Kisspeptin activation of TRPC4 channels in female GnRH neurons requires PIP₂ depletion and cSrc kinase activation

Kisspeptin signaling via its G+q-coupled receptor GPR54 plays a crucial role in modulating GnRH neuronal excitability, which controls pituitary gonadotropins secretion and ultimately reproduction. Kisspeptin potently depolarizes GnRH neurons primarily through the activation of canonical transient receptor potential (TRPC) channels, but the intracellular signaling cascade has not been elucidated. Presently,wehaveestablished that kisspeptin activation ofTRPCchannels requires multiplemembrane and intracellular signaling molecules. First, phosphatidylinositol-4,5-bisphosphate (PIP₂) hydrolysis by phospholipase Cμ is required because whole-cell dialysis of Dioctanoylglycerol-PIP₂ (DiC8-PIP₂) inhibited the kisspeptin activation of TRPC channels, and the phosphatidylinositol 4-kinase inhibitor wortmannin, which attenuates PIP₂ synthesis, prolonged TRPC channel activation. Using single cell RT-PCR, we identified that the mRNA for the PIP₂-interacting TRPC channel subunit, TRPC4+, is expressed in GnRH neurons. Depletion of intracellular Ca²⁺ stores by thapsigargin and inositol 1,4,5-trisphosphate hadnoeffect, indicating thattheTRPCchannelsarenotstore-operated.Neitherremovingextracellular Ca ²⁺ nor buffering intracellular Ca²⁺ with EGTA or BAPTA had any effect on the kisspeptin activation of theTRPCchannels.However,theCa ²⁺channel blocker Ni2+inhibited the kisspeptin-inducedinward current.Moreover,inhibitionofproteinkinaseCbybisindolylmaleimide- IorcalphostinChadnoeffect, but activation of protein kinase C by phorbol 12,13-dibutyrate occluded the kisspeptin-activated current. Finally, inhibition of the cytoplasmic tyrosine kinase cSrc by genistein or the pyrazolo-pyrimidine PP2 blocked the activation of TRPC channels by kisspeptin. Therefore, TRPC channels in GnRH neurons are receptor-operated,andkisspeptin activatesTRPCchannelsthroughPIP₂ depletionandcSrc tyrosine kinase activation, which is a novel signaling pathway for peptidergic excitation of GnRH neurons.