High Impact AMPAkines Induce a Gq‐Protein Coupled Endoplasmic Calcium Release in Cortical Neurons: A Possible Mechanism for Explaining the Toxicity of High Impact AMPAkines

ABSTRACT α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) positive allosteric modulators (AMPAkines) have a multitude of promising therapeutic properties. The pharmaceutical development of high impact AMPAkines has, however, been limited by the appearance of calcium‐dependent neuronal toxicity and convulsions in vivo. Such toxicity is not observed at exceptionally high concentrations of low impact AMPAkines. Because most AMPAR are somewhat impermeable to calcium, the current study sought to examine the extent to which different mechanisms contribute to the rise in intracellular calcium in the presence of high impact ampakines. In the presence of AMPA alone, cytosolic calcium elevation is shown to be sodium‐dependent. In the presence of high impact AMPAkines such as cyclothiazide (CTZ) or CX614, however, AMPAR potentiation also activates an additional mechanism that induces calcium release from endoplasmic reticular (ER) stores. The pathway that connects AMPAR to the ER system involves a Gq‐protein, phospholipase Cβ‐mediated inositol triphosphate (InsP3) formation, and ultimately stimulation of InsP3‐receptors located on the ER. The same linkage was not observed using high concentrations of the low impact AMPAkines, CX516 (Ampalex), and CX717. We also demonstrate that CX614 produces neuronal hyper‐excitability at therapeutic doses, whereas the newer generation low impact AMPAkine CX1739 is safe at exceedingly high doses. Although earlier studies have demonstrated a functional linkage between AMPAR and G‐proteins, this report demonstrates that in the presence of high impact AMPAkines, AMPAR also couple to a Gq‐protein, which triggers a secondary calcium release from the ER and provides insight into the disparate actions of high and low impact AMPAkines. In the presence of high impact AMPAkines, but not low impact AMPAkines, AMPA receptor agonist binding induces activation of a Gq‐protein, whereby PIP2 is converted to inositol triphosphate by phospholipase Cβ that leads to subsequent stimulation of inositol triphosphate receptors located on endoplasmic reticulum. Calcium stored in the endoplasmic reticulum is then released into the cytoplasm of cortical neurons. This pathway is blocked by antagonists of phospholipase Cβ and the inositol triphosphate receptor. This novel pathway, which is specific to high impact AMPAkines, may help explain why high impact AMPAkines are more seizurogenic than low impact AMPAkines.