Linlin Ma

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (ICRAC) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of ICRAC. In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of ICRAC. The results show that cells expressing low Orai1 : STIM1 ratios produce ICRAC with strong fast Ca2+‐dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce ICRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

TRPM2 channels mediate acetaminophen-induced liver damage

Significance Acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. There are limited options for early treatment. Acetaminophen liver toxicity leads to the formation of reactive oxygen and nitrogen species which cause an increase in intracellular Ca2+ and hepatocellular death. We show that acetaminophen-induced liver toxicity depends on Transient Receptor Potential Melanostatine 2 (TRPM2) cation channels in hepatocytes, which are activated in response to oxidative stress and are responsible for Ca2+ overload. Lack of TRPM2 channels in hepatocytes or their pharmacological inhibition protects liver from acetaminophen toxicity. This provides evidence that TRPM2 may present a potential therapeutic target for treatment of oxidative-stress related liver diseases. Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca2+ homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca2+ concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca2+ rise. Here we report that the channel responsible for Ca2+ entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H2O2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H2O2. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.

Divalent cations potentiate TRPV1 channel by lowering the heat activation threshold

Transient receptor potential vanilloid type 1 (TRPV1) channel responds to a wide spectrum of physical and chemical stimuli. In doing so, it serves as a polymodal cellular sensor for temperature change and pain. Many chemicals are known to strongly potentiate TRPV1 activation, though how this is achieved remains unclear. In this study we investigated the molecular mechanism underlying the gating effects of divalent cations Mg2+ and Ba2+. Using a combination of fluorescence imaging and patch-clamp analysis, we found that these cations potentiate TRPV1 gating by most likely promoting the heat activation process. Mg2+ substantially lowers the activation threshold temperature; as a result, a significant fraction of channels are heat-activated at room temperature. Although Mg2+ also potentiates capsaicin- and voltage-dependent activation, these processes were found either to be not required (in the case of capsaicin) or insufficient (in the case of voltage) to mediate the activating effect. In support of a selective effect on heat activation, Mg2+ and Ba2+ cause a Ca2+-independent desensitization that specifically prevents heat-induced channel activation but does not prevent capsaicin-induced activation. These results can be satisfactorily explained within an allosteric gating framework in which divalent cations strongly promote the heat-dependent conformational change or its coupling to channel activation, which is further coupled to the voltage- and capsaicin-dependent processes.

Movement of hClC-1 C-termini during common gating and limits on their cytoplasmic location

Functionally, the dimeric human skeletal muscle chloride channel hClC-1 is characterized by two distinctive gating processes, fast (protopore) gating and slow (common) gating. Of these, common gating is poorly understood, but extensive conformational rearrangement is suspected. To examine this possibility, we used FRET (fluorescence resonance energy transfer) and assessed the effects of manipulating the common-gating process. Closure of the common gate was accompanied by a separation of the C-termini, whereas, with opening, the C-termini approached each other more closely. These movements were considerably smaller than those seen in ClC-0. To estimate the C-terminus depth within the cytoplasm we constructed a pair of split hClC-1 fragments tagged extracellularly and intracellularly respectively. These not only combined appropriately to rescue channel function, but we detected positive FRET between them. This restricts the C-termini of hClC-1 to a position close to its membrane-resident domain. From mutants in which fast or common gating were affected, FRET revealed a close linkage between the two gating processes with the carboxyl group of Glu²³² apparently acting as the final effector for both.

Divalent cations activate TRPV1 through promoting conformational change of the extracellular region

Divalent cations Mg2+ and Ba2+ selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg2+ potentiates channel activation only from the extracellular side; on the intracellular side, Mg2+ inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1–S2 linker, the S3–S4 linker, and the pore turret are all required for Mg2+ potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg2+-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg2+ also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg2+ precedes channel activation, suggesting that Mg2+-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.

Nicotinic Acid is a Common Regulator of Heat-Sensing TRPV1-4 Ion Channels

Nicotinic acid (NA, a.k.a. vitamin B3 or niacin) can reduce blood cholesterol and low-density lipoproteins whereas increase high-density lipoproteins. However, when NA is used to treat dyslipidemias, it causes a strong side effect of cutaneous vasodilation, commonly called flushing. A recent study showed that NA may cause flushing by lowering activation threshold temperature of the heat-sensitive capsaicin receptor TRPV1 ion channel, leading to its activation at body temperature. The finding calls into question whether NA might also interact with the homologous heat-sensitive TRPV2–4 channels, particularly given that TRPV3 and TRPV4 are abundantly expressed in keratinocytes of the skin where much of the flushing response occurs. We found that NA indeed potentiated TRPV3 while inhibited TRPV2 and TRPV4. Consistent with these gating effects, NA lowered the heat-activation threshold of TRPV3 but elevated that of TRPV4. We further found that activity of TRPV1 was substantially prolonged by extracellular NA, which may further enhance the direct activation effect. Consistent with the broad gating effect on TRPV1–4 channels, evidence from the present study hints that NA may share the same activation pathway as 2-aminoethoxydiphenyl borate (2-APB), a common agonist for these TRPV channels. These findings shed new light on the molecular mechanism underlying NA regulation of TRPV channels.

Nicotinic Acid Activates the Capsaicin Receptor TRPV1 Potential Mechanism for Cutaneous Flushing

Objective—Nicotinic acid (also known as niacin or vitamin B3), widely used to treat dyslipidemias, represents an effective and safe means to reduce the risk of mortality from cardiovascular disease. Nonetheless, a substantial fraction of patients discontinue treatment because of a strong side effect of cutaneous vasodilation, commonly termed flushing. In the present study, we tested the hypothesis that nicotinic acid causes flushing partially by activating the capsaicin receptor TRPV1, a polymodal cellular sensor that mediates the flushing response on consumption of spicy food. Approach and Results—We observed that the nicotinic acid–induced increase in blood flow was substantially reduced in Trpv1–/– knockout mice, indicating involvement of the channel in flushing response. Using exogenously expressed TRPV1, we confirmed that nicotinic acid at submillimolar to millimolar concentrations directly and potently activates TRPV1 from the intracellular side. Binding of nicotinic acid to TRPV1 lowers its activation threshold for heat, causing channel opening at physiological temperatures. The activation of TRPV1 by voltage or ligands (capsaicin and 2-aminoethoxydiphenyl borate) is also potentiated by nicotinic acid. We further demonstrated that nicotinic acid does not compete directly with capsaicin but may activate TRPV1 through the 2-aminoethoxydiphenyl borate activation pathway. Using live-cell fluorescence imaging, we observed that nicotinic acid can quickly enter the cell through a transporter-mediated pathway to activate TRPV1. Conclusions—Direct activation of TRPV1 by nicotinic acid may lead to cutaneous vasodilation that contributes to flushing, suggesting a potential novel pathway to inhibit flushing and to improve compliance.Objective— Nicotinic acid (also known as niacin or vitamin B3), widely used to treat dyslipidemias, represents an effective and safe means to reduce the risk of mortality from cardiovascular disease. Nonetheless, a substantial fraction of patients discontinue treatment because of a strong side effect of cutaneous vasodilation, commonly termed flushing. In the present study, we tested the hypothesis that nicotinic acid causes flushing partially by activating the capsaicin receptor TRPV1, a polymodal cellular sensor that mediates the flushing response on consumption of spicy food. Approach and Results— We observed that the nicotinic acid–induced increase in blood flow was substantially reduced in Trpv1 –/– knockout mice, indicating involvement of the channel in flushing response. Using exogenously expressed TRPV1, we confirmed that nicotinic acid at submillimolar to millimolar concentrations directly and potently activates TRPV1 from the intracellular side. Binding of nicotinic acid to TRPV1 lowers its activation threshold for heat, causing channel opening at physiological temperatures. The activation of TRPV1 by voltage or ligands (capsaicin and 2-aminoethoxydiphenyl borate) is also potentiated by nicotinic acid. We further demonstrated that nicotinic acid does not compete directly with capsaicin but may activate TRPV1 through the 2-aminoethoxydiphenyl borate activation pathway. Using live-cell fluorescence imaging, we observed that nicotinic acid can quickly enter the cell through a transporter-mediated pathway to activate TRPV1. Conclusions— Direct activation of TRPV1 by nicotinic acid may lead to cutaneous vasodilation that contributes to flushing, suggesting a potential novel pathway to inhibit flushing and to improve compliance. # Significance {#article-title-49}

Nicotinic Acid Activates the Capsaicin Receptor TRPV1

Objective—Nicotinic acid (also known as niacin or vitamin B3), widely used to treat dyslipidemias, represents an effective and safe means to reduce the risk of mortality from cardiovascular disease. Nonetheless, a substantial fraction of patients discontinue treatment because of a strong side effect of cutaneous vasodilation, commonly termed flushing. In the present study, we tested the hypothesis that nicotinic acid causes flushing partially by activating the capsaicin receptor TRPV1, a polymodal cellular sensor that mediates the flushing response on consumption of spicy food. Approach and Results—We observed that the nicotinic acid–induced increase in blood flow was substantially reduced in Trpv1–/– knockout mice, indicating involvement of the channel in flushing response. Using exogenously expressed TRPV1, we confirmed that nicotinic acid at submillimolar to millimolar concentrations directly and potently activates TRPV1 from the intracellular side. Binding of nicotinic acid to TRPV1 lowers its activation threshold for heat, causing channel opening at physiological temperatures. The activation of TRPV1 by voltage or ligands (capsaicin and 2-aminoethoxydiphenyl borate) is also potentiated by nicotinic acid. We further demonstrated that nicotinic acid does not compete directly with capsaicin but may activate TRPV1 through the 2-aminoethoxydiphenyl borate activation pathway. Using live-cell fluorescence imaging, we observed that nicotinic acid can quickly enter the cell through a transporter-mediated pathway to activate TRPV1. Conclusions—Direct activation of TRPV1 by nicotinic acid may lead to cutaneous vasodilation that contributes to flushing, suggesting a potential novel pathway to inhibit flushing and to improve compliance.Objective— Nicotinic acid (also known as niacin or vitamin B3), widely used to treat dyslipidemias, represents an effective and safe means to reduce the risk of mortality from cardiovascular disease. Nonetheless, a substantial fraction of patients discontinue treatment because of a strong side effect of cutaneous vasodilation, commonly termed flushing. In the present study, we tested the hypothesis that nicotinic acid causes flushing partially by activating the capsaicin receptor TRPV1, a polymodal cellular sensor that mediates the flushing response on consumption of spicy food. Approach and Results— We observed that the nicotinic acid–induced increase in blood flow was substantially reduced in Trpv1 –/– knockout mice, indicating involvement of the channel in flushing response. Using exogenously expressed TRPV1, we confirmed that nicotinic acid at submillimolar to millimolar concentrations directly and potently activates TRPV1 from the intracellular side. Binding of nicotinic acid to TRPV1 lowers its activation threshold for heat, causing channel opening at physiological temperatures. The activation of TRPV1 by voltage or ligands (capsaicin and 2-aminoethoxydiphenyl borate) is also potentiated by nicotinic acid. We further demonstrated that nicotinic acid does not compete directly with capsaicin but may activate TRPV1 through the 2-aminoethoxydiphenyl borate activation pathway. Using live-cell fluorescence imaging, we observed that nicotinic acid can quickly enter the cell through a transporter-mediated pathway to activate TRPV1. Conclusions— Direct activation of TRPV1 by nicotinic acid may lead to cutaneous vasodilation that contributes to flushing, suggesting a potential novel pathway to inhibit flushing and to improve compliance. # Significance {#article-title-49}

Functional study of cytoplasmic loops of human skeletal muscle chloride channel, hClC-1

The membrane-resident domain of chloride channels and transporters of the CLC family is composed of 18 alpha-helices (designated A to R) connected sequentially by extracellular and intracellular loops, whose functional characteristics are generally unclear. To study the relevance of the intracellular loops linking helices D and E, F and G, H and I and J and K, alanine-exchange mutagenesis, split channel strategy, GST (glutathione transferase)-pull-down methods and whole-cell patch-clamp recordings were used. We investigated the possible roles of these loops in binding to the cytoplasmic, carboxyl tail (C-tail) of the protein, as well as their physiological roles in channel function. Although other interacting positions are conceivable, our results indicate that there is unlikely to be significant binding between the C-tail and any one of these individual cytoplasmic loops. Particular loops might, however, be essential for some channel characteristics. For example, alanine-exchange mutation of the loop linking helix D to E eliminated channel currents; of the loop linking helix H to I caused a significant shift of the open probability of fast gating (P(o)(f)), towards more positive voltages; and of the loop linking helix J to K locked the common gating of hClC-1 open. Therefore, the gating mechanisms of hClC-1 might not only involve the helices and the C-tail, but also involve some of the loops.

Analysis of carboxyl tail function in the skeletal muscle Cl- channel hClC-1.

Human ClC-1 (skeletal muscle Cl- channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine beta-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser(720) and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser(720) (but with CBS1 deleted) and a complete C-tail beginning at Leu(598). Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1-CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu(665) and Lys(680).