Protector Role of Cx30.2 in Pancreatic ß-Cell against Glucotoxicity-Induced Apoptosis.

Glucotoxicity may exert its deleterious effects on pancreatic ß-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. ß-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in ß cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of ß cells (RIN-m5F). METHODS: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. RESULTS: High glucose did not induce apoptosis in RIN-m5F ß cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. CONCLUSION: The data suggest that the upregulation of Cx30.2 protects ß cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.

Permeant cations modulate pore dynamics and gating of TRPV1 ion channels.

The transient receptor vanilloid 1 (TRPV1) is a non-selective ion channel, which is activated by several chemical ligands and heat. We have previously shown that activation of TRPV1 by different ligands results in single-channel openings with different conductance, suggesting that the selectivity filter is highly dynamic. TRPV1 is weakly voltage dependent; here, we sought to explore whether the permeation of different monovalent ions could influence the voltage dependence of this ion channel. By using single-channel recordings, we show that TRPV1 channels undergo rapid transitions to closed states that are directly connected to the open state, which may result from structural fluctuations of their selectivity filter. Moreover, we demonstrate that the rates of these transitions are influenced by the permeant ion, suggesting that ion permeation regulates the voltage dependence of these channels. Our data could be the basis for more detailed MD simulations exploring the permeation mechanism and how the occupancy of different ions alters the three-dimensional structure of the pore of TRPV1 channels.

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid.

Voltage-dependent gating of the voltage-gated proton channels (HV1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents. To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependence on voltage and the pH gradient (ΔpH). We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization. The kinetics of the fluorescence signal reveal the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by ΔpH. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. These results shed light on the complexities of voltage-dependent opening of human HV1 channels.

Modes of action of lysophospholipids as endogenous activators of the TRPV4 ion channel.

The Transient Receptor Potential Vanilloid 4 (TRPV4) channel has been shown to function in many physiological and pathophysiological processes. Despite abundant information on its importance in physiology, very few endogenous agonists for this channel have been described, and very few underlying mechanisms for its activation have been clarified. TRPV4 is expressed by several types of cells, such as vascular endothelial, and skin and lung epithelial cells, where it plays pivotal roles in their function. In the present study, we show that TRPV4 is activated by lysophosphatidic acid (LPA) in both endogenous and heterologous expression systems, pinpointing this molecule as one of the few known endogenous agonists for TRPV4. Importantly, LPA is a bioactive glycerophospholipid, relevant in several physiological conditions, including inflammation and vascular function, where TRPV4 has also been found to be essential. Here we also provide mechanistic details of the activation of TRPV4 by LPA and another glycerophospholipid, lysophosphatidylcholine (LPC), and show that LPA directly interacts with both the N- and C-terminal regions of TRPV4 to activate this channel. Moreover, we show that LPC activates TRPV4 by producing an open state with a different single-channel conductance to that observed with LPA. Our data suggest that the activation of TRPV4 can be finely tuned in response to different endogenous lipids, highlighting this phenomenon as a regulator of cell and organismal physiology. KEY POINTS: The Transient Receptor Potential Vaniloid (TRPV) 4 ion channel is a widely distributed protein with important roles in normal and disease physiology for which few endogenous ligands are known. TRPV4 is activated by a bioactive lipid, lysophosphatidic acid (LPA) 18:1, in a dose-dependent manner, in both a primary and a heterologous expression system. Activation of TRPV4 by LPA18:1 requires residues in the N- and C-termini of the ion channel. Single-channel recordings show that TRPV4 is activated with a decreased current amplitude (conductance) in the presence of lysophosphatidylcholine (LPC) 18:1, while LPA18:1 and GSK101 activate the channel with a larger single-channel amplitude. Distinct single-channel amplitudes produced by LPA18:1 and LPC18:1 could differentially modulate the responses of the cells expressing TRPV4 under different physiological conditions.

How to study a highly toxic protein to bacteria: A case of voltage sensor domain of mouse sperm-specific sodium/proton exchanger.

Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.

TRP channels: a journey towards a molecular understanding of pain.

The perception of nociceptive signals, which are translated into pain, plays a fundamental role in the survival of organisms. Because pain is linked to a negative sensation, animals learn to avoid noxious signals. These signals are detected by receptors, which include some members of the transient receptor potential (TRP) family of ion channels that act as transducers of exogenous and endogenous noxious cues. These proteins have been in the focus of the field of physiology for several years, and much knowledge of how they regulate the function of the cell types and organs where they are expressed has been acquired. The last decade has been especially exciting because the 'resolution revolution' has allowed us to learn the molecular intimacies of TRP channels using cryogenic electron microscopy. These findings, in combination with functional studies, have provided insights into the role played by these channels in the generation and maintenance of pain.

Discovery and characterization of Hv1-type proton channels in reef-building corals.

Voltage-dependent proton-permeable channels are membrane proteins mediating a number of important physiological functions. Here we report the presence of a gene encoding Hv1 voltage-dependent, proton-permeable channels in two species of reef-building corals. We performed a characterization of their biophysical properties and found that these channels are fast-activating and modulated by the pH gradient in a distinct manner. The biophysical properties of these novel channels make them interesting model systems. We have also developed an allosteric gating model that provides mechanistic insight into the modulation of voltage-dependence by protons. This work also represents the first functional characterization of any ion channel in scleractinian corals. We discuss the implications of the presence of these channels in the membranes of coral cells in the calcification and pH-regulation processes and possible consequences of ocean acidification related to the function of these channels.

A rationally designed orthogonal synthetase for genetically encoded fluorescent amino acids.

The incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of a novel orthogonal aminoacyl-tRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

KV1.2 channels inactivate through a mechanism similar to C-type inactivation.

Slow inactivation has been described in multiple voltage-gated K+ channels and in great detail in the Drosophila Shaker channel. Structural studies have begun to facilitate a better understanding of the atomic details of this and other gating mechanisms. To date, the only voltage-gated potassium channels whose structure has been solved are KvAP (x-ray diffraction), the KV1.2-KV2.1 "paddle" chimera (x-ray diffraction and cryo-EM), KV1.2 (x-ray diffraction), and ether-à-go-go (cryo-EM); however, the structural details and mechanisms of slow inactivation in these channels are unknown or poorly characterized. Here, we present a detailed study of slow inactivation in the rat KV1.2 channel and show that it has some properties consistent with the C-type inactivation described in Shaker. We also study the effects of some mutations that are known to modulate C-type inactivation in Shaker and show that qualitative and quantitative differences exist in their functional effects, possibly underscoring subtle but important structural differences between the C-inactivated states in Shaker and KV1.2.

A novel origin for calcium selectivity.

A native calcium ion channel has been identified in bacteria for the first time.

The Contribution of the Ankyrin Repeat Domain of TRPV1 as a Thermal Module.

The TRPV1 cation nonselective ion channel plays an essential role in thermosensation and perception of other noxious stimuli. TRPV1 can be activated by low extracellular pH, high temperature, or naturally occurring pungent molecules such as allicin, capsaicin, or resiniferatoxin. Its noxious thermal sensitivity makes it an important participant as a thermal sensor in mammals. However, details of the mechanism of channel activation by increases in temperature remain unclear. Here, we used a combination of approaches to try to understand the role of the ankyrin repeat domain (ARD) in channel behavior. First, a computational modeling approach by coarse-grained molecular dynamics simulation of the whole TRPV1 embedded in a phosphatidylcholine and phosphatidylethanolamine membrane provides insight into the dynamics of this channel domain. Global analysis of the structural ensemble shows that the ARD is a region that sustains high fluctuations during dynamics at different temperatures. We then performed biochemical and thermal stability studies of the purified ARD by the means of circular dichroism and tryptophan fluorescence and demonstrate that this region undergoes structural changes at similar temperatures that lead to TRPV1 activation. Our data suggest that the ARD is a dynamic module and that it may participate in controlling the temperature sensitivity of TRPV1.

TRP ion channels: Proteins with conformational flexibility.

Ion channels display conformational changes in response to binding of their agonists and antagonists. The study of the relationships between the structure and the function of these proteins has witnessed considerable advances in the last two decades using a combination of techniques, which include electrophysiology, optical approaches (i.e. patch clamp fluorometry, incorporation of non-canonic amino acids, etc.), molecular biology (mutations in different regions of ion channels to determine their role in function) and those that have permitted the resolution of their structures in detail (X-ray crystallography and cryo-electron microscopy). The possibility of making correlations among structural components and functional traits in ion channels has allowed for more refined conclusions on how these proteins work at the molecular level. With the cloning and description of the family of Transient Receptor Potential (TRP) channels, our understanding of several sensory-related processes has also greatly moved forward. The response of these proteins to several agonists, their regulation by signaling pathways as well as by protein-protein and lipid-protein interactions and, in some cases, their biophysical characteristics have been studied thoroughly and, recently, with the resolution of their structures, the field has experienced a new boom. This review article focuses on the conformational changes in the pores, concentrating on some members of the TRP family of ion channels (TRPV and TRPA subfamilies) that result in changes in their single-channel conductances, a phenomenon that may lead to fine-tuning the electrical response to a given agonist in a cell.

Role of lysophosphatidic acid in ion channel function and disease.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that exhibits a wide array of functions that include regulation of protein synthesis and adequate development of organisms. LPA is present in the membranes of cells and in the serum of several mammals and has also been shown to participate importantly in pathophysiological conditions. For several decades it was known that LPA produces some of its effects in cells through its interaction with specific G protein-coupled receptors, which in turn are responsible for signaling pathways that regulate cellular function. Among the target proteins for LPA receptors are ion channels that modulate diverse aspects of the physiology of cells and organs where they are expressed. However, recent studies have begun to unveil direct effects of LPA on ion channels, highlighting this phospholipid as a direct agonist and adding to the knowledge of the field of lipid-protein interactions. Moreover, the roles of LPA in pathophysiological conditions associated with the function of some ion channels have also begun to be clarified, and molecular mechanisms have been identified. This review focuses on the effects of LPA on ion channel function under normal and pathological conditions and highlights our present knowledge of the mechanisms by which it regulates the function and expression of N- and T-type Ca++ channels; M-type K+ channel and inward rectifier K+ channel subunit 2.1; transient receptor potential (TRP) melastatin 2, TRP vanilloid 1, and TRP ankyrin 1 channels; and TWIK-related K+ channel 1 (TREK-1), TREK-2, TWIK-related spinal cord K+ channel (TRESK), and TWIK-related arachidonic acid-stimulated K+ channel (TRAAK).

Inhibition of TRPV1 channels by a naturally occurring omega-9 fatty acid reduces pain and itch.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is mainly found in primary nociceptive afferents whose activity has been linked to pathophysiological conditions including pain, itch and inflammation. Consequently, it is important to identify naturally occurring antagonists of this channel. Here we show that a naturally occurring monounsaturated fatty acid, oleic acid, inhibits TRPV1 activity, and also pain and itch responses in mice by interacting with the vanilloid (capsaicin)-binding pocket and promoting the stabilization of a closed state conformation. Moreover, we report an itch-inducing molecule, cyclic phosphatidic acid, that activates TRPV1 and whose pruritic activity, as well as that of histamine, occurs through the activation of this ion channel. These findings provide insights into the molecular basis of oleic acid inhibition of TRPV1 and also into a way of reducing the pathophysiological effects resulting from its activation.

The role of allosteric coupling on thermal activation of thermo-TRP channels.

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH(o)) and entropy (ΔS(o)) upon channel gating. The magnitude, sign, and temperature dependence of ΔH(o) and ΔS(o), the last given by an associated change in heat capacity (ΔCp), can determine a channel's temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔCp makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH(o) and ΔS(o), including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH(o)/ΔS(o) ratio associated with them, can also determine a channel's temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔCp-independent manner.

Ion channels in analgesia research.

Several recent techniques have allowed us to pinpoint the receptors responsible for the detection of nociceptive stimuli. Among these receptors, ion channels play a fundamental role in the recognition and transduction of stimuli that can cause pain. During the last decade, compelling evidence has been gathered on the role of the TRPV1 channel in inflammatory and neuropathic states. Activation of TRPV1 in nociceptive neurons results in the release of neuropeptides and transmitters, leading to the generation of action potentials that will be sent to higher CNS areas, where they will often be perceived as pain. Its activation will also evoke the peripheral release of pro-inflammatory compounds that may sensitize other neurons to physical, thermal, or chemical stimuli. For these reasons, and because its continuous activation causes analgesia, TRPV1 is now considered a viable drug target for clinical use in the management of pain. Using the TRPV1 channel as an example, here we describe some basic biophysical approaches used to study the properties of ion channels involved in pain and in analgesia.

A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic.

Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.