January 2012

Document Type


Degree Name



Oregon Health & Science University


The principal mediators of potassium conductance are potassium ion (K[superscript +]) channels, a superfamily of integral membrane proteins whose subunits assemble to form an aqueous pore. These pores utilize a filter to selectively permeate K[superscript +] ions through passive diffusion down a concentration gradient across the plasma membrane of a cell. Potassium conductance dominates at the resting membrane potential of a cell, and shapes the waveform, duration, and strength of cell repolarization. In order to regulate ion conduction in response to various stimuli, K[superscript +] channels open and close in a process called gating. Gating defines the kinetics of channel currents that determine K[superscript +] flux and thus cell excitability. A major scientific research goal is to develop a complete picture of the motions that occur in the channel molecule during gating to better understand channel function. A remarkable family of K[superscript +] channels is the inward rectifiers (Kir). Their namesake property of rectification originates from block of the channel pore by multivalent cations in the cell cytosol. Kir channels possess the intrinsic capacity to rearrange the K[superscript +] conduction pathway through interactions with phosphatidylinositol-4,5-bisphosphate (PIP[subscript 2]) and other intracellular ligands that bias their gating conformation. Four transmembrane α-helix protein segments (TM2) line the conduction pathway, a single segment contributed from each of the K[superscript +] channel subunits. Extensive scientific literature champions a model in which the TM2 helices partake in a bending motion critical to the gating mechanism. A hypothesis for channel gating was formed that a well-conserved glycine residue in the middle of each TM2 segment acts as a hinge to provide flexibility necessary for the helices to bend. A disease mutation was identified in a patient with congenital hyperinsulinism (CHI). The mutation lies in the gene which encodes the pore subunits of the ATP-sensitive potassium (K[subscript ATP]) channel, and it replaces the glycine hinge with positively-charged arginine. K[subscript ATP] channels are a Kir family member that regulate K[superscript +] conductance through interactions with intracellular nucleotides, coupling cellular metabolic state with excitability. They play a crucial role in glucose-dependent insulin secretion, and loss of channel function causes CHI. Presented in this dissertation are studies using in vitro structure/function methods to understand the role of the TM2 glycine as a hinge in K[subscript ATP] channels. Chapter 1 studies the consequence of the glycine-to-arginine CHI mutation on channel function. Results show the positively-charged mutation eliminates ion conduction by altering the electrostatic environment of the pore. Channels incorporating mutant and wild-type subunits conduct potassium ions, consistent with the heterozygous patient phenotype and clinical outcome. K[superscript +] currents were also recovered when a second negatively-charged mutation was placed in close proximity to stabilize the electrostatic environment in the pore. Position of the second mutation was ideal to establish a direct electrostatic interaction that restores ion conduction and gating. Nucleotide regulation in the double mutant channels was the same as WT, showing the glycine hinge is not essential for K[subscript ATP] channel gating. In Chapter 2, an attempt to reduce TM2 flexibility by mutating glycine to proline uncovered an unexpected phenotype called inactivation. Inactivation was defined as a severe reduction in spontaneous K[subscript ATP] channel activity. Many native K[superscript +] channels exhibit C-type inactivation, a gating process that depends on permeating ions interacting with the selectivity filter. The glycine residue is in close proximity to the selectivity filter in Kir channels, and a glycine mutation could potentially stabilize a closed filter conformation in K[subscript ATP]. However, current decay kinetics of G156P K[subscript ATP] channels did not change when ionic conditions were altered arguing against the mutation inducing C-type inactivation. An alternative hypothesis comes from previously identified inactivation mutations found in the cytoplasmic domain of K[subscript ATP] channels that disrupt intersubunit interactions. Cytoplasmic domains form the binding sites for regulatory molecules PIP[subscript 2] and ATP, and intersubunit interactions between cytoplasmic domains stabilize channel activation. G156P channel inactivation was sensitive to PIP[subscript 2] modulation and more significantly recovers from inactivation after channel inhibition by ATP, suggesting the mutation causes loss of coupling between the ligand-binding cytoplasmic domain and downstream gating apparatus in the transmembrane domain that is required to sustain channel activation. Chapter 3 summarizes additional mutagenesis study of the glycine hinge, and attempts to redress conceptual caveats of a hinge mechanism. The conclusions of this dissertation enhance our understanding of the transmembrane pore environment and its interaction with other channel domains, and they lay the groundwork for future considerations of the hinge hypothesis.




Neuroscience Graduate Program


School of Medicine



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