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Title:Amphotericin B ion channels restore cystic fibrosis airway surface physiology
Author(s):Muraglia, Katrina Anne
Director of Research:Burke, Martin D
Doctoral Committee Chair(s):Burke, Martin D
Doctoral Committee Member(s):Grosman, Claudio; Sligar, Stephen G; Tajkhorshid, Emad
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):molecular prosthetics
cystic fibrosis
small molecules
amphotericin b
ion channels
Abstract:Just as artificial limbs replace the physical function of a missing arm or leg, small molecule surrogates acting as prosthetics on the molecular scale could replace protein functions that are either dysfunctional or absent in humans. These ‘molecular prosthetics’ could build upon and interface with the existing architecture and residual function of the organism to restore normal physiology. Importantly, small molecules with the capacity to move ions across lipid membranes and act as surrogates for channels or transporters have already been shown to restore physiology in protein-deficient cells and animals. This approach could now potentially address diseases caused by a deficiency of protein function, even in cases of severely reduced or absent protein production, such as cystic fibrosis (CF). Amphotericin B (AmB) is a natural product known to self-assemble into nonspecific ion channels in sterol-containing lipid membranes. It was long believed that AmB killed yeast through its inherent ion channel activity, existing primarily in the form of small aggregates that could insert into the lipid bilayer and cause toxic permeabilization. We demonstrate here that AmB actually predominantly forms large, extramembranous aggregates that kill yeast by extracting ergosterol (Erg), a required sterol in yeast membranes. Additionally, AmB is toxic to cells only when the ratio of AmB present exceeds that of membrane sterols. These mechanistic findings led to the development of two strategies to separate the ion channel forming capacity of AmB from its toxicity to eukaryotic cells. The first strategy utilizes low doses of AmB so as not to exceed the total membrane sterol content. The second strategy is to pre-complex AmB with the native sterol (ergosterol or cholesterol) to mitigate toxicity while retaining the channel forming activity. These two strategies proved to be successful in restoring growth to a strain of yeast missing essential potassium transporter proteins. Cystic fibrosis (CF) is caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, some of which result in little to no protein produced, rendering protein substrate-dependent current therapies ineffective in these cases. Loss-of-function mutations in the CFTR anion channel result in reduced apical bicarbonate transport and decreased airway surface liquid (ASL) pH, which impairs respiratory host defenses and leads to chronic lung infections. Because basolateral pumps and channels remain active in the absence of functional CFTR, we hypothesized that an outward-facing bicarbonate gradient would develop across the apical membrane, which could be harnessed by a bicarbonate-permeable small molecule channel. Our current mechanistic understanding of the AmB ion channel allowed us to use it as a probe to test that hypothesis that an imperfect but anion-permeable small molecule ion channel surrogate for CFTR could restore ASL physiology. Here we report that AmB facilitates apical bicarbonate transport thereby increasing ASL pH. This effect is sustained for at least 48 hours and restores ASL viscosity and antimicrobial activity in CF patient-derived human lung epithelia across a range of genotypes, including those with little to no production of CFTR. AmB similarly increases ASL pH in CFTR-/- piglets. Dependence of the AmB-mediated rescue on Na+/K+ ATPase indicates that this unselective CFTR surrogate is functionally interfaced with the endogenous ion transport network driving transepithelial bicarbonate movement. Additionally, non-channel-based activities of CFTR, including regulation of other apical ion transporters, are not required for maintaining these key parameters of ASL physiology. These results suggest a potential CFTR-independent and therefore genotype-agnostic mechanism for addressing CF.
Issue Date:2018-06-14
Type:Thesis
URI:http://hdl.handle.net/2142/101755
Rights Information:Copyright 2017 Katrina Anne Muraglia
Date Available in IDEALS:2018-09-27
Date Deposited:2018-08


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