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

> **NIH NIH R35** · TEXAS TECH UNIVERSITY · 2024 · $428,400

## Abstract

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 f...

## Key facts

- **NIH application ID:** 10827942
- **Project number:** 5R35GM124979-07
- **Recipient organization:** TEXAS TECH UNIVERSITY
- **Principal Investigator:** Benjamin James Wylie
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $428,400
- **Award type:** 5
- **Project period:** 2017-08-10 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10827942

## Citation

> US National Institutes of Health, RePORTER application 10827942, The Functional Interplay of Lipid Membrane Components: Activation, Inhibition, and Raft Formation. (5R35GM124979-07). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10827942. Licensed CC0.

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