Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation.

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues-I696 and W697-with Ala markedly affects TRPV1's response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.

Advances in modulating thermosensory TRP channels.

INTRODUCTION: Thermosensory channels are a subfamily of the transient receptor potential (TRP) channel family that are activated by changes in the environmental temperature. These channels, known as thermoTRPs, cover the entire spectrum of temperatures, from noxious cold (< 15°C) to injurious heat (> 42°C). In addition, dysfunction of these channels contributes to the thermal hypersensitivity that accompanies painful conditions. Moreover, because of their wide tissue and cellular distribution, thermoTRPs are also involved in the pathophysiology of several diseases, from inflammation to cancer. AREAS COVERED: Although the number of thermoTRPs is increasing with the identification of novel members such as TRPM3, we will cover the recent advances in the pharmacology of the classical thermosensory channels, namely TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. This review will focus on the therapeutic progress carried out for all these channels and will highlight the tenet that TRPV1, TRPM8 and TRPA1 are the most exploited channels, and that the interest on TRPV3 and TRPV4 is growing with the first TRPV3 antagonist that moves into Phase-II clinical trials. In contrast, the pharmacology of TRPV2 is yet in its infancy. EXPERT OPINION: Despite the tremendous academic and industrial investment to develop therapeutic modulators of thermoTRPs, it apparently seems that we are still far from the first successful product, although hope is maintained high for all compounds currently in clinical trials. A major concern has been the appearance of side effects. A better knowledge of the thermosensory protein networks (signal-plexes), along with the application of system biology approaches may provide novel strategies to modulate thermoTRPs activity with improved therapeutic index. A case in point is TRPV1, where acting on interacting proteins is providing new therapeutic opportunities.

Mutation of Ser-50 and Cys-66 in Snapin modulates protein structure and stability.

Snapin is a 15 kDa protein present in neuronal and non-neuronal cells that has been implicated in the regulation of exocytosis and endocytosis. Protein kinase A (PKA) phosphorylates Snapin at Ser-50, modulating its function. Likewise, mutation of Cys-66, which mediates protein dimerization, impairs its cellular activity. Here, we have investigated the impact of mutating these two positions on protein oligomerization, structure, and thermal stability, along with the interaction with SNARE proteins. We found that recombinant purified Snapin in solution appears mainly as dimers in equilibrium with tetramers. The protein exhibits modest secondary structure elements and notable thermal stability. Mutation of Cys-66 to Ser abolished subunit dimerization, but not higher-order oligomers. This mutant augmented the presence of α-helical structure and slightly increased the protein thermal stability. Similarly, the S50A mutant, mimicking the unphosphorylated protein, also exhibited a higher helical secondary structure content than the wild type, along with greater thermal stability. In contrast, replacement of Ser-50 with Asp (S50D), emulating the protein-phosphorylated state, produced a loss of α-helical structure, concomitant with a decrease in protein thermal stability. In vitro, the wild type and mutants weakly interacted with SNAP-25 and the reconstituted SNARE complex, although S50D exhibited the strongest binding to the SNARE complex, consistent with the observed higher cellular activity of PKA-phosphorylated Snapin. Our observations suggest that the stronger binding of S50D to SNAREs might be due to a destabilization of tetrameric assemblies of Snapin that favor the interaction of protein dimers with the SNARE proteins. Therefore, phosphorylation of Ser-50 has an important impact on the protein structure and stability that appears to underlie its functional modulation.

Ionic channels as targets for drug design: a review on computational methods.

Ion channels are involved in a broad range of physiological and pathological processes. The implications of ion channels in a variety of diseases, including diabetes, epilepsy, hypertension, cancer and even chronic pain, have signaled them as pivotal drug targets. Thus far, drugs targeting ion channels were developed without detailed knowledge of the molecular interactions between the lead compounds and the target channels. In recent years, however, the emergence of high-resolution structures for a plethora of ion channels paves the way for computer-assisted drug design. Currently, available functional and structural data provide an attractive platform to generate models that combine substrate-based and protein-based approaches. In silico approaches include homology modeling, quantitative structure-activity relationships, virtual ligand screening, similarity and pharmacophore searching, data mining, and data analysis tools. These strategies have been frequently used in the discovery and optimization of novel molecules with enhanced affinity and specificity for the selected therapeutic targets. In this review we summarize recent applications of in silico methods that are being used for the development of ion channel drugs.

Interaction of the C-terminal region of the Ggamma protein with model membranes.

Heterotrimeric G-proteins interact with membranes. They accumulate around membrane receptors and propagate messages to effectors localized in different cellular compartments. G-protein-lipid interactions regulate G-protein cellular localization and activity. Although we recently found that the Gbetagamma dimer drives the interaction of G-proteins with nonlamellar-prone membranes, little is known about the molecular basis of this interaction. Here, we investigated the interaction of the C-terminus of the Ggamma(2) protein (P(gamma)-FN) with model membranes and those of its peptide (P(gamma)) and farnesyl (FN) moieties alone. X-ray diffraction and differential scanning calorimetry demonstrated that P(gamma)-FN, segregated into P(gamma)-FN-poor and -rich domains in phosphatidylethanolamine (PE) and phosphatidylserine (PS) membranes. In PE membranes, FN increased the nonlamellar phase propensity. Fourier transform infrared spectroscopy experiments showed that P(gamma) and P(gamma)-FN interact with the polar and interfacial regions of PE and PS bilayers. The binding of P(gamma)-FN to model membranes is due to the FN group and positively charged amino acids near this lipid. On the other hand, membrane lipids partially altered P(gamma)-FN structure, in turn increasing the fluidity of PS membranes. These data highlight the relevance of the interaction of the C-terminal region of the Ggamma protein with the cell membrane and its effect on membrane structure.

Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.