Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains

The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons

Molecular Evolution of the Infrared Sensory Gene TRPA1 in Snakes and Implications for Functional Studies

TRPA1 is a calcium ion channel protein recently identified as the infrared receptor in pit organ-containing snakes. Therefore, understanding the molecular evolution of TRPA1 may help to illuminate the origin of “heat vision” in snakes and reveal the molecular mechanism of infrared sensitivity for TRPA1. To this end, we sequenced the infrared sensory gene TRPA1 in 24 snake species, representing nine snake families and multiple non-snake outgroups. We found that TRPA1 is under strong positive selection in the pit-bearing snakes studied, but not in other non-pit snakes and non-snake vertebrates. As a comparison, TRPV1, a gene closely related to TRPA1, was found to be under strong purifying selection in all the species studied, with no difference in the strength of selection between pit-bearing snakes and non-pit snakes. This finding demonstrates that the adaptive evolution of TRPA1 specifically occurred within the pit-bearing snakes and may be related to the functional modification for detecting infrared radiation. In addition, by comparing the TRPA1 protein sequences, we identified 11 amino acid sites that were diverged in pit-bearing snakes but conserved in non-pit snakes and other vertebrates, 21 sites that were diverged only within pit-vipers but conserved in the remaining snakes. These specific amino acid substitutions may be potentially functional important for infrared sensing

The Ankyrin Repeat Domain of the TRPA Protein Painless Is Important for Thermal Nociception but Not Mechanical Nociception

The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception

Evolution of Vertebrate Transient Receptor Potential Vanilloid 3 Channels: Opposite Temperature Sensitivity between Mammals and Western Clawed Frogs

Transient Receptor Potential (TRP) channels serve as temperature receptors in a wide variety of animals and must have played crucial roles in thermal adaptation. The TRP vanilloid (TRPV) subfamily contains several temperature receptors with different temperature sensitivities. The TRPV3 channel is known to be highly expressed in skin, where it is activated by warm temperatures and serves as a sensor to detect ambient temperatures near the body temperature of homeothermic animals such as mammals. Here we performed comprehensive comparative analyses of the TRPV subfamily in order to understand the evolutionary process; we identified novel TRPV genes and also characterized the evolutionary flexibility of TRPV3 during vertebrate evolution. We cloned the TRPV3 channel from the western clawed frog Xenopus tropicalis to understand the functional evolution of the TRPV3 channel. The amino acid sequences of the N- and C-terminal regions of the TRPV3 channel were highly diversified from those of other terrestrial vertebrate TRPV3 channels, although central portions were well conserved. In a heterologous expression system, several mammalian TRPV3 agonists did not activate the TRPV3 channel of the western clawed frog. Moreover, the frog TRPV3 channel did not respond to heat stimuli, instead it was activated by cold temperatures. Temperature thresholds for activation were about 16 °C, slightly below the lower temperature limit for the western clawed frog. Given that the TRPV3 channel is expressed in skin, its likely role is to detect noxious cold temperatures. Thus, the western clawed frog and mammals acquired opposite temperature sensitivity of the TRPV3 channel in order to detect environmental temperatures suitable for their respective species, indicating that temperature receptors can dynamically change properties to adapt to different thermal environments during evolution

TRPA1 Is a Polyunsaturated Fatty Acid Sensor in Mammals

Fatty acids can act as important signaling molecules regulating diverse physiological processes. Our understanding, however, of fatty acid signaling mechanisms and receptor targets remains incomplete. Here we show that Transient Receptor Potential Ankyrin 1 (TRPA1), a cation channel expressed in sensory neurons and gut tissues, functions as a sensor of polyunsaturated fatty acids (PUFAs) in vitro and in vivo. PUFAs, containing at least 18 carbon atoms and three unsaturated bonds, activate TRPA1 to excite primary sensory neurons and enteroendocrine cells. Moreover, behavioral aversion to PUFAs is absent in TRPA1-null mice. Further, sustained or repeated agonism with PUFAs leads to TRPA1 desensitization. PUFAs activate TRPA1 non-covalently and independently of known ligand binding domains located in the N-terminus and 5th transmembrane region. PUFA sensitivity is restricted to mammalian (rodent and human) TRPA1 channels, as the drosophila and zebrafish TRPA1 orthologs do not respond to DHA. We propose that PUFA-sensing by mammalian TRPA1 may regulate pain and gastrointestinal functions

Inorganic Polyphosphate Modulates TRPM8 Channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity

Role of the electrostatic potential on the BK potassium channel conductance

Fernando D. Gonzalez-Nilo and Wendy Gonzalez. Centro de Bioinformatica, Universidad de Talca, Talca, Chile.The family of K channels presents a highly conserved structural motif (TVGYG), known as the selectivity filter. Despite the fact that the selectivity filter is conserved in all K channels, they show different conductances. We observed that the number of negative charged aminoacids in the outer vestibule of BK (high conductance), KvAP (medium conductance) and Shaker (low conductance) K channels, is proportional to the conductance of each channel. In particular, BK channel has a loop bearing two charged aminoacids in the outer vestibule. These negative charges would increase the local K ion concentration in the outer vestibule of the pore, diminishing the required energy to access the selectivity filter. To verify experimentally this hypothesis a series of BK channel mutants were constructed and the single channel conductances were measured. In agreement with our predictions, the removal of the loop charges (D261N/E264Q) leads to a reduced inward conductance. Deleting the outer loop does not affect inward conductance unless another charge is removed (Δloop/E276Q). This suggests that E276 can compensate the electrostatic contribution of the loop charges when the loop is deleted. The results are in agreement with electrostatic potential calculations (Poisson-Boltzmann) carried out on a molecular model of the BK channel pore. In conclusion, we have identified residues involved in the electrostatic tuning of the BK channel inward conductance. Acknowledgment: FONDECYT 1040254 (FG) and 1030830 (RL). Fundación Andes (FG)Meeting Abstract, Presentation Number: 483-Pos Poster Board Number: B322

External surface charge neutralization induces outward rectification on the Calcium- and Voltage- activated potassium channel BK

Gonzalez Nilo, Danilo and Gonzalez, Wendy. Centro de Bioinformatica y Simulacion Molecular, Universidad de Talca, Talca, Chile.There are two acidic amino acid residues in an extracellular loop (D326 and E329) of the large-conductance Calcium- and voltage- activated K+ channel (BK).Those are not present in lower conductance K+ channels. In order to determine the effect of these charges on channel conductance, we expressed BK mutants D326N, E329Q and D326N-E329Q on Xenopus oocytes and measured the expressed currents under path clamp. The double mutation D326N-E329Q induces outward rectification evidenced by a small decrease on a lower channel conductance at negative voltages (12 % at -100 mV, 110 mM K+ symmetrical). The outward rectification induced by charge neutralization increases to 22% at 30 mM symmetrical K+ concentration and disappears at 1 M symmetrical K+ suggesting an electrostatic mechanism. Surface charge effect of D326 on outward rectification accounts for the total effect of the double mutant. On the other hand, E329 charge neutralization promotes no rectification. To understand the differential effect of D326 and E329, we built a molecular model for the open pore of the BK channel using the MthK channel as template. Electrostatic potential calculations (Poisson-Boltzmann) were carried out on the molecular model embedded in a lipid bilayer. Our calculations indicate that the differential effect of the charges on the electrostatic potential at the selectivity filter is due to a differential dielectric shielding. The electrostatic potential induced E329 is more strongly shielded that D326 by the high dielectric constant of the external solution. (Supported by FONDECYT grants 103-0830 (RL), 104-0254 (FG) and D-200518 DID U.Austral (IC)).CECS hosts a Millennium Institute

Exploring the Shaker K+ channel S3b segment using deletion mutants

Wendy Gonzalez and Danilo Gonzalez Nilo. University of Talca, Talca, Chile.To asses the role of S3 region during the activation of the Shaker K+ channels we constructed Shaker K+ channel deletion mutants. A complete deletion of S3-S4 linker and part of the S3b segment Shaker Δ(6-46)Δ(327-360) produced a dramatic decrease in the maximum open probability. If we continue eliminating residues from the S3b segment, the resultant proteins do not express functional channels. Surprisingly, channel expression and functionality are recovered when both the S3-S4 linker and the entire S3b segment have been eliminated (Shaker Δ(6-46)Δ(312-360)). This result indicates that neither the S3-S4 linker nor the S3b segment is essential for the activation of Shaker K+ channels. When characterizing the S3b deletion mutants, we found a periodic relationship of the activation time constants and the number of the residues in this region of the protein Shaker Δ(6-46)Δ(326-360) through Shaker Δ(6-46)Δ(333-360). This periodic behavior can be represented by two sine functions with an angular frequency of 100o per residue, typical of α-helices but with an angular phase difference of 160o. This result suggests a break in the α-helix that occurs at threonine 329. The secondary structure break at Thr 329 was also found on a molecular dynamics simulation of this region (Iso 321 -Asp 336) done in H2O and for 1 ns time duration also showed a break point of the helix at threonine-329. These results are consistent with an extracellular location of segment S3b. Supported by Fondecyt 103-0830 and Fundacion Andes. The CECS is a Millenium InstitutePresentation Number: 2245-Pos Poster Board Number: B35