Figure 2.

(A) Laser-pulse photolysis experiments (23, 24) with BC₃H1 cells containing the muscle-type AChR (33). The laser-pulse photolysis method was used as has been described in detail (23, 24, 27, 28). Seventy micromolar carbamoylcholine was photolytically released from the 1-mM caged carbamoylcholine with which the cell was preequilibrated in the absence (Top) and presence (Bottom) of 500 μM MK-801 or in the presence of both 500 μM MK-801 and 2 μM aptamer II-3 (Middle). The cell was preequilibrated (using a device described in ref. 27) for 200 ms with the compounds used in the experiments, and then a 9-ns laser pulse at 337 nm released free carbamoylcholine. At the concentration used, MK-801 does not contribute to the absorption of light at 337 nm. The whole-cell current thus induced was recorded at ≈22°C, −60 mV, and pH 7.4. The desensitization reaction, which occurs on a slower time scale, is not shown. In the upper trace, the current amplitude is 2.6 nA and in the lowest trace, it is 0.5 nA. In the middle trace, 2 μM aptamer II-3 restores the current amplitude (2.3 nA) to about 90% of the noninhibited receptor amplitude (upper trace). The spikes seen in all three traces represent an instrument artifact. Nonlinear least-squares fitting (Marquardt algorithm) was performed by using Microcal origin. (B) Cell-flow experiments (22) with BC₃H1 cells containing the muscle-type AChR (33). The whole-cell current was recorded for 2 s at ≈22°C, −60 mV, and pH 7.4. The upper traces parallel to the abscissa represent the current amplitudes corrected for receptor desensitization that occurs during the rising phase. For each concentration of carbamoylcholine used, control experiments without inhibitor were performed, and the current amplitude corrected for receptor desensitization was taken as the 100% response. All experiments were carried out in the presence of a constant concentration (0.5 μM) of aptamer I-14 and increasing concentrations of carbamoylcholine [100 μM, 250 μM, 400 μM, 500 μM corresponding to 0.30, 0.55, 0.65, and 0.75 fractions of open receptor channels as calculated from published cell-flow experiments relating the concentration of open receptor channels in BC₃H1 cells to carbamoylcholine concentration (22, 24)]. The cell was preincubated for 2 s with the compounds used in the experiments. Similar results were obtained with a mixture of 12 Class I aptamers (Class I aptamers 1, 5, 6, 7, 9, 11, 13, 16, 18, 19, 20 and 22) (32). In the presence of a constant concentration (4 μM) of the aptamers, with which the cell was also preequilibrated for 2 s, over 50% of the receptors were inhibited in the presence of 100 μM carbamoylcholine. They were not inhibited in the presence of 500 μM carbamoylcholine, when the fraction of the receptors in the open-channel conformation is more than 2-fold larger than at 100 μM carbamoylcholine.