Modulation of Kir3 by lipids and tyrosine phosphorylation

Abstract

Neurotransmitters, hormones and growth factors act on ion channels to modulate excitable tissue. When a ligand binds a receptor it may activate ion channels in the membrane to initiate multiple intracellular events. One family of ion channels that are of particular interest is the G-protein gated-inwardly rectifying potassium channel (Kir3), the target of a variety of effectors. Activation of Kir3 produces distinct downstream events that modulate cellular function. An understanding of these pathways may elucidate the molecular mechanisms of excitability.Kir3 channel gating requires Gbetagamma, Na, PIP2 and ATP. I tested the hypothesis that Kir3 gating components have unique roles in downstream signal transduction events. Using in vitro expression systems I have determined that agonist-activation of the Gq-coupled human endothelin A receptor (HETAR) by endothelin-1 (Et-1) inhibits the Gi-coupled mu opioid receptor when coexpressed with Kir3 heteromultimers. Similarly, arachidonic acid mimics endothelin-inhibition of Kir3, suggesting that eicosanoids modulate Kir3. The response to MOR-activation was significantly inhibited by Et-1 activation of HETAR in homomeric channels composed of either Kir3.2 or Kir3.4. In contrast, homomeric channels of Kir3.1 were insensitive. Domain deletion and channel chimera studies suggested that the sites within the channel sensitive to Et-1-induced inhibition were within the region responsible for channel gating. Mutation of a single amino acid in the homomeric Kir3.1 to produce Kir3.1(F137S)(N217D) converted the channel from an arachidonic acid and endothelin insensitive channel to an eicosanoid-sensitive channel. The critical aspartate residue required for eicosanoid sensitivity is the same residue required for Na regulation of PIP2 gating. The results suggest a model of Kir3 gating that incorporates a series of regulatory steps including Gbetagamma, PIP2, Na and arachidonic acid binding to the channel gating domain.In another study, I identified specific tyrosine residues in the amino-terminus of Kir3 subunits that are the target of tyrosine kinase following BDNF activation of the TrkB receptor. I determined that electrostatic interactions may define a consensus site for tyrosine phosphorylation of Kir3.My thesis provides a new framework for the molecular basis of how Kir3 regulation activates downstream signal transduction pathways to modulate excitability.