The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl− channel

Abstract

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.

Strategies to investigate the mechanism of action of CFTR modulators

“…To identify CFTR modulators and investigate their mechanism of action, we use the patch-clamp technique to study CFTR Cl À currents in excised inside-out membrane patches from mammalian cells expressing recombinant wild-type (wt) human CFTR [13]. Following the activation of CFTR Cl À currents by cAMP-dependent phosphorylation, we add drugs to the solution bathing the intracellular side of the membrane.…”

“…Noise analysis is required to quantify CFTR inhibition by drugs that block with ''very fast speed'' [25 -27], whereas single-channel studies may be employed to quantify CFTR inhibition by drugs that block with ''intermediate speed'' [11,13,15]. For the latter studies, we generate events lists, create open-and closed-time histograms and fit histograms with one or more component exponential functions using the maximum likelihood method.…”

Microelectrodes and their use to assess ion channel function

“…To date, the most popular approaches are either to isolate the membrane of interest by mechanical force (excised-patch, see Ref. [1]) or to remove one membrane by selective permeabilization (see Ref. [2]).…”

Section D (cell physiology): an armamentarium to investigate CFTR function

“…The activity of wild-type and mutant CFTR is studied in native tissues and heterologous expression systems at the molecular, cellular and tissue levels. Many of the methods are electrophysiological ranging from high-resolution recording of individual CFTR Cl À channels using the patch-clamp technique [7] to Cl À flow across polarised epithelia monitored with the Ussing chamber technique [8]. Other methods employ planar lipid bilayers and ATPases assays to understand, at the molecular level, how ATP hydrolysis drives conformation changes in the CFTR Cl À channel [7,9].…”

“…Many of the methods are electrophysiological ranging from high-resolution recording of individual CFTR Cl À channels using the patch-clamp technique [7] to Cl À flow across polarised epithelia monitored with the Ussing chamber technique [8]. Other methods employ planar lipid bilayers and ATPases assays to understand, at the molecular level, how ATP hydrolysis drives conformation changes in the CFTR Cl À channel [7,9]. Yet other methods employ fluorescent dyes and radioisotopes to assay the function of CFTR in populations of cells [10,11].…”