Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.The CFTR chloride channel regulates salt and water transport across epithelial tissues (1). Mutations that reduce the synthesis or functional activity of this channel cause cystic fibrosis (CF), 2 the most common lethal genetic disorder among Caucasions (2). CFTR is a member of the ATP-binding cassette (ABC) transporter superfamily (3) based on its general domain structure that includes two transmembrane domains (which form the pore) and two nucleotide-binding domains (NBD1 and NBD2, which bind and hydrolyze ATP). CFTR also has a large centrally localized regulatory domain (R domain) that is a special feature of this ABC transporter. The R domain has many sites for phosphorylation by cyclic nucleotide-dependent protein kinases, which are the main physiologic activators of this channel (4). CFTR channel opening typically requires both kinase-mediated phosphorylation of the R domain and ATP binding to both NBDs (reviewed in Refs. 5 and 6). Regarding the role of ATP in promoting channel opening, the available evidence supports a model in which two ATP molecules bind in pockets at an interface between the two NBDs (i.e. at the interface of an NBD1-NBD2 dimer) (7,8). This model is based in part on structural and functional information that has been...

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

“…3C). This decrease in the channel current may be due to the side effect of curcumin (24). It is interesting that ATP and curcumin only activated the K946D mutant by 40% but activated the D835R/ D836R/E838R mutant completely (Fig.…”

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

“…The ⌬NBD2-CFTR construct was a generous gift from Dr. K. Kirk (University of Alabama). In this construct amino acids from NBD2 and the C-terminal are deleted (32).…”

“…It has been shown that CFTR channels completely missing NBD2 (i.e. ⌬NBD2-CFTR) exhibit ATP-independent openings similar to those observed in G551D channels (32). Thus, the opening of the gate may not absolutely require the action of NBD2.…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…In particular, it is known that CFTR channels can open, albeit with a very low frequency, in a complete absence of ATP (Bompadre et al 2005a). This ATP-independent gating is readily seen with mutants with dysfunctional site 2 (e.g., the G551D mutation) (Bompadre et al 2007), or interestingly constructs that lack the entire NBD2 (Cui et al 2007;Wang et al 2007). Furthermore, gain-of-function (GOF) mutations that enhance ATP-free CFTR gating and increase the otherwise low activities of constructs that are insensitive to ATP (G551D and NBD-deletion mutants) have been produced (Szollosi et al 2010;Wang et al 2010).…”

“…As noted earlier CFTR truncation mutants that lack NBD2 show detectable channel activity (Cui et al 2007). Wang et al (2007Wang et al ( , 2010 observed that the channel activity of an NBD2-deletion mutant was strongly dependent on PKA phosphorylation but not ATP binding, an effect that required the R domain. Similarly, G551D -CFTR activity is dependent on PKA phosphorylation but not ATP binding (Bompadre et al 2007;Wang et al 2010).…”