The Channel–Enzyme Interface: Structure–Function of Single CFTR and TRPM2 Ion Channel Proteins
Summary: László Csanády studies the structure and mechanism of two medically important ion channels, CFTR and TRPM2, which are unique examples of channels that also function as active enzymes.
My lab studies the structure and function of ion channels: transmembrane protein pores that allow passage of some type of ion across a biological membrane. Our experimental approaches include recording and analysis of single-channel and macroscopic patch-clamp currents, site-directed mutagenesis, protein biochemistry, and mathematical modeling. Our studies are focused on two target proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and the transient receptor potential melastatin 2 (TRPM2) cation channel.
The CFTR Chloride Channel
The ultimate aim of this research is to help cure patients suffering from cystic fibrosis (CF) and secretory diarrheas such as cholera. CF is the most common life-threatening inherited disease among Caucasians, caused by malfunction of the CFTR protein. 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 incurable for now, and despite recent advances in symptomatic treatment, the expected life span 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 loss of salt and water through the gut.
CFTR is located 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, or 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. Most channels have equilibrium kinetic schemes; that is, they close by reversing the opening transition. In contrast, our work offers 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, which has lost the terminal phosphate) before it can close. In other words, CFTR's kinetic scheme is cyclical, consistent with its transporter ancestry.
We study the various steps of this enzymatic cycle and the conformational coupling between the catalytic site and the channel gate. Because of CFTR's unusual cyclical scheme, the rates of two transitions most robustly affect overall channel activity: the opening step and the O1→O2 transition. Deepening our mechanistic understanding of these steps could guide the rational design of new drugs that can efficiently alter CFTR activity: speeding up opening and slowing the O1→ O2 transition to stimulate, or affecting these transitions in the opposite way to inhibit, CFTR activity—as required to treat CF or secretory diarrheas, respectively.
More broadly, because ABC proteins related to CFTR determine tissue distribution and oral bioavailability of most therapeutic drugs, our studies on CFTR function might also yield information (1) to help improve drug bioavailability and/or tissue distribution in brain tumor or HIV/AIDS therapy and (2) to fight multidrug resistance in cancer patients. During cancer chemotherapy, overexpression of these proteins results in multidrug resistance. Moreover, mutations in ABC genes cause a variety of inherited diseases. Thus, influencing ABC protein activity could benefit broader cohorts of patients. Because CFTR is the only ion channel in the family, we can use CFTR as a unique "model," exploiting the resolution that single-channel recording affords to study a basic mechanism that other, harder-to-study ABC proteins also share.
The TRPM2 Cation Channel
The TRPM2 protein is a recently identified transmembrane cation channel permeable to Ca2+. It is abundantly expressed in the brain, in the pancreas, and in phagocytic cells. Ca2+ influx through the TRPM2 pore is a key step in immunocyte activation, insulin secretion, and neuronal cell death after stroke. Thus, TRPM2 is an emerging target in the treatment of multiple diseases, including neurological and immunological conditions and diabetes. Although under certain conditions inhibition of TRPM2 activity would seem beneficial (e.g., stroke, myocardial infarction, Alzheimer's disease, chronic inflammatory diseases, and hyperinsulinism), in other cases hopes for a clinical benefit are set on TRPM2 stimulation (e.g., diabetes and diseases associated with TRPM2 loss-of-function mutations, such as western Pacific amyotrophic lateral sclerosis, Parkinsonism-dementia, and certain forms of bipolar disorder). Understanding TRPM2 structure and mechanism will be vital for designing drugs that specifically target this protein.
TRPM2 channels are coactivated by intracellular ADP-ribose (ADPR) and Ca2+: the channels open only in the presence of both ligands. In recent work we have clarified the mechanism of Ca2+ activation and the location of the activating sites. In the presence of ADPR, activation by intracellular Ca2+ follows the Monod–Wyman–Changeux mechanism; binding of each of four Ca2+ ions incrementally increases the open–closed equilibrium constant by ~33-fold. The Ca2+ binding sites are located intracellularly from the gate, but in a shielded crevice near the pore entrance. Because of the positive feedback by Ca2+ ions' entering through the pore, a single brief intracellular Ca2+ signal seems sufficient to trigger prolonged, self-sustained TRPM2 activity in intact cells, provided that ADPR is available. Thus, although Ca2+ is the trigger, ADPR controls the timing of TRPM2 channel activity in living cells.
ADPR is produced under conditions of oxidative stress, such as the respiratory burst in immune cells or ischemia in the brain. ADPR binds to the carboxy-terminal NUDT9-H domain of the channel, which functions as an enzyme that cleaves ADPR. This enzymatic activity is rather surprising, because ion channels are passive devices that facilitate the flux of ions down their electrochemical gradients and therefore, unlike active transporters, do not require energy input to operate their gates. Thus, just as with CFTR, TRPM2 ranks among the few known ion channels whose gating is linked to an enzymatic activity ("chanzymes").
What is the best strategy to manipulate TRPM2 activity? Because TRP-family channels are involved in diverse physiological processes, any useful TRPM2 agonists/antagonists would need to be highly selective. This requirement singles out the NUDT9-H domain, the only protein segment unique to TRPM2, as the most attractive pharmacological target. And yet, at the moment, our understanding of the role of this chanzyme domain in TRPM2 gating is extremely limited. Thus, the goal of our research is to understand how ADPR's binding to the NUDT9-H domain drives TRPM2 gating. Specifically, we would like to identify how strictly enzymatic activity and gating are coupled, which step of the catalytic cycle is linked to opening and closing of the ion pore, and which are the key amino acid residues responsible for ADPR binding/hydrolysis. Using this information, we hope to design ADPR analogs that specifically activate or inhibit TRPM2.
Grants from the National Institutes of Health (Fogarty International Center and National Institute of Diabetes and Digestive and Kidney Diseases), the Wellcome Trust, and the Országos Tudományos Kutatási Alapprogramok provided partial support for these projects.
As of January 17, 2012