Depletion of PIP2, Rather than Calcium Rise or PKC Activation, is Responsible for Inhibition of KCNQ2/3 Channels by Gq-Coupled Receptors in TSA201 Cells

Abstract

Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyses the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) into the second messengers diacylgycerol and inositoltrisphosphate, leading to (i) protein kinase C (PKC) activation, (ii) calcium release, and in some cases (iii) PIP2 depletion. Which of these three events is responsible for the inhibition of KCNQ2/3 (Kv7.2/7.3) potassium channels? We measure in tsA201 cells PKC activity by a FRET-based sensor (CKAR), calcium by Fura4F, PIP2 by PH domains (PH-PLCδ1), and find that activating endogenous purinergic receptors by UTP leads to PKC activation and calcium release, but not PIP2 depletion. With activation of overexpressed M1 muscarinic receptors by 10 µM oxotremorine-M (oxo-M), the amplitude and time course of the CKAR and Fura responses are much like with UTP, but PIP2 is depleted. KCNQ2/3 channels are inhibited only by oxo-M (85%) and not by UTP (<1%), indicating that it is the PIP2 depletion that inhibits KCNQ2/3 in response to oxo-M. PLC activation by UTP can be boosted by overexpressing G-proteins or P2Y2 receptors. Then UTP depletes PIP2 and does inhibit KCNQ2/3 (92%). Overexpression of AKAP79 or CaM-YFP does not increase KCNQ2/3 inhibition by UTP, but with AKAP79, KCNQ2/3 can be partly inhibited (15%) by adding OAG to maximally activate PKC.We conclude that: (1) PLC activation can elicit full-size calcium and PKC responses without depleting PIP2. (2) G-protein abundance regulates downstream effects of purinergic receptors in tsA201 cells. (3) In response to UTP and oxo-M the PIP2 depletion inhibits KCNQ2/3 in tsA201 cells, but PIP2 depletion is not necessary for KCNQ2/3 inhibition under all circumstances.Supported by NIH grant R01 NS08174 and the Human Frontier Science Program Organization