Figure 4.

Experimental protocol used for the determination of sustained exposure to AT-1001. Traces recorded in presence of three concentrations of AT-1001 are superimposed. As described for Figure 3, the cell is first exposed for 45 s to a steady concentration of AT-1001 (here 0.01, 0.1, and 1 μM) and current recorded. The response evoked by a brief acetylcholine (ACh) test pulse (50 μM, 10 s) is then recorded in presence of the same concentration of compound. The bars above the current traces indicate timing of AT-1001 and ACh exposures. ACh and compound are rinsed for 15 s before returning into the same concentration of AT-1001. Arrows indicate the measurement of current amplitude for these three concentrations. Note that returning into 1 μM of AT-1001 causes an inward current corresponding to the activation of the α3β4 at this concentration. The small amplitude of the current during the second exposure is attributed to receptor desensitization, as shown by the dashed line that is in continuation of the response evoked by the first exposure (at the 1 μM of AT-1001).