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Author Notes:

N. A. McCarty, Email: namccar@emory.edu

B.B.S. and N.A.M. designed the research. B.B.S., G.C., K.A.C., and D.T.I. performed experiments.

B.B.S. analyzed the data. B.B.S. and N.A.M. interpreted the results. B.B.S. and N.A.M. drafted the manuscript. B.B.S., N.A.M., G.C., and D.T.I. edited the manuscript.

The authors thank Criss Hartzell, Andrew Jenkins, Randy Hall, and Edward Balog for guidance and many insightful discussions.

We would also like to thank Joanna Goldberg and Jeffrey Meisner for their assistance with Sphingomyelinase C purification.

We would like to thank Sam Molina for his assistance with the confocal microscopy and the Emory University CF@LANTA RDP Experimental Models Support Core for providing HBEs.

The authors declare that they have no competing interests.

Subjects:

Research Funding:

This research project was supported in part by the Emory University Integrated Cellular Imaging Microscopy Core of the Emory + Children’s Pediatric Research Center.

The mCherry-NT-lysenin construct was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.

This work was supported by National Institutes of Health [Grants DK-056481, DK-075016 to N.A.M.] and the Cystic Fibrosis Foundation [CFF MCCART14GO to N.A.M. and the CF@LANTA CFF-RDP predoctoral fellowship to B.B.S.].

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • GREEN FLUORESCENT PROTEIN
  • CANINE KIDNEY-CELLS
  • EPITHELIAL-CELLS
  • R DOMAIN
  • INTERMOLECULAR INTERACTIONS
  • ACID SPHINGOMYELINASE
  • PHOSPHORYLATION SITES
  • PORE ARCHITECTURE
  • CHLORIDE CHANNEL
  • ATP HYDROLYSIS

Bacterial Sphingomyelinase is a State-Dependent Inhibitor of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR)

Tools:

Journal Title:

Scientific Reports

Volume:

Volume 7, Number 1

Publisher:

, Pages 2931-2931

Type of Work:

Article | Final Publisher PDF

Abstract:

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.

Copyright information:

© The Author(s) 2017.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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