A PDZ-Binding Motif is Essential but Not Sufficient to Localize the C Terminus of CFTR to the Apical Membrane

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L\u3e for apical localization. However, two other C-terminal regions, encompassing amino acids 1370-1394 and 1404-1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif

Pulmonary Function and Clinical Observations in Men With Congenital Bilateral Absence of the Vas Deferens

Congenital bilateral absence of the vas deferens (CBAVD) was once thought to be a distinct clinical entity, but genetic similarities in men with cystic fibrosis (CF) and CBAVD are described increasingly. We evaluated the clinical status, growth and nutritional state, and respiratory function of 18 men with CBAVD to determine whether these men with different CF transmembrane regulator (CFTR) genotypes may have clinical evidence of mild CF. Following a thorough history and examination, pulmonary function tests, sweat test, and renal ultrasound were performed. Genetic evaluation for 50 known CF mutations, screening for private mutations (single-strand conformational polymorphism and direct sequencing), and assay of the length of the polypyrimidine tract in the splice site acceptor of intron 8 was performed. A history of pulmonary disease was present in three, and an additional man had some features suggestive of malabsorption. Results of general physical examination and anthropomorphic measurements were unremarkable in all patients, with a mean (SD) body mass index of 26 (3). Pulmonary function tests of large and small airway function as well as lung volumes were normal in all except one whose results were consistent with moderate asthma. Five men were compound heterozygotes for CFTR mutations, four of whom had positive sweat tests (sweat chloride >60 mEq/L). Twelve men were heterozygotes for CFTR mutations while no mutations were identified in one man. Although putative etiologic factors may suggest that men with CBAVD and CFTR mutations could be considered within the spectrum of clinical CF, the authors suggest that in men with CBAVD without any other clinical features of CF, the diagnosis of CF may not be made

A PDZ-interacting domain in CFTR is an apical membrane polarization signal

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain–containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain–containing proteins play a key role in the apical polarization of ion channels in epithelial cells. J. Clin. Invest. 104:1353–1361 (1999)

Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain

Abstract Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of nociceptive sensory afferents in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain

Domestic-wild hybridization to improve aquaculture performance in Chinook salmon

Salmon farming is one of Canada\u27s fastest growing industries and contributes to Canada\u27s economy as well as creating jobs in rural areas; however, the industry is challenged by the need to balance production economics against environmental impacts. While Atlantic salmon (Salmo salar) are the most commonly farmed species on the west coast of Canada, Chinook salmon (Oncorhynchus tshawytscha) are a valuable alternative, as they fill a niche market and generate reduced environmental concerns because they are a native species. However, Chinook salmon have not been systematically domesticated, and their performance remains highly variable. Here we report on the results of a research program designed to develop a performance-enhanced hybrid Chinook salmon stock. Growth and survival were estimated for seven domestic-wild hybrid Chinook salmon crosses at various freshwater stages and during 15 months of saltwater rearing at a British Columbia Chinook salmon farm and compared with domestic-domestic crosses (control). The project included 8640 individually (PIT) tagged offspring from the domestic stock and seven domestic-wild hybrid stocks originating from the Lower Fraser Valley, Lower Mainland Vancouver, and Vancouver Island, British Columbia, Canada. Within each stock, milt from 10 sires was used to fertilize eggs pooled from 15 highly inbred domestic females to produce 80 half-sib families. Our breeding design allows the partitioning of stock and sire effects, and minimises maternal genetic and maternal environment effects. Replicates of all families were reared under common environmental conditions in both fresh- and salt water and monitored for body size and survival. There was significant variation in survival, body size, and saltwater biomass among the Chinook salmon hybrid stocks. The performance of some of the hybrid crosses exceeded that of the fully domesticated stock, although the pattern of performance varied with rearing stage. Overall, two hybrid stocks consistently outperformed the domestic stock in terms of survival, growth, and biomass estimates. We systematically assess production performance across a wide range of wild-domestic hybrid crosses in a Pacific salmon species, and our results highlight opportunities to improve the production performance of Chinook salmon culture