Combined phosphoinositide and Ca²⁺ signals mediating receptor specificity toward neuronal Ca²⁺ channels

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Ca ²⁺ (I_Ca) and M-type K⁺ currents in superior cervical ganglion sympathetic neurons. In those cells,M₁ muscarinic and AT₁ angiotensin types do not elicit Ca²⁺_i signals and suppress both currents via depletion of PIP₂, whereas the B₂ bradykinin and P2Y purinergic types elicit robust IP ₃-mediated [Ca²⁺]_i rises and neither deplete PIP₂ nor inhibit I_Ca. We have suggested that this specificity arises from differential Ca²⁺_i signals underlying receptor-specific stimulation of PIP₂ synthesis by phosphatidylinositol (PI) 4-kinase. Here, we investigate which PI 4-kinase isoform underlies this signal, whether stimulation of PI 4-phosphate 5-kinase is also required, and the origin of receptor-specific Ca²⁺_i signals. Recordings of ICa were used as a PIP₂ "biosensor." In control, stimulation of M₁, but not B₂ or P2Y, receptors robustly suppressed I_Ca. However, when PI 4-kinase IIIβ, diacylglycerol kinase, Rho, or Rho-kinase was blocked, agonists of all three receptors robustly suppressed I_Ca. Overexpression of exogenous M₁ receptors yielded large [Ca²⁺]_i rises by muscarinic agonist, and transfection of wild-type IRBIT decreased Ca²⁺_i signals, whereas dominant negative IRBIT-S68A had little effect on B₂ or P2Y responses but greatly increased muscarinic responses. We conclude that overlaid on microdomain organization is IRBIT, setting a "threshold" for [IP₃], assisting in fidelity of receptor specificity.