Figure 5
From: Identification of critical residues in loop E in the 5-HT3ASR binding site
Whole cell patch clamp recording from tsA 201 cells transfected with wildtype and Y152A cDNA. Data were obtained using whole cell patch clamp on tsA201 cells transfected with wt or Y152A mutant 5-HT3ASR cDNA. Cells were clamped at a holding potential of -60 mV and 5-HT applied with a rapid perfusion system. The application time for 5-HT is indicated by the bar above the curves. The responses indicated in A and B are representative examples of at least 4 sets of experiments. A. Wildtype 5-HT3ASR. 5-HT was applied as indicated for a 4 s duration. The concentrations of 5-HT used and the response amplitudes are: a. 0.3 μM (0.03 nA) b. 1.0 μM (0.96 nA) c. 3.0 μM (2.5 nA) d. 30.0 μM (4.4 nA), e. 100.0 μM (3.9 nA). B. Y152A mutant receptors. 5-HT was applied as indicated for an 8 s duration. The curves in B were offset vertically for clarity. The dashed line in each curve indicates the baseline level for that response. The concentrations of 5-HT used and the response amplitudes are: f. 3.0 μM (0.10 nA) g. 100 μM (0.14 nA) h. 300.0 μM (0.57 nA) i. 1.0 mM (1.0 nA). C. Concentration response curve for 5-HT activation of wt 5-HT3ASRs. Each data point represents the combined results of 4 experiments. Responses were normalized to the response obtained at 30 μM and the curves fit as described in the methods section. EC50 = 2.7 ± 0.25 μM, n = 1.6 ± 0.22. D. Concentration response curve for 5-HT activation of Y152A 5-HT3ASRs. Each data point represents the combined results of 4 experiments. The response did not plateau in the range of concentrations used and hence responses were normalized to the maximum concentration of 5-HT (1 mM). The EC50 was estimated by fitting the data to the following equation: I = Imax/(1 +(EC50/ [C])n), where is the normalized current at 5-HT concentration [C], EC50 is the concentration of 5-HT needed to obtain half maximal activation and n is the apparent Hill coefficient. Imax in this case represents the estimated maximum current relative to the response obtained at 1 mM. To obtain the curve shown in the figure, the Hill coefficient was fixed at the wt value of 1.6. The EC50 value obtained from this fit is 370 ± 27 μM.