Steady-state and time-resolved fluorescent methodologies to characterize the conformational landscape of the selectivity filter of K+ channels.

A variety of equilibrium and non-equilibrium methods have been used in a multidisciplinary approach to study the conformational landscape associated with the binding of different cations to the pore of potassium channels. These binding processes, and the conformational changes resulting therefrom, modulate the functional properties of such integral membrane properties, revealing these permeant and blocking cations as true effectors of such integral membrane proteins. KcsA, a prototypic K+ channel from Streptomyces lividans, has been extensively characterized in this regard. Here, we revise several fluorescence-based approaches to monitor cation binding under different experimental conditions in diluted samples, analyzing the advantages and disadvantages of each approach. These studies have contributed to explain the selectivity, conduction, and inactivation properties of K+ channels at the molecular level, together with the allosteric communication between the two gates that control the ion channel flux, and how they are modulated by lipids.

Anionic Phospholipids Shift the Conformational Equilibrium of the Selectivity Filter in the KcsA Channel to the Conductive Conformation: Predicted Consequences on Inactivation.

Here, we report an allosteric effect of an anionic phospholipid on a model K+ channel, KcsA. The anionic lipid in mixed detergent-lipid micelles specifically induces a change in the conformational equilibrium of the channel selectivity filter (SF) only when the channel inner gate is in the open state. Such change consists of increasing the affinity of the channel for K+, stabilizing a conductive-like form by maintaining a high ion occupancy in the SF. The process is highly specific in several aspects: First, lipid modifies the binding of K+, but not that of Na+, which remains unperturbed, ruling out a merely electrostatic phenomenon of cation attraction. Second, no lipid effects are observed when a zwitterionic lipid, instead of an anionic one, is present in the micelles. Lastly, the effects of the anionic lipid are only observed at pH 4.0, when the inner gate of KcsA is open. Moreover, the effect of the anionic lipid on K+ binding to the open channel closely emulates the K+ binding behaviour of the non-inactivating E71A and R64A mutant proteins. This suggests that the observed increase in K+ affinity caused by the bound anionic lipid should result in protecting the channel against inactivation.

Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements.

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel's selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+-KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67-W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+-bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

Modulation of Function, Structure and Clustering of K+ Channels by Lipids: Lessons Learnt from KcsA.

KcsA, a prokaryote tetrameric potassium channel, was the first ion channel ever to be structurally solved at high resolution. This, along with the ease of its expression and purification, made KcsA an experimental system of choice to study structure-function relationships in ion channels. In fact, much of our current understanding on how the different channel families operate arises from earlier KcsA information. Being an integral membrane protein, KcsA is also an excellent model to study how lipid-protein and protein-protein interactions within membranes, modulate its activity and structure. In regard to the later, a variety of equilibrium and non-equilibrium methods have been used in a truly multidisciplinary effort to study the effects of lipids on the KcsA channel. Remarkably, both experimental and "in silico" data point to the relevance of specific lipid binding to two key arginine residues. These residues are at non-annular lipid binding sites on the protein and act as a common element to trigger many of the lipid effects on this channel. Thus, processes as different as the inactivation of channel currents or the assembly of clusters from individual KcsA channels, depend upon such lipid binding.

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites.

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

Conformational plasticity in the KcsA potassium channel pore helix revealed by homo-FRET studies.

Potassium channels selectivity filter (SF) conformation is modulated by several factors, including ion-protein and protein-protein interactions. Here, we investigate the SF dynamics of a single Trp mutant of the potassium channel KcsA (W67) using polarized time-resolved fluorescence measurements. For the first time, an analytical framework is reported to analyze the homo-Förster resonance energy transfer (homo-FRET) within a symmetric tetrameric protein with a square geometry. We found that in the closed state (pH 7), the W67-W67 intersubunit distances become shorter as the average ion occupancy of the SF increases according to cation type and concentration. The hypothesis that the inactivated SF at pH 4 is structurally similar to its collapsed state, detected at low K+, pH 7, was ruled out, emphasizing the critical role played by the S2 binding site in the inactivation process of KcsA. This homo-FRET approach provides complementary information to X-ray crystallography in which the protein conformational dynamics is usually compromised.

Mechanisms Underlying the Strong Inhibition of Muscle-Type Nicotinic Receptors by Tetracaine.

Nicotinic acetylcholine (ACh) receptors (nAChRs) are included among the targets of a variety of local anesthetics, although the molecular mechanisms of blockade are still poorly understood. Some local anesthetics, such as lidocaine, act on nAChRs by different means through their ability to present as both charged and uncharged molecules. Thus, we explored the mechanisms of nAChR blockade by tetracaine, which at physiological pH is almost exclusively present as a positively charged local anesthetic. The nAChRs from Torpedo electroplaques were transplanted to Xenopus oocytes and the currents elicited by ACh (IACh s), either alone or co-applied with tetracaine, were recorded. Tetracaine reversibly blocked IACh , with an IC50 (i.e., the concentration required to inhibit half the maximum IACh ) in the submicromolar range. Notably, at very low concentrations (0.1 µM), tetracaine reduced IACh in a voltage-dependent manner, the more negative potentials produced greater inhibition, indicating open-channel blockade. When the tetracaine concentration was increased to 0.7 µM or above, voltage-independent inhibition was also observed, indicating closed-channel blockade. The IACh inhibition by pre-application of just 0.7 µM tetracaine before superfusion of ACh also corroborated the notion of tetracaine blockade of resting nAChRs. Furthermore, tetracaine markedly increased nAChR desensitization, mainly at concentrations equal or higher than 0.5 µM. Interestingly, tetracaine did not modify desensitization when its binding within the channel pore was prevented by holding the membrane at positive potentials. Tetracaine-nAChR interactions were assessed by virtual docking assays, using nAChR models in the closed and open states. These assays revealed that tetracaine binds at different sites of the nAChR located at the extracellular and transmembrane domains, in both open and closed conformations. Extracellular binding sites seem to be associated with closed-channel blockade; whereas two sites within the pore, with different affinities for tetracaine, contribute to open-channel blockade and the enhancement of desensitization, respectively. These results demonstrate a concentration-dependent heterogeneity of tetracaine actions on nAChRs, and contribute to a better understanding of the complex modulation of muscle-type nAChRs by local anesthetics. Furthermore, the combination of functional and virtual assays to decipher nAChR-tetracaine interactions has allowed us to tentatively assign the main nAChR residues involved in these modulating actions.

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel.

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1-S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+ These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity.

Towards understanding the molecular basis of ion channel modulation by lipids: Mechanistic models and current paradigms.

Research on ion channel modulation has become a hot topic because of the key roles these membrane proteins play in both prokaryotic and eukaryotic organisms. In this respect, lipid modulation adds to the overall modulatory mechanisms as a potential via to find new pharmacological targets for drug design based on interfering with lipid/channel interactions. However, our knowledge in this field is scarce and often circumscribed to the sites where lipids bind and/or its final functional consequences. To fully understand this process it is necessary to improve our knowledge on its molecular basis, from the binding sites to the signalling pathways that derive in structural and functional effects on the ion channel. In this review, we have compiled information about such mechanisms and established a classification into four different modes of action. Afterwards, we have revised in more detail the lipid modulation of Cys-loop receptors and of the potassium channel KcsA, which were chosen as model channels modulated by specific lipids. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Differential binding of monovalent cations to KcsA: Deciphering the mechanisms of potassium channel selectivity.

This work explores whether the ion selectivity and permeation properties of a model potassium channel, KcsA, could be explained based on ion binding features. Non-permeant Na+ or Li+ bind with low affinity (millimolar KD's) to a single set of sites contributed by the S1 and S4 sites seen at the selectivity filter in the KcsA crystal structure. Conversely, permeant K+, Rb+, Tl+ and even Cs+ bind to two different sets of sites as their concentration increases, consistent with crystallographic evidence on the ability of permeant species to induce concentration-dependent transitions between conformational states (non-conductive and conductive) of the channel's selectivity filter. The first set of such sites, assigned also to the crystallographic S1 and S4 sites, shows similarly high affinities for all permeant species (micromolar KD's), thus, securing displacement of potentially competing non-permeant cations. The second set of sites, available only to permeant cations upon the transition to the conductive filter conformation, shows low affinity (millimolar KD's), thus, favoring cation dissociation and permeation and results from the contribution of all S1 through S4 crystallographic sites. The differences in affinities between permeant and non-permeant cations and the similarities in binding behavior within each of these two groups, correlate fully with their permeabilities relative to K+, suggesting that binding is an important determinant of the channel's ion selectivity. Conversely, the complexity observed in permeation features cannot be explained just in terms of binding and likely relates to reported differences in the occupancy of the S2 and S3 sites by the permeant cations.

Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine.

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine's hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine's hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.

Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine.

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (I ACh ) in a dose-dependent manner (IC 50 close to 70 µM), but unlike lidocaine, DEA did not affect I ACh desensitization. I ACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced I ACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA).

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp(67)-Glu(71)-Asp(80) inactivation triad within the channel structure and its bearing on the selectivity filter.

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives.

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.

Detergent-labile, supramolecular assemblies of KcsA: relative abundance and interactions involved.

In this work, we illustrate the ability of the prokaryotic potassium channel KcsA to assemble into a variety of supramolecular clusters of defined sizes containing the tetrameric KcsA as the repeating unit. Such clusters, particularly the larger ones, are markedly detergent-labile and thus, disassemble readily upon exposure to the detergents commonly used in protein purification or conventional electrophoresis analysis. This is a reversible process, as cluster re-assembly occurs upon detergent removal and without the need of added membrane lipids. Interestingly, the dimeric ensemble between two tetrameric KcsA molecules are quite resistant to detergent disassembly to individual KcsA tetramers and along with the latter, are likely the basic building blocks through which the larger clusters are organized. As to the proteins domains involved in clustering, we have observed disassembly of KcsA clusters by SDS-like alkyl sulfates. As these amphiphiles bind to inter-subunit, "non-annular" sites on the protein, these observations suggest that such sites also mediate channel-channel interactions leading to cluster assembly.

Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant.

Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl ß-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.

Triazine-based vanilloid 1 receptor open channel blockers: design, synthesis, evaluation, and SAR analysis.

The thermosensory transient receptor potential vanilloid 1 channel (TRPV1) is a polymodal receptor activated by physical and chemical stimuli. TRPV1 activity is drastically potentiated by proinflammatory agents released upon tissue damage. Given the pivotal role of TRPV1 in human pain, there is pressing need for improved TRPV1 antagonists, the development of which will require identification of new pharmacophore scaffolds. Uncompetitive antagonists acting as open-channel blockers might serve as activity-dependent blockers that preferentially modulate the activity of overactive channels, thus displaying fewer side effects than their competitive counterparts. Herein we report the design, synthesis, biological evaluation, and SAR analysis of a family of triazine-based compounds acting as TRPV1 uncompetitive antagonists. We identified the triazine 8aA as a potent, pure antagonist that inhibits TRPV1 channel activity with nanomolar efficacy and strong voltage dependency. It represents a new class of activity-dependent TRPV1 antagonists and may serve as the basis for lead optimization in the development of new analgesics.

Solid-phase synthesis of a library of amphipatic hydantoins. Discovery of new hits for TRPV1 blockade.

Some heterocyclic systems, called privileged scaffolds, appear frequently in bioactive products and marketed drugs. The combination of a recognized privileged scaffold (hydantoin) and a functional group with high incidence in bioactive molecules (guanidine) guided the design of a library of amphipatic compounds, which allowed the discovery of novel TRPV1 ion channel blockers. The library was synthesized by parallel solid-phase synthesis from an orthogonally protected resin-bound Lys-Lys skeleton. Key steps of the synthetic procedure were the construction of the hydantoin ring, by reaction of the N-terminal amino group with N,N-disuccinimidyl carbonate (DSC) and subsequent base-induced cyclization, and the guanidinylation of the C-terminal Lys side-chain after removal of the Alloc protecting-group. The preliminary biological studies have allowed the identification of some of the key structural features directing the blockage of capsaicin-induced Ca(2+) influx through TRPV1 channels, particularly, the strong preference showed for highly lipophilic acyl groups and substituted guanidine moieties. Active compounds based on this new pharmacophoric scaffold that display in vitro and in vivo inhibitory activity.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)<10 µM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC(50)>100 µM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.