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

Abstract

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 µM and 5 µM. 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 µM and 100 µM in the pipette, the half-maximal activation voltage (V _1/2) was shifted by +28.5±1.7 mV. The activation time constant (τ_a) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. τ_a changed from 1106±60 ms at 0 µM cAMP to 265±7 ms at a saturating concentration of 1 mM cAMP. A dose–response relationship yielded values of 0.6 µM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 µM forskolin induced an elevation of the cAMP level by about 1.6±0.2 µM. Co-expressions of HvCNG channels in combination with the mouse 5-HT_4(a)- or 5-HT_1A- 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-HT_4(a)-receptors by 0.1 µM 5-HT induced an increase of cAMP of about 4.6±1.5 µM, whereas cAMP levels decreased from a control value of 1±0.2 µM to 0.41±0.1 µM after stimulation of the m5-HT_1A-receptors.