Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(ß,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.

Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

HRT1 modulates vascular smooth muscle cell proliferation and apoptosis.

The Notch signaling pathway plays vital roles in vascular development and homeostasis. However, the functional role of HRT1, a primary downstream effector of Notch signaling in VSMC, is poorly characterized. In the present study, we postulated that HRT1 plays fundamental roles in modulating VSMC fate. To test the hypothesis that HRT1 is coupled to growth regulation, we generated VSMC lines constitutively overexpressing HRT1 (HRT1SMC) and demonstrated an exaggerated growth behavior compared to its control cell line. The lack of cell cycle arrest at confluence in HRT1SMC was associated with an attenuated up-regulation of the cell cycle inhibitor, p21(WAF1/CIP1). We further established that both transient and constitutive HRT1 signaling promoted VSMC survival in response to serum deprivation and pro-apoptotic Fas ligand. Resistance to apoptosis was associated with the induction of Akt expression/activity, a well-described anti-apoptotic mediator. Overall, these findings provide initial evidence that HRT1 functions as a critical determinant of VSMC proliferation and survival.

Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis.

Mutations in the Notch3 receptor result in the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome, a heritable arteriopathy predisposing to early onset stroke. Based upon clinical evidence that CADASIL arteriopathy results in degeneration and loss of vascular smooth muscle cells (VSMC) from the arterial wall, we postulated that Notch3 signaling is a critical determinant of VSMC survival. We initially established that both transient and constitutive Notch3 signaling promoted VSMC survival in response to the proapoptotic Fas ligand (FasL). Resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the FasL signaling pathway. We determined that Notch3's regulation of c-FLIP was independent of the activity of the classical DNA-binding protein, RBP-Jk, but dependent upon cross-talk activation of the ERK/MAPK pathway. We extended our observations to the in vivo context by determining a coordinate regulation of Notch3 and c-FLIP within the arterial wall in response to injury. Furthermore, we defined that expression levels of Notch3 and c-FLIP are coordinately up-regulated within the neointima of remodeled arteries. Taken together, these findings provide initial evidence that Notch3 signaling may be a critical determinant of VSMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis via signaling cross-talk with the ERK/MAPK pathway.

Coordinate Notch3-hairy-related transcription factor pathway regulation in response to arterial injury. Mediator role of platelet-derived growth factor and ERK.

The Notch family of receptors and downstream effectors plays a critical role in cell fate determination during vascular ontogeny. Moreover, the human cerebral autosomal dominant artriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) syndrome of premature stroke and dementia is a heritable arteriopathy with alterations in vascular smooth muscle cells (VSMCs) resulting from mutations within Notch3. However, the expression and regulation of the Notch and hairy-related transcription factor (HRT) pathway in adult VSMCs in vitro and in vivo remain poorly characterized. The present study documents that the well-described modulation of VSMC fate in response to vascular injury and growth factor activation involves a coordinate regulation of the Notch and HRT pathways. Furthermore, platelet-derived growth factor promotes a similar coordinate down-regulation of the Notch receptors and HRT genes in cultured VSMCs via an ERK-dependent signaling pathway. Moreover, we established that HRT1 and HRT2 are direct downstream target genes of Notch3 signaling in VSMCs and determined that the activity of the nuclear protein RBP-Jk is essential for their regulation. These findings provide initial insight into the context- and cell type-dependent coordinate regulation of Notch3 and downstream HRT transcriptional pathway effector genes in VSMCs in vitro and in vivo that may have important implications for understanding the role of Notch signaling in human health and vascular disease.