Experiments to test the role of phosphatidylinositol 4,5-bisphosphate in neurotransmitter-induced M-channel closure in bullfrog sympathetic neurons

Various neurotransmitters excite neurons by suppressing a ubiquitous, voltage-dependent, noninactivating K+ conductance called the M-conductance (g(M)). In bullfrog sympathetic ganglion neurons the suppression of g(M) by the P2Y agonist ATP involves phospholipase C (PLC). The present results are consistent with the involvement of the lipid and inositol phosphate cycles in the effects of both P2Y and muscarinic cholinergic agonists on g(M). Impairment of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) with the phosphatidylinositol 4-kinase inhibitor wortmannin (10 mum) slowed or blocked the recovery of agonist-induced g, suppression. This effect could not be attributed to an action of wortmannin on myosin light chain kinase or on phosphatidylinositol 3-kinase. Inhibition of PIP2 synthesis at an earlier point in the lipid cycle by the use of R59022 (40 mum) to inhibit diacylglycerol kinase also slowed the rate of recovery of successive ATP responses. This effect required several applications of agonist to deplete levels of various phospholipid intermediates in the lipid cycle. PIP2 antibodies attenuated the suppression of g, by agonists. Intracellular application of 20 muM PIP2 slowed the rundown of KCNQ2/3 currents expressed in COS-1 or tsA-201 cells, and 100 muM PIP2 produced a small potentiation of native M-current bullfrog sympathetic neurons. These are the results that might be expected if agonist-induced activation of PLC and the concomitant depletion of PIP2 contribute to the excitatory action of neurotransmitters that suppress g,.