The First Extracellular Linker Is Important for Several Aspects of the Gating Mechanism of Human TRPA1 Channel.

Transient receptor potential ankyrin 1 (TRPA1) is an excitatory ion channel involved in pain, inflammation and itching. This channel gates in response to many irritant and proalgesic agents, and can be modulated by calcium and depolarizing voltage. While the closed-state structure of TRPA1 has been recently resolved, also having its open state is essential for understanding how this channel works. Here we use molecular dynamics simulations combined with electrophysiological measurements and systematic mutagenesis to predict and explore the conformational changes coupled to the expansion of the presumptive channel's lower gate. We show that, upon opening, the upper part of the sensor module approaches the pore domain of an adjacent subunit and the conformational dynamics of the first extracellular flexible loop may govern the voltage-dependence of multimodal gating, thereby serving to stabilize the open state of the channel. These results are generally important in understanding the structure and function of TRPA1 and offer new insights into the gating mechanism of TRPA1 and related channels.

N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

C-terminal acidic cluster is involved in Ca2+-induced regulation of human transient receptor potential ankyrin 1 channel.

The transient receptor potential ankyrin 1 (TRPA1) channel is a Ca(2+)-permeable cation channel whose activation results from a complex synergy between distinct activation sites, one of which is especially important for determining its sensitivity to chemical, voltage and cold stimuli. From the cytoplasmic side, TRPA1 is critically regulated by Ca(2+) ions, and this mechanism represents a self-modulating feedback loop that first augments and then inhibits the initial activation. We investigated the contribution of the cluster of acidic residues in the distal C terminus of TRPA1 in these processes using mutagenesis, whole cell electrophysiology, and molecular dynamics simulations and found that the neutralization of four conserved residues, namely Glu(1077) and Asp(1080)-Asp(1082) in human TRPA1, had strong effects on the Ca(2+)- and voltage-dependent potentiation and/or inactivation of agonist-induced responses. The surprising finding was that truncation of the C terminus by only 20 residues selectively slowed down the Ca(2+)-dependent inactivation 2.9-fold without affecting other functional parameters. Our findings identify the conserved acidic motif in the C terminus that is actively involved in TRPA1 regulation by Ca(2+).

Pore helix domain is critical to camphor sensitivity of transient receptor potential vanilloid 1 channel.

BACKGROUND: The recent discovery that camphor activates and strongly desensitizes the capsaicin-sensitive and noxious heat-sensitive channel transient receptor potential vanilloid subfamily member 1 (TRPV1) has provided new insights and opened up new research paths toward understanding why this naturally occurring monoterpene is widely used in human medicine for its local counter-irritant, antipruritic, and anesthetic properties. However, the molecular basis for camphor sensitivity remains mostly unknown. The authors attempt to explore the nature of the activation pathways evoked by camphor and narrow down a putative interaction site at TRPV1. METHODS: The authors transiently expressed wild-type or specifically mutated recombinant TRPV1 channels in human embryonic kidney cells HEK293T and recorded cation currents with the whole cell, patch clamp technique. To monitor changes in the spatial distribution of phosphatidylinositol 4,5-bisphosphate, they used fluorescence resonance energy transfer measurements from cells transfected with the fluorescent protein-tagged pleckstrin homology domains of phospholipase C. RESULTS: The results revealed that camphor modulates TRPV1 channel through the outer pore helix domain by affecting its overall gating equilibrium. In addition, camphor, which generally is known to decrease the fluidity of cell plasma membranes, may also regulate the activity of TRPV1 by inducing changes in the spatial distribution of phosphatidylinositol-4,5-bisphosphate on the inner leaflet of the plasma membrane. CONCLUSIONS: The findings of this study provide novel insights into the structural basis for the modulation of TRPV1 channel by camphor and may provide an explanation for the mechanism by which camphor modulates thermal sensation in vivo.

A "cute" desensitization of TRPV1.

Capsaicin and other vanilloids selectively excite and subsequently desensitize pain-conducting nerve fibers (nociceptors) and this process contributes to the analgesic (and thus therapeutically relevant) effects of these compounds. Such a desensitization process is triggered by the activation of the transient receptor potential vanilloid subtype 1 receptor channels (TRPV1) that open their cationic pores, permeable to sodium, potassium and calcium (Ca(2+)) ions. Depending on the duration of capsaicin exposure and the external calcium concentration, the Ca(2+) influx via TRPV1 channels desensitizes the channels themselves, which, from the cellular point of view, represents a feedback mechanism protecting the nociceptive neuron from toxic Ca(2+) overload. The 'acute desensitization' accounts for most of the reduction in responsiveness occurring within the first few (~20) seconds after the vanilloids are administered to the cell for the first time. Another form of desensitization is 'tachyphylaxis', which is a reduction in the response to repeated applications of vanilloid. The wealth of pathways following TRPV1 activation that lead to increased intracellular Ca(2+) levels and both forms of desensitization is huge and they might utilise just about every known type of signalling molecule. This review will not attempt to cover all historical aspects of research into all these processes. Instead, it will try to highlight some new challenging thoughts on the important phenomenon of TRPV1 desensitization and will focus on the putative mechanisms that are thought to account for the acute phase of this process.

Superoxide dismutase and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase polymorphisms and pancreatic cancer risk.

OBJECTIVES: Pancreatic carcinoma etiology and molecular pathogenesis is weakly understood. According to the assumption that genetic variation in carcinogen metabolism further modifies the risk of exposure-related cancers, an association of functional polymorphisms in oxidative stress-modifying genes superoxide dismutase 2 (SOD2 [Ala16Val, rs4880]), SOD3 (Arg231Gly, rs1799895), nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase (NQO1 [Pro187Ser, rs1800566], and NQO2 (Phe47Leu, rs1143684) with pancreatic cancer risk was studied. METHODS: Polymorphisms were studied by allelic discrimination. RESULTS: In a hospital-based case-control study on 500 individuals (235 cases and 265 controls) of Czech white origin, SOD2, SOD3, NQO1, and NQO2 polymorphisms showed no significant association with pancreatic cancer risk. Major lifestyle factors such as smoking and alcohol, coffee, or tea consumption did not modify the effect of the studied polymorphisms. CONCLUSIONS: The first European study of the SOD2, SOD3, NQO1, and NQO2 roles in pancreatic cancer etiology did not find significant associations. Despite this observation, other populations with different lifestyle(s) may be at risk and should be further studied.

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels.

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.