Combined Phosphoinositide and Ca2+ Signals Mediating Receptor Specificity toward Neuronal Ca2+ Channels

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Ca2+ (I-Ca) and M-type K+ currents in superior cervical ganglion sympathetic neurons. In those cells, M-1 muscarinic and AT(1) angiotensin types do not elicit Ca-i(2+) signals and suppress both currents via depletion of PIP2, whereas the B-2 bradykinin and P2Y purinergic types elicit robust IP3-mediated [Ca2+](i) rises and neither deplete PIP2 nor inhibit I-Ca. We have suggested that this specificity arises from differential Ca-i(2+) 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 Ca-i(2+) signals. Recordings of I-Ca were used as a PIP2 "biosensor." In control, stimulation of M-1, but not B-2 or P2Y, receptors robustly suppressed I-Ca. However, when PI 4-kinase III beta, diacylglycerol kinase, Rho, or Rho-kinase was blocked, agonists of all three receptors robustly suppressed I-Ca. Overexpression of exogenous M-1 receptors yielded large [Ca2+](i) rises by muscarinic agonist, and transfection of wildtype IRBIT decreased Ca-i(2+) signals, whereas dominant negative IRBIT-S68A had little effect on B-2 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.