Heterologously expressed serotonin 1A receptors couple to muscarinic K+ channels in heart.

Abstract

In cardiac atrial cells, muscarinic acetylcholine receptors activate a K+ current directly via a guanine nucleotide-binding protein (G protein). Serotonin type 1A receptors may activate a similar pathway in hippocampal neurons. To develop a system in which receptor/G protein/K+ channel coupling can be experimentally manipulated, we have used a highly efficient recombinant vaccinia virus vector system to express human serotonin 1A receptors in primary cultures of rat atrial myocytes. The expressed 1A receptors activated the inwardly rectifying K+ conductance that is normally activated by the endogenous muscarinic acetylcholine receptors. Maximal responses to either agonist occluded further activation by the other agonist. The average activation time constants for serotonin were about 5 times slower than for acetylcholine. The data support suggestions that the intracellular signaling pathway from seven-helix receptors to G proteins and directly to ion channels is widespread in excitable cells. After a fraction of the G proteins are activated irreversibly by guanosine 5'-(ythioltriphosphate, subsequent transduction proceeds more efficiently. One possible interpretation is that multiple G-protein molecules are required to activate each channel. Vaccinja virus expression vectors are thus useful for expressing seven-helix receptors in primary cultures of postmitotic cells and have provided a heterologous expression system for the signaling pathway from seven-helix receptors to G proteins and directly to ion channels.

Expression of an atrial G-protein-activated potassium channel in Xenopus oocytes.

“…To avoid the possibility that a low level of expression of the muscarinic receptor will make a well-expressed KG channel undetectable, atrial RNA was usually supplemented with SHT1A-R mRNA; oocytes injected with this RNA mixture will be termed RNA-injected oocytes throughout the paper. When expressed in atrial myocytes, the SHT1A-R efficiently coupled to the KG channel normally existing in these cells (23), and it was expected to do so in the oocytes.…”

“…The similarity of the channels and of the signaling pathways in atrium and some nerve-cell preparations was strengthened by the demonstration of the coupling of a neuronal 5HT1A receptor (5HT1A-R), transiently expressed in atrial myocytes, to the atrial KG channels (23). By electrophysiological and pharmacological criteria, the atrial KG channel belongs to a family of inward rectifiers that conduct K+ much better in the inward than the outward direction, are blocked by extracellular Na+, Cs+, and Ba2+, and are believed to possess a single-file pore with several permeant and blocking ion binding sites (24).…”

The role of conserved aspartate and serine residues in ligand binding and in function of the 5‐HT1A receptor: A site‐directed mutation study

“…Constructioa of the plasmid pTMI-SHTIAR [4], containing tile 5-FIT~AR eDNA was described previously [4,15]. The receptor was cxpresr~l in ¢OS-7 ¢¢11s by the infection-translation protocol [3]: 6 x 10 ~ cells were first infected with the helper virus, vTF7-3 (multiplicity of infection of 5) which expresses the bacteriophage "1"7 RNA polymera~, and transferal ruing Lipofeetia (Ilethesda R~earch Laboratories) with 10 pg of recombinant plasmids containing the wild-type or mutant 5-HTtAR eDNA driven by a T7 promoter in the specialized V'V vector pTMI.…”

“…Recently the 5-HTtAR was expressed in a variety of mammalian cell types [3] using the vaccinia virus (VV)/ T7 polymerase vector system; in cardiac atrial cells, the expressed receptor activated K ~ channels within 1 s [4]. Endogenous 5-HT~AR also activates K + channels in hippocampal neurons without the involvement of known soluble second messengers [5], a property of the 5-HT~AR which may be relevant for its moderately rapid neurotransmitter signaling (i.e.…”

Activation of heteromeric G protein-gated inward rectifier K + channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons

“…Current and voltage commands, data acquisition, and analysis were performed using PCLAMP 6.0. Recordings in myocytes, CHO, and ␤TC3 cells were performed as described (23). For neurons, the bath solution contained 120 mM NaCl, 2.5 mM KCl, 5 mM Hepes, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose (pH 7.3 with NaOH, Ϸ260 mOsm); the pipette solution contained 5 mM KCl, 125 mM K gluconate, 5 mM Hepes, 5 mM Na 2 phosphocreatine, 1.1 mM EGTA, 0.1 mM CaCl 2 , 2 mM MgCl 2 , 4 mM MgATP, 0.3 mM Na 2 GTP (pH 7.2 with KOH, Ϸ250 mOsm).…”