in (A). A fit of the Hill equation resulted in apparent K1/2 values of 3.4–8.3 nM for KD values of 0.03–1,000 nM, respectively. (D) Relative deviations from the calculated K1/2 values from the experimentally determined K1/2 value in dependence of genuine assumed KD values.

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