How to Measure CFTR-Dependent Bicarbonate Transport: From Single Channels to the Intact Epithelium

Abstract

Bicarbonate serves many functions in our body. It is the predominant buffer maintaining a physiological pH in the blood and within our cells. It is also essential for proper digestion of nutrients and solubilization of complex protein mixtures, such as digestive enzymes and mucins, in epithelial secretions. Transepithelial HCO3- transport also drives net fluid secretion in many epithelial tissues including those in the gastrointestinal and reproductive tracts as well as the airways. Indeed, defective bicarbonate secretion is a hallmark of the pathophysiology in the pancreas of most patients suffering from cystic fibrosis. Some, but not all, disease-causing mutations in the CF gene lead to impaired bicarbonate transport when expressed in heterologous systems. Recently developed pharmacological modulators of mutant CFTR have demonstrated an ability to activate chloride transport but little is known about whether they also increase the secretion of bicarbonate. It is therefore essential to assay bicarbonate transport when studying the effect of small molecules on CFTR function. However, due to the chaotropic nature of the ion, the measurement of the absolute bicarbonate concentration and its permeability through CFTR is far from trivial. In this chapter we will review some of the techniques available to measure bicarbonate transport through single ion channels, individual cells, and intact epithelial layers.

The Role of pHi in Intestinal Epithelial Proliferation–Transport Mechanisms, Regulatory Pathways, and Consequences

“…The ability to assess pH i in mammalian gastrointestinal cells with the use of fluorescent dyes (Thomas, 1986) made the study of pH i -recovery after an acidic or alkaline insult possible, as well as the delineation of the involved ion transporters (Flemstrom and Isenberg, 2001;Kaunitz and Akiba, 2006;Seidler, 2013). pH i measurements have also been utilized to outline the transport proteins involved in intestinal absorptive and secretory processes (Zachos et al, 2005;Hug et al, 2011;Seidler and Nikolovska, 2019).…”

New ISE-Based Apparatus for Na+, K+, Cl−, pH and Transepithelial Potential Difference Real-Time Simultaneous Measurements of Ion Transport across Epithelial Cells Monolayer–Advantages and Pitfalls

“…From the radiochemical measurements, one can estimate the sodium and chloride fluxes to be in the order of 1 μM∙cm −2 ∙h −1 = 280 pM∙cm −2 ∙s −1 , and thus it will produce a change of ion concentration of only 28 nM∙s −1 in 10 ml medium, a change that is too small to be detected by ISE, except for the pH electrode [18]. Hug et al [20] estimated bicarbonate secretion by epithelia using a pH electrode assuming that the change in pH is only caused by bicarbonate secretion, which is incorrect. Nair et al [21] introduced silver wire that was coated with silver chloride 50 µm apart from the apical surface of epithelial cells that are known to secrete chloride anions.…”

Rapid therapeutic advances in CFTR modulator science

“…Like all members of this protein family, it has two transmembrane domains (TMD‐1 and ‐2) which anchor the protein in the plasma membrane and two nucleotide binding domains (NBD‐1 and ‐2) which are cytoplasmic, bind, and hydrolyze ATP as a heterodimer, and control gating (opening and closing) of the channel . CFTR is unique among traffic ATPases in that it has a regulatory domain that imparts phosphorylation dependent regulation (protein kinase A), and that it, functions as a chloride and bicarbonate channel . Traffic ATPases typically function as small molecule pumps which move their cargo across the plasma membrane at speeds that are orders of magnitude slower than ion channels .…”

“…30,31 CFTR is unique among traffic ATPases in that it has a regulatory domain that imparts phosphorylation dependent regulation (protein kinase A), and that it, functions as a chloride and bicarbonate channel. [29][30][31][32] Traffic ATPases typically function as small molecule pumps which move their cargo across the plasma membrane at speeds that are orders of magnitude slower than ion channels. 33 New insights into the molecular (human) and atomic (zebrafish) structure of CFTR have recently been published, providing more information regarding relationships between its structure and unique functions.…”