Study type: In vitro · Status: Evidence verified by machine against declared source

Agonists that stimulate secretion promote the recruitment of CFTR into membrane lipid microdomains.

The Journal of general physiology · 2019

Study scale: Images are representative of n = 20–40 cells in at least two experiments under each condition.

Product or molecular entity relationships

VIP: Exact entity relationship. Legacy citation custody associates this source with the catalog record; no product-relevance conclusion is implied.

Public plain-language summary

The published text is limited to the declared source and preserves reported uncertainty. It does not establish efficacy, safety, suitability, or evidence strength.

Research question

CFTR channels are assumed to be static during physiological regulation due to tethering by sodium-hydrogen exchanger regulatory factor (NHERF) and other scaffold and adapter proteins, and the factors determining their distribution on the surface of well-differentiated airway epithelial cells are not well understood.

Study design

In addition to increasing CFTR abundance at the cell surface, secretagogue-induced partitioning into microdomains may modulate its regulation and the pharmacological rescue of mutant CFTR by corrector drugs used to treat CF. Sequestering of CFTR into lipid microdomains may bring it into proximity with different cohort of regulatory proteins in a macromolecular complex (Li and Naren, 2010) if proteins are differentially excluded into platforms as described for caveolin.

Participants or experimental system

CFTR forms macromolecular complexes with many proteins; however, little is known regarding its associations with membrane lipids or the regulation of its distribution and mobility at the cell surface.

Study scale

Images are representative of n = 20–40 cells in at least two experiments under each condition.

Intervention or exposure

Platform induction is prevented by pretreating cells with cholesterol oxidase to disrupt lipid rafts or by exposure to the ASMase functional inhibitor amitriptyline or the membrane-impermeant reducing agent 2-mercaptoethanesulfonate.

Comparator

To visualize endogenous CFTR and the effects of VIP, cells were exposed bilaterally to control medium or medium containing 200 nM VIP (Sigma-Aldrich) for 1 h, then gently washed and scraped from the insert into PBS and centrifuged onto glass coverslips (500 rpm, 5 min; Cytospin 4; Thermo Fisher Scientific).

Outcomes examined

We identified two distinct CFTR channel populations in the plasma membrane of primary human bronchial epithelial cells that had been transduced with adenovirus containing enhanced GFP (EGFP)-CFTR, one population that is diffusely distributed and another that is in cholesterol-dependent clusters (Abu-Arish et al., 2015).

Key findings

CFTR clusters are closely associated with motile cilia and with the enzyme acid sphingomyelinase (ASMase) that is constitutively bound on the cell surface.

Limitations and uncertainty

Although the clusters (in contrast to the platforms) were optical resolution–limited size in the images and this approach provides only an upper estimate of microdomain area, measuring the subdiffraction-sized aggregates more accurately would only strengthen our conclusion regarding the different dimensions of rafts versus platforms.

Product relevance and evidence boundary

This record is a published study, held here with its citation and review status. It is not a statement that any catalog item is effective, safe, or suitable for any use, and nothing in it is a dose or a protocol.

Evidence verified by machine against declared source. Verification is limited to the declared source and review scope. It does not mean independent replication or establish efficacy, safety, or suitability.