| dc.description.abstract | PATHOLOGY
LOCAL SIGNALING MICRODOMAINS IN EXCITABLE CELLS:
DEFINING NOVEL ROLES FOR ANKYRIN-B IN ION
CHANNEL TARGETING AND REGULATION
CRYSTAL FAITH KLINE
Dissertation under the direction of Professor Peter J. Mohler
The focus of this project was to evaluate the role of ankyrin-B in the targeting and regulation of ion channels to membrane microdomains in excitable cells. Specifically, the project evaluates the role of ankyrin-B for the targeting and metabolic regulation of the KATP channel and defines the minimal binding region of the InsP3 receptor for ankyrin-B binding. Alterations in KATP channel localization and regulation are associated with severe abnormalities in glucose homeostasis; namely, neonatal diabetes and congenital hyperinsulinemia of infancy. Disruption in the proper localization of the InsP3 receptor to the ER/SR membrane has been linked to abnormal intracellular Ca2+ dynamics in cardiomyocytes, resulting in arrhythmia.
Using an array of in vivo and in vitro techniques, this project presents evidence supporting a role for ankyrin-B in the targeting and metabolic regulation of Kir6.2, the pore-forming subunit of the KATP channel. Specifically, in vitro binding analysis demonstrates that an eight amino acid motif in the C-terminal domain of Kir6.2 is both necessary and sufficient for interaction with ankyrin-B. Furthermore, when three key acidic residues in this motif are mutated to an opposite charge, association with ankyrin-B is lost. Most exciting is the discovery that a known human mutation, E322K, resides in this motif and is associated with permanent neonatal diabetes mellitus. In vivo work demonstrates that the E322K mutation results in two phenotypes (disruption of Kir6.2 targeting to the plasma membrane and a decrease in ATP sensitivity) that can be resolved using a mathematical model of beta cell electrical activity. The observation of an ankyrin-B/Kir6.2 interaction represents a novel discovery, but more importantly, provides a potential target for therapeutic modulation of KATP channel activity.
Similar in vitro and in vivo techniques were used to determine a minimal binding region on the InsP3 receptor necessary for association with ankyrin-B. This determination resolves a long-standing debate regarding the validity of in vivo ankyrin-B/InsP3 receptor interactions. Furthermore, identification of a plausible ankyrin-B-binding domain on the InsP3 receptor has far-reaching implications with regard to the formation of macromolecular ER/SR signaling complexes that are involved in the targeting and regulation of InsP3 receptor activity in vivo.
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