5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 muM and 5 muM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 muM and 100 muM in the pipette. the half-maximal activation voltage (V-1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 muM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 muM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 muM forskolin induced an elevation of the cAMP level by about 16+/-0.2 muM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4(a)- or 5-HT1A-receptors and the corresponding G(s)- or G(i)-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4(a)-receptors by 0.1 muM 5-HT induced an increase of cAMP of about 4.6+/-1.5 muM. whereas cAMP levels decreased from a control value of 1+/-0.2 muM to 0.41+/-0.1 muM after stimulation of the m5-HT1A-receptors.