The ultimate aim of this research is to help cure patients suffering from the diseases cystic fibrosis (CF), and secretory diarrheas like cholera. CF is the most common life-threatening inherited disease among Caucasians, caused by malfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein (Ref. 1). A plethora of inherited mutations can lead to reduced CFTR function. When this happens, children are born with CF, a complex debilitating disorder originating from defective salt-water balance at the epithelial surfaces of the lung, gut, pancreas, and sweat ducts. The disease is presently incurable, and despite recent advances in symptomatic treatment the expected lifespan of CF patients is still only ~30 years. Secretory diarrheas, still common in developing countries, are caused by bacterial toxins that cause CFTR hyperactivity which leads to excessive salt-water loss through the gut.
CFTR is localized at the apical surfaces of epithelia, where it regulates transepithelial salt and water movement. It belongs to the superfamily of ABC proteins, which couple ATP hydrolysis cycles at conserved nucleotide-binding domains to active transport of diverse small molecules across biological membranes into higher concentration compartments. CFTR is unique among ABC proteins in that it is a channel protein: it allows chloride ions to move passively across cell membranes. It consists of several parts, or domains. One part (transmembrane domains, TMDs) forms a pore, a pathway through which ions can cross the membrane. The pore opens only when binding of ATP at CFTR’s two cytosolic nucleotide-binding domains (NBDs) triggers the formation of a stable intramolecular NBD1/NBD2 heterodimer. Inside this dimer a phosphate group is cleaved off the end of one of the bound ATP molecules. Hence destabilized the dimer dissociates and the pore closes (Fig. 2D). Most channels have equilibrium kinetic schemes i.e. close by reversing the opening transition. In contrast, we have provided evidence that CFTR closes through a pathway distinct from that used for opening: once opened (to state O1), a channel must move to a second open state (state O2, that has lost the terminal phosphate) before it can close. In other words: CFTR’s kinetic scheme is cyclical consistent with its transporter ancestry (Fig. 2; Ref. 2).
We study the various steps of this enzymatic cycle, and the conformational coupling between the catalytic site and the channel gate. Thanks to CFTR’s unusual cyclical scheme, overall channel activity is most robustly affected by the rates of two transitions: the opening step and the O1O2 transition. Deepening our mechanistic understanding of these steps has the potential to guide the rational design of novel drugs capable of efficiently altering CFTR activity: speeding up opening and slowing the O1O2 transition to stimulate, or affecting these transitions in the opposite way to inhibit, CFTR activity – as required for the treatment of CF and secretory diarrheas, respectively.
From a broader perspective, our studies on CFTR function might also yield information to help improve drug bioavailability and/or tissue distribution in brain tumor or HIV/AIDS therapy, and fight multidrug resistance in cancer patients. This is because ABC proteins related to CFTR determine tissue distribution and oral bioavailability of most therapeutic drugs. During cancer chemotherapy, their overexpression results in multidrug resistance. Moreover, a variety of inherited diseases is caused by mutations in ABC genes. Thus, influencing ABC protein activity could benefit broader cohorts of patients. Since CFTR is the only ion channel in the family we can use CFTR as a unique “model”, exploiting the resolution afforded by single channel recording to study a basic mechanism shared with other, harder to study, ABC proteins
Fig. 2. Distributions of dwell times of single CFTR chloride ion channels in the open (bursting) state report on the mechanism of channel gating.
The peaked wild-type (WT) burst distribution (top left) signals that CFTR channel gating is a non-equilibrium cycle (bottom right) strictly (95%) coupled to ATP hydrolysis. The altered shapes of the burst distributions of catalytic site mutants K464A (top right) and D1370N (bottom left) attest to partial and full disruption, respectively, of this coupling.