Yohei Norimatsu

Cystic Fibrosis Transmembrane Conductance Regulator: Using Differential Reactivity toward Channel-Permeant and Channel-Impermeant Thiol-Reactive Probes To Test a Molecular Model for the Pore

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5−6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Exploring the determinants of trace amine-associated receptor 1's functional selectivity for the stereoisomers of amphetamine and methamphetamine

Amphetamines are widely abused drugs that interfere with dopamine transport and storage. Recently, however, another mechanism of action was identified: stereoselective activation of the GαS protein-coupled trace amine-associated receptor 1 (TAAR1). To identify structural determinants of this stereoselectivity, we functionally evaluated six mutant receptors in vitro and then used homology modeling and dynamic simulation to predict drug affinities. Converting Asp102 to Ala rendered mouse and rat TAAR1 (mTAAR1 and rTAAR1, respectively) insensitive to β-phenylethylamine, amphetamine (AMPH), and methamphetamine (METH). Mutating Met268 in rTAAR1 to Thr shifted the concentration-response profiles for AMPH and METH isomers rightward an order of magnitude, whereas replacing Thr268 with Met in mTAAR1 resulted in profiles leftward shifted 10-30-fold. Replacing Asn287 with Tyr in rTAAR1 produced a mouselike receptor, while the reciprocal mTAAR1 mutant was rTAAR1-like. These results confirm TAAR1 is an AMPH/METH receptor in vitro and establish residues 102 (3.32) and 268 (6.55) as major contributors to AMPH/METH binding with residue 287 (7.39) determining species stereoselectivity.

Lubiprostone Activates CFTR, but not ClC-2, via the Prostaglandin Receptor (EP4)

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP(4) receptor has been described [2-5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP(4) receptor or β adrenergic receptor (βAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP(4) receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP(4) did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR(inh)172. Co-expression of CFTR and EP(4) resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR(inh)172. The EC(50) for lubiprostone mediated CFTR activation was ~10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP(4) receptor in oocytes.

Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

High-throughput screening has led to the identification of small-molecule blockers of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, but the structural basis of blocker binding remains to be defined. We developed molecular models of the CFTR channel on the basis of homology to the bacterial transporter Sav1866, which could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker such as N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101), which is thought to act by entering the channel from the extracellular side. As a more-stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual, ligand-docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest-scoring docked position was near two pore-lining residues, Phe337 and Thr338, and the rates of reactions of anionic, thiol-directed reagents with cysteines substituted at these positions were slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (Phe342) was replaced with alanine, the apparent affinity of the blocker was increased ∼200-fold. A molecular mechanics-generalized Born/surface area analysis of GlyH-101 binding predicted that substitution of Phe342 with alanine would substantially increase blocker affinity, primarily because of decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

Limited Efficacy of α-Conopeptides, Vc1.1 and RgIA, To Inhibit Sensory Neuron CaV Current

Better analgesic drugs are desperately needed to help physicians to treat pain. While many preclinical studies support the analgesic effects of α-conopeptides, Vc1.1 and RgIA, the mechanism is controversial. Abstract Chronic pain is very difficult to treat. Thus, novel analgesics are a critical area of research. Strong preclinical evidence supports the analgesic effects of α-conopeptides, Vc1.1 and RgIA, which block α9α10 nicotinic acetylcholine receptors (nAChRs). However, the analgesic mechanism is controversial. Some evidence supports the block of α9α10 nAChRs as an analgesic mechanism, while other evidence supports the inhibition of N-type CaV (CaV2.2) current via activation of GABAB receptors. Here, we reassess the effect of Vc1.1 and RgIA on CaV current in rat sensory neurons. Unlike the previous findings, we found highly variable effects among individual sensory neurons, but on average only minimal inhibition induced by Vc1.1, and no significant effect on the current by RgIA. We also investigated the potential involvement of GABAB receptors in the Vc1.1-induced inhibition, and found no correlation between the size of CaV current inhibition induced by baclofen (GABAB agonist) versus that induced by Vc1.1. Thus, GABAB receptors are unlikely to mediate the Vc1.1-induced CaV current inhibition. Based on the present findings, CaV current inhibition in dorsal root ganglia is unlikely to be the predominant mechanism by which either Vc1.1 or RgIA induce analgesia.

External Zn2+ binding to cysteine‐substituted cystic fibrosis transmembrane conductance regulator constructs regulates channel gating and curcumin potentiation

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by ATP binding‐induced dimerization of nucleotide‐binding domains, the interaction between the phosphorylated regulatory (R) domain and the curcumin‐sensitive interface between intracellular loop (ICL) 1 and ICL4, and the resultant inward‐to‐‘outward’ reorientation of transmembrane domains. Although transmembrane helices (TM) 2 and TM11 link the ICL1–ICL4 interface with the interface between extracellular loop (ECL) 1 and ECL6, it is unknown whether both interfaces are gating‐coupled during the reorientation. Herein, R334C and T1122C mutations were used to engineer two Zn2+ bridges near and at the ECL1–ECL6 interface, respectively, and the gating effects of a Zn2+ disturbance at the ECL1–ECL6 interface on the stimulatory ICL1/ICL4‐R interaction were determined. The results showed that both Zn2+ bridges inhibited channel activity in a dose‐ and Cl−‐dependent manner, and the inhibition was reversed by a washout or suppressed by thiol‐specific modification. Interestingly, their Cl−‐dependent Zn2+ inhibition was weakened at higher Zn2+ concentrations, their Zn2+ affinity was stronger in the resting state than in the activated state, and their activation current noises were decreased by external Zn2+ binding. More importantly, the external Zn2+ inhibition was reversed by internal curcumin in the R334C construct but not in the T1122C mutant. Therefore, although both Zn2+ bridges may promote channel closure, external Zn2+ may disturb the ECL1–ECL6 interface and thus prevent the stimulatory ICL1/ICL4‐R interaction and curcumin potentiation via a gating coupling between these two interfaces.

Electrostatic Basis of Anion Over Cation Selectivity in the CFTR Chloride Channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated, anion-selective channel that is the product of the gene mutated in the inherited disease, cystic fibrosis. Chloride conductance via CFTR is a key component of the salt and water secretory system that maintains mucus hydration in the airway lumen. CFTR belongs to the large family of ABC transporter proteins, but it is the only member known to function as an ion channel. The channel is highly selective for anions over cations, but the structural basis for the observed charge selectivity is unknown. Individual point mutations generally do not seem to strongly affect charge selectivity, suggesting the possibility of a mechanism that is not highly dependent on local structural specialization such as that seen in the K-channel selectivity filter. We used a recent structural model of CFTR, based on the crystal structure of the Sav1866 ABC transporter and experimentally validated by cysteine scanning [1], to begin a computational investigation of the structural basis of the selectivity of the CFTR channel for anions over cations. Poisson-Boltzmann calculations suggest that an excess of basic residues in the transmembrane domain creates a large, anion-stabilizing region that includes the channel pore and extends towards the extracellular vestibule. The results indicate that electrostatic interactions affecting a large volume of the protein are a major contributor to anion over cation selectivity of the CFTR channel, similar to the situation observed in OprP [2].[1] C Alexander et al. Biochemistry 48 (2009) 10078-10088.[2] P Pongprayoon et al PNAS 106 (2009), 21614-21618.

Binding of GlyH-101 in the Pore of the CFTR Chloride Channel

GlyH-101 is a small molecule (MW: 493) that carries a single negative charge (pKa: 5.5) under physiological conditions (∼ pH 7.4) and blocks the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by entering from the extracellular side and binding to a site or sites within the pore (Muanprasat et al., J. Gen. Physiol. 124: 125-137). However, the precise binding sites for this molecule have yet to be identified. We used virtual ligand docking software, “Glide” (Schrodinger Inc.) to identify potential GlyH-101 binding sites within molecular models of CFTR derived by means of molecular dynamics simulation from a homology model based on Sav 1866 (Alexander et al., Biochemistry 48: 10078-10088). These sites were evaluated by determining the extent of occlusion of reactive cysteines by the blocker. The results suggest that the binding of GlyH-101 near a narrow portion of the pore could reduce the reactivity of T338C with [Au(CN)2]- and MTSES- by a repulsive charge-charge interaction. We tested the efficacy and potency of GlyH-101 for CFTR mutant channels and discovered a single amino acid substitution that significantly increases the potency of GlyH-101. Supported by NIH, Cystic Fibrosis Foundation, American Lung Association, the Wellcome Trust, and the BBSRC.