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Cystic Fibrosis and Ion Channel Research
Summary: Michael Welsh's research is focused on the pathophysiology of airway disease in cystic fibrosis and on the DEG/ENaC family of Na+ channels.
Cystic Fibrosis and CFTR
Cystic fibrosis (CF) is a common, lethal genetic disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). CFTR is an anion channel located in the apical membrane of epithelia, where it mediates transepithelial salt transport. Loss of CFTR in airway epithelia leads to airway infections, the current major cause of morbidity and mortality. Our lab is interested in how CFTR works, the pathophysiology of airway disease, and the development of new therapies.
CFTR. CFTR belongs to the ATP-binding cassette (ABC) transporter family and contains its defining features, including two nucleotide-binding domains (NBDs). Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <—> ADP + ADP) that regulates gating. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, and our results indicate that they share a common ATP-binding site in the second NBD. Our data suggest that at physiologic nucleotide concentrations the predominant enzymatic reaction is adenylate kinase rather than ATPase, and therefore gating involves little or no energy consumption. These results reveal novel transport energetics for an ABC transporter and suggest potential new therapeutic targets (Figure 1).
Pathogenesis of CF airway infection. Airway infections, especially with Pseudomonas aeruginosa, cause most CF mortality. Development of airway disease involves impaired local pulmonary host defense mechanisms, followed by adaptation of bacteria and host to a chronic infection state. Once P. aeruginosa colonize CF airways, they become impossible to eradicate. We recently showed that in CF lungs, P. aeruginosa exist as biofilms, matrix-encased communities specialized for surface persistence. Biofilms notoriously resist killing, which explains the antimicrobial resistance that plagues patients. These findings also underscore the need for therapeutic strategies focused on biofilms (Figure 2).
Our earlier work showed that the thin layer of liquid covering the airway surface contains a soup of antimicrobial factors. These factors can rapidly kill bacteria deposited on mucosal surfaces and prevent acute invasive infections. We wondered if the innate immune system also possesses specific antibiofilm defenses that might prevent chronic bacterial infections. We found that lactoferrin, an abundant constituent of the airway surface liquid, blocks P. aeruginosa biofilm development at concentrations well below those that prevent growth. By chelating iron, lactoferrin stimulates twitching, a specialized form of surface motility, causing the bacteria to wander across the surface instead of forming biofilms. These findings reveal a mucosal defense mechanism acting at a critical juncture in biofilm development, when bacteria stop roaming as individuals and aggregate into durable communities. This defense may prevent bacteria that survive initial killing from developing intractable biofilms (Figure 3).
Airway remodeling. A hallmark of CF is airway remodeling. To understand the mechanisms, we are focusing on growth factors. We found that human airway epithelia constitutively express both a ligand, heregulin-α, and its receptors, erbB2 and erB3–4. Although heregulin stimulates proliferation, airway epithelia divide at a low rate, suggesting heregulin-α does not bind its receptor under basal conditions. These observations raised the question of how epithelia control ligand-receptor interactions. In differentiated airway epithelia, we localized heregulin-α exclusively in airway surface liquid and the apical membrane, physically separated from erbB2/3–4, which segregated to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation the instant epithelial integrity is disrupted. Indeed, immediately following mechanical injury, heregulin-α activates erbB2 in cells at the edge of the wound, and this hastens restoration of epithelial integrity. This process may be important in the development of mucosal cancers, where loss of epithelial polarity or mislocalization of growth factor receptors could continuously expose receptors to ligand, thereby stimulating proliferation. This model also suggests a mechanism for abnormal receptor activation, when diseases such as CF, chronic bronchitis, and asthma increase epithelial permeability (Figure 4).
Adenovirus pathogenesis in airway. While attempting to develop gene transfer to CF airways, we found that recombinant adenoviruses are inefficient because the coxsackievirus and adenovirus receptor (CAR) lies on the basolateral surface of airway epithelia, inaccessible to apically applied vector. This finding raised the question of why adenovirus uses a basolateral receptor. During its life cycle, adenovirus binds CAR, enters cells, and replicates. It must then escape to the environment to infect a new host. Following infection, we found that human airway epithelia first release adenovirus basolaterally. Virus then courses between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced in excess during viral replication, facilitates apical escape. When fiber binds CAR, it disrupts junctional integrity, allowing virus to filter between cells and emerge apically. Thus, adenovirus exploits its receptor for two distinct steps in its life cycle: entry into host cells and escape across epithelial barriers (Figure 5).
Biology of DEG/ENaC Channels
Of all the vertebrate senses, touch and pain are the least understood at the molecular level. It has long been postulated that ion channels are core components of these sensors. Early clues identifying ion channel mechanosensors came from a mechanosensory screen in Caenorhabditis elegans that identified degenerin/epithelial Na+ channels (DEG/ENaC).
To understand the molecular bases of these sensations, we studied the acid-sensing ion channels (ASIC1, -2, and -3) in vertebrates and Pickpocket channel subunits in Drosophila. To learn whether DEG/ENaC channels are positioned to detect tactile stimuli, we localized subunits in specialized cutaneous mechanosensory structures, including pacinian corpuscles, Meissner corpuscles, Merkel cell–neurite complexes, and lanceolate fibers surrounding hair shafts. We also identified their interactions with cytosolic proteins that determine their localization, surface expression, and function, and may tether subunits to the cytoskeleton. Disrupting ASIC genes in the mouse produced mechanosensory and nociceptor defects. For example, ASIC2- and ASIC3-null mice showed specific abnormalities in the dynamic sensitivity of mechanoreceptors critical for light-touch perception and discrimination and the sensitivity of mechanoreceptors responding to noxious pinch (Figure 6).
Acid sensitivity suggested ASIC channels might also contribute to nociception. Consistent with this, we discovered ASIC3 in free nerve endings in the epidermis. Loss of ASIC3 blunted the response of a population of C fibers to acid and blocked development of secondary mechanical hyperalgesia induced by acid injected into skeletal muscle. Thus, our data suggest that ASIC subunits participate in heteromultimeric channel complexes in sensory neurons and that in different cellular contexts they may respond to mechanical stimuli or to low pH to mediate normal touch and pain sensation.
ASICs are also expressed in the central nervous system, and large acid-evoked currents have long been observed in central neurons. We established the molecular basis of those currents when we found that ASIC1 is required for H+-gated currents and ASIC2 modulates them. Our data suggest that ASIC1 is distributed to synapses and contributes to synaptic plasticity. Because we found ASIC1 heavily expressed in amygdala, we examined its effect on fear conditioning. ASIC1-null mice had less fear-related behavior than wild-type littermates, whereas mice overexpressing ASIC1a had more fear-related freezing. Our overall hypothesis is that ASIC1 contributes to the generation of fear, and understanding its role may help unravel the molecular underpinnings of human anxiety disorders (Figure 7).
Dr. Welsh also receives support for his research from the National Heart, Lung, and Blood Institute and the Cystic Fibrosis Foundation.
Last updated: September 25, 2008
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