An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels
Figure 1
Development of a cAMP sensor with decreased pH sensitivity.
(A) and (B) The sensitivity of the global cAMP sensors was determined in vitro by addition of increasing concentrations of cAMP to lysates prepared from HEK293 cells expressing the sensor. EC50 presented as mean±SD (n=3 or 4). (C) BCECF was used to measure the pH change produced by addition of 10 mM propanoic acid (PA) or 10 mM trimethylamine (TMA). The pH change is quantified in the lower panel (mean±SEM; n=107 cells from two independent experiments). (D) and (E) Normalised fluorescent ratios from HEK293 cells expressing ‘dead’ global sensors treated with 10 mM PA or 10 mM TMA. The area under the curve (AUC) is shown in the lower panel (mean±SEM; n=33 (YFP/CFP (PA)), 63 (Ci/Ce (PA)), 25 (YFP/CFP (TMA)) and 43 (Ci/Ce (TMA)) cells. Data from 4-6 independent experiments). ** p<0.001, *** p<0.0001 compared to YFP/CFP measured by t test.