Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore.

We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.

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.

Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP(4)).

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 ß adrenergic receptor (ßAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either ßAR or the EP(4) receptor were also studied. In oocytes co-expressing ClC-2 and ßAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the ß 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 ßAR 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.

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.

External Zn(2+) 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 Zn(2+) bridges near and at the ECL1-ECL6 interface, respectively, and the gating effects of a Zn(2+) disturbance at the ECL1-ECL6 interface on the stimulatory ICL1/ICL4-R interaction were determined. The results showed that both Zn(2+) 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 Zn(2+) inhibition was weakened at higher Zn(2+) concentrations, their Zn(2+) affinity was stronger in the resting state than in the activated state, and their activation current noises were decreased by external Zn(2+) binding. More importantly, the external Zn(2+) inhibition was reversed by internal curcumin in the R334C construct but not in the T1122C mutant. Therefore, although both Zn(2+) bridges may promote channel closure, external Zn(2+) 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.

Limited efficacy of α-conopeptides, Vc1.1 and RgIA, to inhibit sensory neuron CaV current.

Chronic pain is very difficult to treat. Thus, novel analgesics are a critical area of research. Strong pre-clinical 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) vs. 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. SIGNIFICANCE STATEMENT: Better analgesic drugs are desperately needed to help physicians to treat pain. While many pre-clinical studies support the analgesic effects of α-conopeptides, Vc1.1 and RgIA, the mechanism is controversial. The development of improved α-conopeptide analgesics would be greatly facilitated by a complete understanding of the analgesic mechanism. However, we show that we cannot reproduce one of the proposed analgesic mechanisms, which is an irreversible inhibition of CaV current in a majority of sensory neurons.

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 ß-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.