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 microM and 5 microM. 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 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) 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 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.
Heine
Martin
M
Institute of Physiology and Pathophysiology, Department of Neuro- and Sensory Physiology Humboldtallee 23, Georg-August-University Göttingen, Germany.
Ponimaskin
Evgeni
E
Bickmeyer
Ulf
U
Richter
Diethelm W
DW
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D054815:Cyclic Nucleotide-Gated Cation Channels; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D044422:Receptors, Serotonin, 5-HT4; D015398:Signal Transduction / Q000502:physiology; D018411:Spodoptera
D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D044422:Receptors, Serotonin, 5-HT4; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases