The Functional Interplay of Lipid Membrane Components: Activation, Inhibition, and Raft Formation.

向内整流K+ (Kir)通道和G蛋白偶联受体（gpcr）是膜蛋白

Inward-rectifier K+ (Kir) channels and G protein-coupled receptors (GPCRs) are membrane proteins that are regulated by cholesterol and anionic lipids found in their native membranes. We will use solid-state NMR (SSNMR) to study proteins with functional lipids in bilayer environments ranging from proteoliposomes to biological membranes. These measurements will compliment functional assays, fluorescence techniques, and molecular dynamics (MD) simulations under identical conditions. Kir channels are involved in long-QT syndrome, hypoglycemia, Bartter’s syndrome, epilepsy, substance abuse, and periodic paralysis. Kir Channels are ligand gated, but details of the structure and dynamics of gated channels are largely unknown. The Kir2 channel family is gated by the anionic lipid phosphatidylinositol 4,5-bisphosphate (PIP2) but inactivated by cholesterol which competes with PIP2 to access the protein. G protein-activated Kir channels (GIRK, Kir3) are gated by the coaction of PIP2 and Gbγ protein heterodimers. In the Kir3 family, cholesterol increases rather than suppresses activity. Here we will explore the differing roles of functional lipids and quantify the structure and dynamics of the observed active and inactivated states. We will continue our studies of the Kir channel, KirBac1.1. We assigned 90% of the 15N and 13C chemical shifts in this protein (over 1600 unique heavy atoms) and used these assignments to identify allostery, the activation mechanism, the inactivated structure bound to a cholesterol dimer, refined the structure of the closed state, and solved the structure of the open state of the channel. Now we will measure the channel dynamics and identify the multiple gated states of the channel reflected in our data. We will study structural changes in the channel under voltage and identify discrete channel states and lipid contacts using freeze-trapped Dynamic Nuclear Polarization. In tandem, we will also study the Kir3.1-KirBac1.3 channel chimera. Preliminary data identifies PIP2 binding residues and membrane-water interfacial residues key for channel function. The eventual goal will be the mammalian Kir3.2 (GIRK2) channel and its full complement of functional activators. In a second project we will study the CC motif chemokine receptor CCR3 with the CCL11 chemokine in lipid bilayers. No drug trial targeting CCR3 has succeeded, which is unfortunate as it is involved in cancer metastasis, HIV entry, and the COVID19 cytokine storm. To date, we identified both CCL11 docking, and signal transduction are dose dependent upon bilayer cholesterol. Preliminary SSNMR studies found cholesterol conformationally selects for optimal ligand binding configurations of the receptor. We plan to fully assign the 15N and 13C chemical shifts of CCR3 in cholesterol and anionic lipid enriched membranes. The structures of this protein with CCLL11 in different functional states will be solved, and regional dynamics measured following a similar workflow established for KirBac1.1. NMR will also be used to solve the structures of CCL11 in solution and in complex with CCR3. We will pursue cholesterol oligomerization, CCR3 dimerization, and the relationship between these events. Throughout we will examine lipid oligomerization, dynamics, and protein affinity.