Abstract:The CFTR channel is an essential mediator of electrolyte transport across epithelial tissues. CFTR opening is promoted by ATP binding and dimerization of its two nucleotide binding domains (NBDs). Phosphorylation of its R domain (e.g. by PKA) is also required for channel activity. The CFTR structure is unsolved but homology models of the CFTR closed and open states have been produced based on the crystal structures of evolutionarily related ABC transporters. These models predict the formation of a tetrahelix b… Show more
“…Additionally, they provide evidence that inhibition does not rely on catalysis of sphingomyelin molecules that may be directly associated with gating CFTR channels.Figure 2Inhibition is not membrane delimited: ( a ) CFTR currents recorded in the cell-attached configuration after activation with 0.2 mM IBMX were inhibited by 10 μg/ml SMase in the recording chamber suggesting activation of a signaling pathway that impinged upon the channels in the patch. ( b ) CFTR channels activated with 25 U PKA (expected activation of ~50% max) 36 , and recorded in the excised inside-out patch configuration were insensitive to 10 μg/mL SMase backfilled into the recording pipette. ( c ) Summary data showing fractional inhibition of currents in each configuration (p = 0.0021, unpaired t-test).
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Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR’s regulatory “R” domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.
“…Additionally, they provide evidence that inhibition does not rely on catalysis of sphingomyelin molecules that may be directly associated with gating CFTR channels.Figure 2Inhibition is not membrane delimited: ( a ) CFTR currents recorded in the cell-attached configuration after activation with 0.2 mM IBMX were inhibited by 10 μg/ml SMase in the recording chamber suggesting activation of a signaling pathway that impinged upon the channels in the patch. ( b ) CFTR channels activated with 25 U PKA (expected activation of ~50% max) 36 , and recorded in the excised inside-out patch configuration were insensitive to 10 μg/mL SMase backfilled into the recording pipette. ( c ) Summary data showing fractional inhibition of currents in each configuration (p = 0.0021, unpaired t-test).
…”
Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR’s regulatory “R” domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.
“…Single-channel currents were recorded from inside-out patches using the Molecular Devices Axopatch 200B/pClamp 10 system (29). Currents were obtained under symmetrical Cl − at room temperature.…”
Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments — ivacaftor and lumacaftor — can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to “precision” medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.
“…The exact mechanistic origin of these effects is not clear but is consistent with the ICLs acting as a physical and functional link between the cytoplasmic NBDs and the channel gate within the MSDs (12,32,33). Because the present study was focused on the functional role of positively charged amino acid side chains within the ICLs in contributing to the Cl Ϫ permeation pathway, we sought to isolate the effects of MTS modification on Cl Ϫ permeation.…”
Section: Methodsmentioning
confidence: 99%
“…Early homology modeling suggested that the TM extensions that make up the ICLs may form a narrow central "funnel" that forms a pathway for Cl Ϫ movement between the cytoplasm and the transmembrane pore formed by the TMs (9,10). However, such a funnel model was not supported by experiments investigating the functional effects of mutations within the ICLs on Cl Ϫ permeation (11,12). The cytoplasmic entrance to the pore therefore remains the final unexplored region of the Cl Ϫ permeation pathway.…”
Background:The cytoplasmic entrance to the CFTR channel pore is not defined. Results: Functionally important positively charged amino acid side chains attract Cl Ϫ to the cytoplasmic entrance to the pore.
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