ABSTRACT
1. The effect of serotonin (5-HT) on the hyperpolarization-activated cation current (IH) was studied in small-, medium- and large-diameter acutely isolated rat dorsal root ganglion (DRG) cells, including cells categorized as type 1, 2, 3 and 4 based on membrane properties. 5-HT increased IH in 91 % of medium-diameter DRG cells (including type 4) and in 67 % of large-diameter DRG cells, but not other DRG cell types. 2. The increase of IH by 5-HT was antagonized by spiperone but not cyanopindolol, and was mimicked by 5-carboxyamidotryptamine, but not (+)-8-hydroxydipropylaminotetralin (8-OH-DPAT) or cyanopindolol. These data suggested the involvement of 5-HT7 receptors, which were shown to be expressed by medium-diameter DRG cells using RT-PCR analysis. 3. 5-HT shifted the conductance-voltage relationship of IH by +6 mV without changing peak conductance. The effects of 5-HT on IH were mimicked and occluded by forskolin, but not by inactive 1,9-dideoxy forskolin. 4. At holding potentials negative to -50 mV, 5-HT increased steady-state inward current and instantaneous membrane conductance (fast current). The 5-HT-induced inward current and fast current were blocked by Cs+ but not Ba2+ and reversed at -23 mV, consistent with the properties of tonically activated IH. 5. In medium-diameter neurons recorded from in the current clamp mode, 5-HT depolarized the resting membrane potential, decreased input resistance and facilitated action potential generation by anode-break excitation. 6. The above data suggest that in distinct subpopulations of DRG neurons, 5-HT increases cAMP levels via activation of 5-HT7 receptors, which shifts the voltage dependence of IH to more depolarized potentials and increases neuronal excitability.
Subject(s)
Ganglia, Spinal/metabolism , Ion Channels/metabolism , Neurons, Afferent/metabolism , Serotonin/pharmacology , 8-Hydroxy-2-(di-n-propylamino)tetralin/pharmacology , Action Potentials/drug effects , Animals , Cell Size , Cyclic Nucleotide-Gated Cation Channels , Dopamine Antagonists/pharmacology , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , In Vitro Techniques , Ion Channels/drug effects , Ion Channels/genetics , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neurons, Afferent/drug effects , Neurons, Afferent/ultrastructure , Patch-Clamp Techniques , Pindolol/analogs & derivatives , Pindolol/pharmacology , Potassium Channels , Rats , Rats, Sprague-Dawley , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serotonin/analogs & derivatives , Serotonin Antagonists/pharmacology , Serotonin Receptor Agonists/pharmacology , Spiperone/pharmacologyABSTRACT
A trimeric enzyme chloramphenicol acetyltransferase (CATI) has been synthesized in the Zubay system genetically depleted from DnaK and DnaJ. Most of CAT formed in the system fail to assemble into an active trimer. Instead CAT is accumulated in either a GroEL-bound complex or as an inactive monomer. Addition of purified DnaK and DnaJ to the system prior to the start of protein synthesis leads to the increase of the specific activity of formed CAT. A portion of exogenous DnaK and DnaJ added to the system associate with nascent polypeptide chains in the ribosomes. DnaK also comigrates with 50S-ribosomal subunits.
