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.
Highlights
Lu and coworkers[3, 4] found that two catalytically distinct orthologs of sphingomyelinase C (SMase) inhibited CFTR channel function in the Xenopus oocyte expression system
We began to characterize the inhibitory effect of sphingomyelinase C (SMase) on the chloride channel function of CFTR in two systems
We presented evidence that SMase is a novel gating modifier of CFTR that inhibits membrane embedded channels in Xenopus oocytes without relying on catalysis of CFTR-associated sphingomyelin or internalization of the channel
Summary
Lu and coworkers[3, 4] found that two catalytically distinct orthologs of SMase inhibited CFTR channel function in the Xenopus oocyte expression system. SMase activity leads to multiple biochemical and biophysical changes in cells; catalysis of sphingomyelin reduces the prevalence of positive charge in the outer-leaflet of the cell membrane[5], activates various signaling pathways in numerous cell types[6,7,8,9,10,11], facilitates the formation of membrane microdomains[12,13,14], and induces plasma membrane internalization[15, 16] It is currently unclear which of these processes may be responsible for inhibition of CFTR chloride currents in the Xenopus expression system. These results suggest that SMase inhibits CFTR channels via a novel, state-dependent mechanism and that the presence of SMase in the pulmonary interstitial space may diminish the ability to activate CFTR
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