The Roles of Focal Adhesion Assembly in Membrane Localization of Ion Channels and Action Potential Shape
Action potential firing in various excitable cells (cardiomyocytes, neurons, smooth and skeletal muscle cells) is enabled by the presence of inwardly rectifying potassium channels, such as Kir2.1, which set the resting membrane potential to a negative value allowing the activation of depolarizing voltage-gated currents. In the heart, Kir2.1-mediated IK1 current is upregulated during development, but downregulated in certain pathologies, including myocardial infarction, heart failure, and the Anderson-Tawil syndrome, a congenital disease characterized by periodic paralysis and polymorphic tachycardias. Such electrophysiological changes in the heart are often associated with profound alterations in cardiomyocyte size and shape, extracellular matrix (ECM), as well as cytoskeletal tension. Cardiomyocytes sense physical changes in their environment through integrins, heterodimeric receptors for ECM proteins that relay information to focal adhesions (FAs), force-sensitive sub-cellular structures connected to actin cytoskeleton. In different systems, dynamic changes in FA proteins as well as current flow through ion channels, including Kir2.1, are known to govern important cellular processes, including survival, proliferation, differentiation, migration, and matrix remodeling. Still, relationships between ion channels, FAs, and mechanosensitive cell electrophysiology are not well-understood. The objective of this thesis has been to explore how changes in integrin engagement and FA assembly within excitable cells affect Kir2.1 membrane localization, IK1 amplitude, and action potential characteristics. We hypothesized that integrin engagement will increase the number of active Kir2.1 channels to the membrane, thus increasing IK1 amplitude and changing action potential characteristics.
To accomplish this objective, we utilized a monoclonal line of HEK293 cells engineered to express fluorescently tagged Kir2.1 to visualize channels, a monoclonal line of HEK293 cells (“Ex293”) engineered to express Kir2.1, cardiac sodium channel Nav1.5, and gap junctional channel Connexin-43 as a well-defined excitable cell source, and neonatal rat cardiomyocytes as native excitable cells. Using microfabrication techniques, we created a platform for robustly and precisely controlling cell shape and size. Combining this technique with single cell electrophysiology and quantitative image analysis, we characterized local and global membrane distributions of Kir2.1, IK1 amplitude, and FAs. We established that membrane-bound Kir2.1 localizes in proximity to FAs, giving a non-uniform distribution of IK1 in the cell membrane.
Next we applied micropatterning of ECM proteins, pharmacological, and environmental manipulations to alter FA size and distribution in individual cells. Combining these techniques with single cell electrophysiology, confocal and total internal reflection fluorescence (TIRF) microscopy, and quantitative image analysis, we provide evidence that FAs and active integrins play a critical role in regulating Kir2.1 membrane localization, IK1 amplitude, and action potential morphology in excitable cells.
Furthermore, by studying Kir2.1 turnover dynamics using fluorescence recovery after photobleaching (FRAP), we show that the channels are uniformly transported to the membrane, where they preferentially accumulate near FA-rich sites via the local inhibition of dynamin-mediated endocytosis. We conclude this thesis with a modeling summary of the Kir2.1 trafficking and localization near FAs at the membrane.
Overall, this thesis shows links between the electrophysiology of ion channels and FA biology, coupling action potential dynamics to changes in the cellular environment and cytoskeletal mechanics. Our results propose a novel mechanism whereby engaged integrins are an important regulator of the membrane localization of Kir2.1 channels via the local inhibition of dynamin-dependent endocytosis. This mechanism renders the IK1 and cellular electrophysiology indirectly mechanosensitive to various intra- and extracellular signals affecting integrin engagement and FA dynamics. The work in this thesis warrants future in-depth studies of how cell-matrix interactions modulate the function of various ion channels across diverse cell types and pathophysiological conditions.
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