The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein

Abstract

Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the “signature” sequence LSG$\text{G}\text{̄}$Q and LSH$\text{G}\text{̄}$H of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC₅₀) of MPB-91 (22–36 μM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis–Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.

Introduction

ATP-binding cassette (ABC) transporters are members of a large family of membrane proteins that catalyse the active transport of a variety of solutes across biological membranes [1]. ABC tranporters are composed of two repeated units joined by a linker region [1]. Each part has six transmembrane segments and a hydrophilic domain containing an ATP-binding site, the nucleotide binding domain (NBD). NBD contains highly conserved Walker A and B motifs in common to a variety of ATP-binding proteins [2]. The LSGGQ motif (also named “linker peptide” or “signature sequence”) shows remarkable conservation in NBDs of all ABC transporters and is interposed between the two Walker domains [1]. The function of this sequence is still largely unknown. Recent studies revealed however that the conserved sequence in each NBD interacts with the Walker A consensus nucleotide-binding domain in the opposite NBD [3], [4], [5], [6], [7], [8], [9].

The cystic fibrosis transmembrane conductance regulator (CFTR), the protein product of the CFTR gene, is an ABC transporter (ABCC7) with unique properties of cAMP-regulated chloride channel [10]. Cystic fibrosis (CF), one of the most common lethal autosomal recessive genetic disease, is caused by mutations of the CFTR gene at the origin of defective Cl⁻ transport across the affected epithelium [11], [12]. CFTR mutations (http://www.genet.sickkids.on.ca/cftr) can be assigned to one of five classes of mutations [13], leading to a protein which chloride channel function is altered (classes III and IV) or lacking (classes I, II and V) at the apical membrane. The glycine-to-aspartic acid missense mutations G551D and G1349D are class III mutations located within the signature sequence LSG$\text{G}\text{̄}$Q in NBD1 and LSH$\text{G}\text{̄}$H in NBD2, respectively [13], [14], [15]. G551D is one of the five most frequent CF mutation with a frequency of 2–5% depending of the population of origin. Class III mutations disrupt activation and regulation of CFTR at the plasma membrane and lead to a severe CF phenotype [13], [14], [16]. However, class III CFTR channels are fully glycosylated, correctly located at the apical plasma membrane and normally phosphorylated at the R domain by cAMP-dependent protein kinases [14], [15], [16], [17], [18]. The most common CF mutation (delF508, class II), leading to a severe CF phenotype, is a three base pair deletion resulting in loss of a phenylalanine residue at position 508 in the protein [10], [11], [13], [14]. Trafficking of delF508 mutants from the endoplasmic reticulum (ER) to the apical plasma membrane of epithelial cells is extremely inefficient. Therefore, cells expressing delF508-CFTR show severely reduced Cl⁻ channel activity compared to wt, since the protein is incorrectly processed (no glycosylation) and retained within the ER [10], [11], [13].

We have demonstrated recently that the glycine G551 in the LSG$\text{G}\text{̄}$Q motif of CFTR is the key amino acid for inhibition of CFTR chloride channel activity by high concentration of genistein [19]. In this study, we used site-directed mutagenesis and pharmacological analysis of CFTR channel activity to search for the activatory genistein binding site. Our results show that mutation of the symmetrical glycine G1349 into aspartate in the LSH$\text{G}\text{̄}$H motif of the second NBD abolished CFTR activation by genistein demonstrating that G1349 is part of the activatory genistein binding site. The opposite role of both symmetrical glycines of the signature sequences is discussed at the light of the recent model proposed for the NBD dimerization of ABC transporters [3], [4], [5], [6], [7], [8], [9], [20].