Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Roderick MacKinnon
Keywords
G protein-coupled receptors (GPCRs), ion channels, cell signaling, Kir2.2, M2R-GIRK signaling, protein clustering
Abstract
G-protein-coupled receptors (GPCRs) regulate a variety of downstream effector proteins, making them essential for many physiological processes. For example, in the heart, the activation of beta-adrenergic receptors (βARs) and the stimulatory G protein (Gαs) leads to the opening of downstream hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, resulting in an accelerated heart rate. In contrast, activation of M2 muscarinic receptors (M2Rs) and the inhibitory G protein (Gαi) opens G protein-gated inward rectifier K⁺ (GIRK) channels, slowing the heart rate. In addition to the Gαs and Gαi pathways, there is also the Gαq signaling pathway. In this pathway, activation of Gq-coupled GPCRs can lead to the activation of phospholipase Cβ (PLCβ). This enzyme can then hydrolyze phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) and thus decrease inward rectifier K+(Kir) channel activity. While the major components of these signaling pathways have been extensively studied, many unknowns remain regarding how GPCRs regulate channels. How do different signaling lipids regulate the activity of ion channels? To address this question, the first part of the thesis presents an analysis of how phosphatidylinositol lipids regulate the gating of Kir2.2 channels. In this study, I employed the planar lipid bilayer system to study how PI(4,5)P2 concentration affects the single-channel kinetics of Kir2.2. I show that Kir2.2 displays bursting behavior in the presence of PI(4,5)P2. Increasing PI(4,5)P2 concentration shortens Kir2.2 interburst duration and lengthens burst duration, without affecting the kinetics within the burst. To study the contribution of each phosphate on PI(4,5)P2 in activating the channel, I tested the response of Kir2.2 to different phosphoinositides (PIPs). I show that 5' phosphate is essential to Kir2.2 gating. Other PIPs without 5' phosphate can compete with PI(4,5)P2 but cannot activate Kir2.2. A cell typically has multiple signaling pathways operating simultaneously, often even sharing the same components. All these proteins constantly move within the cell membrane. How does each protein recognize its downstream targets and ensure that the signaling process functions specifically? In the second part of my thesis, using electron microscopy I show that five membrane proteins – three GPCRs, an ion channel, and an enzyme – form self-clusters under natural expression levels in a cardiac-derived cell line. The cluster size distributions imply that these proteins self-oligomerize through weak interactions. I then investigated the biological function of protein clustering. In this part, I examined the role of protein distributions in the M2R-GIRK channel signaling. I found that a positive bias exists for GIRK clusters to be near M2R clusters. I conclude that M2R clusters increase Gβγ concentration locally. The correlation between the electron microscopy findings and electrophysiology suggests that only a small fraction of GIRK channels—those close to M2R clusters—can be regulated by M2R. By invoking weak interactions that permit transient binding of M2R to M2R and GIRK to GIRK and M2R to GIRK the distribution patterns and electrophysiological properties of the HL-1 cell membrane are replicated in a reaction-diffusion simulation. Based on the simulation, I propose that both protein clustering and the positive bias are crucial to the M2R-GIRK channel signaling pathway.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Zhang, Yuxi, "GPCR to Channel Communication in a Membrane Signaling Pathway" (2025). Student Theses and Dissertations. 832.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/832
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy