Transfection with 5-HT1A receptor gene and antisense directed against muscarinic M2 receptors reveal a mutual influence between Gi/o-coupled receptors in rat atrial myocytes

A recently described reduction in sensitivity of G protein-activated inward-rectifying K+ (GIRK) channels to stimulation of muscarinic M2 receptors (M2AChR) in atrial myocytes overexpressing purinergic A1 receptors (A1AdoR) was further investigated by heterologous expression of a 5-HT1A receptor (5-HT1AR) and by reducing the expression level of endogenous M2AChR receptors using antisense. In 5-HT1AR-expressing myocytes, in line with previous studies, sizable GIRK currents could be activated by 5-HT. In these cells, the mean current density and activation rate of M2AChR-activated current were significantly reduced, supporting the notion that signalling via this receptor is negatively regulated by other G protein-coupled receptors (GPCR) coupling to the same class (Gi/o) of G proteins. To study if reducing M2AChR expression affects sensitivity of GIRK current to stimulation of A1AdoR, antisense oligodinucleotides (AsODN) against the M2AChR were used. Incubation of myocytes with M2AChR-specific AsODN resulted in a significant reduction in mean amplitude and activation rate of ACh-induced currents. This was paralleled by an increase in mean amplitude and activation rate of current activated by stimulation of A1AdoR. Plotting amplitudes of 5-HT- or Ado-induced currents from individual manipulated cells against the amplitude of ACh-induced current yielded a positive correlation between these data. Although difficult to interpret in mechanistic terms, this argues against a competition of receptors for a common pool of Gi/o. The mutual interaction between Gi/o-coupled receptors depends on manipulation of the expression level, since long-term desensitization or down regulation of M2AChR by treatment with carbachol did not affect sensitivity of GIRK current to A1AdoR stimulation, despite a substantial reduction in amplitude and activation rate of M2AChR-activated currents. These data suggest a novel crosstalk between parallel receptors converging on the same class of G proteins.