Analysis of Phosphoinositide-Dependence of Action Potential Firing in Sympathetic Neurons by Electrophysiological Recordings and Mathematical Modeling

Abstract

Superior cervical ganglion (SCG) neurons play an important role for the innervation of the pineal gland. Due to their essential role in controlling the circadian rhythm, we analyzed action potential firing and its dependence on the rare phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in SCG neurons isolated from adult male rats by a combination of electrophysiological, biochemical, and mathematical approaches. Previously, we found action potential firing to be controlled by KCNQ2/3 and Maxi-K potassium channels, and a decrease in their activities by more than 80% was necessary to evoke action potential firing. Physiologically, action potential firing in SCG neurons is induced by activation of muscarinic acetylcholine receptors, which triggers activation of phospholipase C and hydrolysis of PI(4,5)P2. While KCNQ2/3 channel activity has been shown to be strongly dependent on PI(4,5)P2, a regulation of Maxi-K channel activity by phosphoinositides is under debate and has been speculated to be dependent on the subunit composition of Maxi-K channels. To analyze which Maxi-K subunits are expressed in SCGs, we isolated total RNA from SCGs and performed RT-PCR with oligonucleotides for both α- and β-subunits of Maxi-K channels. Our results showed expression of only the Maxi-K α-subunit. Expression of Maxi-K α-subunits in HEK293 cells together with either muscarinic acetylcholine receptors or a voltage-sensitive lipid phosphatase to transiently deplete PI(4,5)P2 showed no decrease in Maxi-K channel activity upon depletion of PI(4,5)P2. We incorporated our electrophysiological and biochemical results into a model of phosphoinositide metabolism of SCG neurons previously published by our group to develop a mathematical description of phosphoinositide metabolism and PI(4,5)P2-dependent ion channel activity in SCG neurons. In conclusion, our model allows for the first time a simulation of phosphoinositide metabolism and its regulation of action potential firing of SCG neurons.