THESIS
2014
xiii, 78 pages : illustrations ; 30 cm
Abstract
Regulatory volume decrease (RVD) is crucial for cells to survive swelling. The volume
sensitive outwardly rectifying anion channel (VSOR) is considered to be the major anion
channel governing RVD in many tissues and cells. The cystic fibrosis transmembrane
conductance regulator (CFTR) functions as both an intracellular ligand-gated channel
and a regulator of other channels and transporters in many epithelial tissues. The loss of
CFTR impaired RVD of epithelial cells with elusive mechanism. Our lab found that
stretch activates CFTR and thus we hypothesize that CFTR may mediate epithelial RVD
by directly responding to the mechanical stress induced by cell-swelling. Here we
examined the molecular identity of volume-sensing anion channel in epithelia RVD by
blocking VSOR and CFTR a...[
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Regulatory volume decrease (RVD) is crucial for cells to survive swelling. The volume
sensitive outwardly rectifying anion channel (VSOR) is considered to be the major anion
channel governing RVD in many tissues and cells. The cystic fibrosis transmembrane
conductance regulator (CFTR) functions as both an intracellular ligand-gated channel
and a regulator of other channels and transporters in many epithelial tissues. The loss of
CFTR impaired RVD of epithelial cells with elusive mechanism. Our lab found that
stretch activates CFTR and thus we hypothesize that CFTR may mediate epithelial RVD
by directly responding to the mechanical stress induced by cell-swelling. Here we
examined the molecular identity of volume-sensing anion channel in epithelia RVD by
blocking VSOR and CFTR activities. In Calu-3 cells, an airway epithelial cell-line with highly expressed CFTR, hypotonicity-induced short circuit current (I
sc) is sensitive to
CFTR specific inhibitor but not to VSOR inhibitors. The l
sc was independent of
cytoplasmic ca
2+ levels and cAMP levels, suggesting the mechanosensitivity of CFTR
played a role. Moreover, genetic suppression or ablation of CFTR markedly reduced
swelling induced l
sc. In cultured CHO cells and freshly isolated intestinal crypts
expressing CFTR, blocking CFTR abolished RVD triggered by hypotonicity. RVD is
important in cell migration. We found that exogenous CFTR expression accelerated cell
migration in HEK293T cells. Surprisingly, GSS1D, a CFTR mutation lacking cAMP-induced
chloride channel activity, functioned like wild-type CFTR in swelling- or stretch-induced
l
sc, RVD and cell migration. Furthermore, cell swelling also activated CFTR in
cell-attached membrane patches in Calu-3 cells. Thus, our studies showed that both
wild-type and G551D CFTR might be involved in epithelial RVD by responding to the
mechanical stress induced by cell-swelling. Our findings also might explain the relative
milder phenotype of cystic fibrosis (CF) patients carrying G551D mutation and shed
light on developing novel mechanical treatment for CF.
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