Cary R. Boyd

Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl- channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.

The Endoplasmic Reticulum–associated Degradation of the Epithelial Sodium Channel Requires a Unique Complement of Molecular Chaperones

This study describes new yeast expression systems for each subunit of the heterotrimeric epithelial sodium channel (ENaC). We found that a significant amount of each subunit resides in the ER and is destroyed via ERAD. We also found that the chaperone requirements for ENaC subunit degradation were unlike any other ERAD substrate examined.

Novel determinants of epithelial sodium channel gating within extracellular thumb domains

Activity of the epithelial Na+ channel (ENaC) is modulated by Na+ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na+. We searched for determinants of Na+ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at γMet438 altered the Na+ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na+ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na+ self-inhibition. Mutations at the homologous site in the α subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na+ self-inhibition. Mutations at the homologous βAla422 resulted in minimal or no change in Na+ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing αG481Mβγ, αβγM438V, and αG481M/βγM438V, respectively. This pair of residues map to theα5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both αG481Cβγ and αβγM438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that αGly481 and γMet438 are functional determinants of Na+ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.

Allosteric Inhibition of the Epithelial Na Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na+ channel (ENaC) mediates the rate-limiting step in transepithelial Na+ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

Regulation of epithelial sodium transport by promyelocytic leukemia zinc finger protein

Aldosterone is the principal regulator of Na homeostasis, and thereby blood pressure. One of the main targets of aldosterone is the epithelial Na channel (ENaC) located in the apical membrane of target cells. Previous studies identified several genes involved in the regulation of ENaC such as SGK1; however, SGK1 knockout mice have only a mild salt-losing phenotype, indicating that further genes must be involved in the action of aldosterone. In our search for further aldosterone-regulated genes, we discovered that aldosterone, at physiological concentrations, induces the expression of the promyelocytic leukemia zinc finger protein (PLZF) in renal cortical collecting duct (CCD) cell lines that stably express mineralocorticoid receptors (MRs). This effect is rapid and does not require de novo protein synthesis, suggesting a direct action. Surprisingly, stable overexpression of human or mouse PLZF isoforms significantly decreased transepithelial Na transport in CCD cells while having no effect on the integrity of the monolayers. In parallel with the decline in Na transport, PLZF suppressed the mRNA levels of beta- and gamma-ENaC subunits. These observations suggest that PLZF is a negative regulator of ENaC in renal epithelial cells and might be part of a negative feedback loop that limits aldosterones stimulatory effects on sodium reabsorption.

Sodium Retention and Volume Expansion in Nephrotic Syndrome: Implications for Hypertension

Sodium retention is a major clinical feature of nephrotic syndrome. The mechanisms responsible for sodium retention in this setting have been a subject of debate for years. Excessive sodium retention occurs in some individuals with nephrotic syndrome in the absence of activation of the renin-angiotensin-aldosterone system, suggesting an intrinsic defect in sodium excretion by the kidney. Recent studies have provided new insights regarding mechanisms by which sodium transporters are activated by factors present in nephrotic urine. These mechanisms likely have a role in the development of hypertension in nephrotic syndrome, where hypertension may be difficult to control, and provide new therapeutic options for the management of blood pressure and edema in the setting of nephrotic syndrome.