Mice lacking histidine decarboxylase exhibit abnormal mast cells

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.     

Mice lacking the transient receptor vanilloid potential 1 channel display normal thirst responses and central Fos activation to hypernatremia

Neurons of the organum vasculosum of the lamina terminalis (OVLT) are necessary for thirst and vasopressin secretion during hypersmolality in rodents. Recent evidence suggests the osmosensitivity of these neurons is mediated by a gene product encoding the transient receptor potential vanilloid-1 (TRPV1) channel. The purpose of the present study was to determine whether mice lacking the TRPV1 channel had blunted thirst responses and central Fos activation to acute and chronic hyperosmotic stimuli. Surprisingly, TRPV1-/- vs. wild-type mice ingested similar amounts of water after injection (0.5 ml sc) of 0.5 M NaCl and 1.0 M NaCl. Chronic increases in plasma osmolality produced by overnight water deprivation or sole access to a 2% NaCl solution for 48 h produced similar increases in water intake between wild-type and TRPV1-/- mice. There were no differences in cumulative water intakes in response to hypovolemia or isoproterenol. In addition, the number of Fos-positive cells along the lamina terminalis, including the OVLT, as well as the supraoptic nucleus and hypothalamic paraventricular nucleus, was similar between wild-type and TRPV1-/- mice after both acute and chronic osmotic stimulation. These findings indicate that TRPV1 channels are not necessary for osmotically driven thirst or central Fos activation, and thereby suggest that TRPV1 channels are not the primary ion channels that permit the brain to detect changes in plasma sodium concentration or osmolality.     

Mice lacking the proton channel Hv1 exhibit sex-specific differences in glucose homeostasis

Sex as a physiologic factor has a strong association with the features of metabolic syndrome. Our previous study showed that loss of the voltage-gated proton channel Hv1 inhibits insulin secretion and leads to hyperglycemia and glucose intolerance in male mice. However, there are significant differences in blood glucose between male and female Hv1-knockout (KO) mice. Here, we investigated the differences in glucose metabolism and insulin sensitivity between male and female KO mice and how sex steroids contribute to these differences. We found that the fasting blood glucose in female KO mice was visibly lower than that in male KO mice, which was accompanied by hypotestosteronemia. KO mice in both sexes exhibited higher expression of gluconeogenesis-related genes in liver compared with WT mice. Also, the livers from KO males displayed a decrease in glycolysis-related gene expression and an increase in gluconeogenesis-related gene expression compared with KO females. Furthermore, exogenous testosterone supplementation decreased blood glucose levels in male KO mice, as well as enhancing insulin signaling. Taken together, our data demonstrate that knockout of Hv1 results in higher blood glucose levels in male than female mice, despite a decreased insulin secretion in both sexes. This sex-related difference in glucose homeostasis is associated with the glucose metabolism in liver tissue, likely due to the physiological levels of testosterone in KO male mice.     

Dynamic regulation of ryanodine receptor type 1 (RyR1) channel activity by Homer 1

Homer, a family of scaffolding proteins originally identified in neurons, is also expressed in skeletal muscle. Previous studies showed that splice variants of Homer 1 (H1) amplify the gain of the ryanodine receptor type 1 (RyR1) channel complex. Using [3H]ryanodine ([3H]Ry) to probe the conformational state of RyR1, the actions of long- and short-forms of H1 are examined singly and in combination. At < or =200 nM, H1 long-forms (H1b or H1c possessing coiled-coil (CC) domains) and short-forms (H1a or H1EVH1 lacking CC domains) enhance specific [3H]Ry binding to RyR1. However, at a concentration > 200 nM, either H1 form completely inhibited [3H]Ry binding. Importantly, the combinations of H1c+H1EVH1, or H1b+H1a acted in an additive manner to enhance or inhibit [3H]Ry-binding activity. H1a and H1c individually or in combination produced the same dynamic pattern in regulating purified RyR1 channels reconstituted in planar lipid bilayers. In combination, their net action on RyR1 channels depends on total concentrations of H1. These data provide a mechanism by which constitutively and transiently expressed H1 forms can tightly regulate RyR1 channel activity in response to changing levels of expression and degradation of H1 proteins.     

Inhibition of transient receptor potential A1 channel by phosphatidylinositol-4,5-bisphosphate

Membrane phosphatidylinositol-4,5-bisphosphate (PIP2) is critical for the function of many transient receptor potential (TRP) ion channels. The role of PIP2 in TRPA1 function is not well known. The effect of PIP2 on TRPA1 was investigated by direct application of PIP2 and by using polylysine and PIP2 antibody that sequester PIP2. In inside-out patches from HeLa cells expressing mouse TRPA1, polytriphosphate (PPPi) was added to the bath solution to keep TRPA1 sensitive to allyl isothiocyanate (AITC; mustard oil). Direct application of PIP2 (10 microM) to inside-out patches did not activate TRPA1, but AITC and Delta(9)-tetrahydrocannabinol (THC) produced strong activation. In inside-out patches in which TRPA1 was first activated with AITC (in the presence of PPPi), further addition of PIP2 produced a concentration-dependent inhibition of TRPA1 [agonist concentration producing half-maximal activity (K(1/2)), 2.8 microM]. Consistent with the inhibition of TRPA1 by PIP2, AITC activated a large whole cell current when polylysine or PIP2 antibody was added to the pipette but a markedly diminished current when PIP2 was added to the pipette. In inside-out patches with PPPi in the bath solution, application of PIP2 antibody or polylysine caused activation of TRPA1, and this was blocked by PIP2. However, TRPA1 was not activated by polylysine and PIP2 antibody under whole cell conditions, suggesting a more complex regulation of TRPA1 by PIP2 in intact cells. These results show that PIP2 inhibits TRPA1 and reduces the sensitivity of TRPA1 to AITC.     

Electrophysiological characterization of a schistosome transient receptor potential channel activated by praziquantel

Ion channels have proved to be productive targets for anthelmintic chemotherapy. One example is the recent discovery of a parasitic flatworm ion channel targeted by praziquantel (PZQ), the main clinical therapy used for treatment of schistosomiasis. The ion channel activated by PZQ – a transient receptor potential ion channel of the melastatin subfamily, named TRPM_PZQ – is a Ca²⁺-permeable ion channel expressed in all parasitic flatworms that are PZQ-sensitive. However, little is currently known about the electrophysiological properties of this target that mediates the deleterious action of PZQ on many trematodes and cestodes. Here, we provide a detailed biophysical characterization of the properties of Schistosoma mansoni TRPM_PZQ channel (Sm.TRPM_PZQ) in response to PZQ. Single channel electrophysiological analysis demonstrated that Sm.TRPM_PZQ when activated by PZQ is a non-selective, large conductance, voltage-insensitive cation channel that displays distinct properties from human TRPM paralogs. Sm.TRPM_PZQ is Ca²⁺-permeable but does not require Ca²⁺ for channel gating in response to PZQ. TRPM_PZQ from Schistosoma japonicum (Sj.TRPM_PZQ) and Schistosoma haematobium (Sh.TRPM_PZQ) displayed similar characteristics. Profiling Sm.TRPM_PZQ responsiveness to PZQ has established a biophysical signature for this channel that will aid future investigation of endogenous TRPM_PZQ activity, as well as analyses of endogenous and exogenous regulators of this novel, druggable antiparasitic target.     

Mice lacking Ca(v)2.3 (alpha1E) calcium channel exhibit hyperglycemia.

To investigate the functional role of Ca(v)2.3 channel in glucose homeostasis, we performed in vivo glucose tolerance and insulin tolerance tests together with stress-induced glucose release tests using mice deficient in Ca(v)2.3 channel (Ca(v)2.3-/-). The Ca(v)2.3-/- mice were significantly heavier than wild-type mice. In glucose tolerance and insulin tolerance tests, Ca(v)2.3-/- mice showed a significantly higher blood glucose level compared to wild-type mice. However, stress-induced blood glucose changes in Ca(v)2.3-/- mice were similar to those in wild-type mice. These results suggest that Ca(v)2.3 channel plays a role in glucose homeostasis by reducing insulin sensitivity and that Ca(v)2.3-/- mice exhibit symptoms resembling non-insulin-dependent diabetes mellitus.     

Mice lacking a CDK inhibitor, p57Kip2, exhibit skeletal abnormalities and growth retardation

p57Kip2, one of the cyclin-dependent kinase (CDK) inhibitors, has been suggested to be a tumor suppressor candidate. To elucidate its biological roles in mouse development and tumorigenesis, we created p57Kip2-deficient mice. The p57Kip2-deficient mice exhibited a cleft palate and defective bone formation resulting in severe dyspnea. Most of the p57Kip2-deficient mice died within 24 h after birth, while about 10% of them survived beyond the weaning period. All of the surviving mice showed severe growth retardation. The males showed immaturity of the testes, prostate and seminal vesicles, and the females showed vaginal atresia, immaturity of the uterus, and an increased number of atretic follicles. Although Yan et al. and Zhang et al. have already reported p57Kip2-deficient mice, they could not investigate the phenotypes of the surviving p57Kip2-deficient mice. Also, most of the symptoms of Beckwith-Wiedemann syndrome could not be reproduced in the mutant mice. Embryonic fibroblasts prepared from p57Kip2-deficient mice showed no differences in the proliferation rate and saturation density, suggesting that G1 arrest is largely independent of p57Kip2 function. Our results suggest that p57Kip2 plays a critical role in development, but do not support the hypothesis that the p57Kip2 gene is a tumor-suppressor gene or is responsible for Beckwith-Wiedemann syndrome.     

Homer regulates gain of ryanodine receptor type 1 channel complex

Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors with intracellular inositol trisphosphate receptors and is modified by neuronal activity. Here, we demonstrate that Homer proteins also physically associate with ryanodine receptors type 1 (RyR1) and regulate gating responses to Ca(2+), depolarization, and caffeine. In contrast to the prevailing notion of Homer function, Homer1c (long form) and Homer1-EVH1 (short form) evoke similar changes in RyR activity. The EVH1 domain mediates these actions of Homer and is selectively blocked by a peptide that mimics the Homer ligand. 1B5 dyspedic myotubes expressing RyR1 with a point mutation of a putative Homer-binding domain exhibit significantly reduced (approximately 33%) amplitude in their responses to K(+) depolarization compared with cells expressing wild type protein. These results reveal that in addition to its known role as an adapter protein, Homer is a direct modulator of Ca(2+) release gain. Homer is the first example of an "adapter" that also modifies signaling properties of its target protein. The present work reveals a novel mechanism by which Homer directly modulates the function of its target protein RyR1 and excitation-contraction coupling in skeletal myotubes. This form of regulation may be important in other cell types that express Homer and RyR1.     

Modulation of transient receptor potential melastatin related 7 channel by presenilins

Presenilins (PS1 and PS2) are multifunctional proteins involved in a diverse array of molecular and cellular functions, including proteolysis, development, neurogenesis, synaptic plasticity, ion channel regulation and phospholipid metabolism. Mutations in presenilin genes are responsible for the majority of Familial Alzheimer disease (FAD). Consequently, FAD-associated mutations in genes encoding PS1 or PS2 lead to several key cellular phenotypes, including alterations in proteolysis of β-amyloid precursor protein (APP) and Ca(2+) entry. The mechanism underlying presenilin (PS)-mediated modulation of Ca(2+) entry remains to be determined. Our previous studies showed that the PS-dependent down-regulation of phosphatidylinositol-4,5-bisphosphate (PIP2) is attributable to the observed Ca(2+) deficits. In this study, we attempted to identify the ion channel that is subject to the PIP2 and PS-dependent modulation. We found that Ca(2+) or Zn(2+) entry via the transient receptor potential melastatin 7 (TRPM7) channel was attenuated by the presence of FAD-associated PS1 mutants, such as ΔE9 and L286V. TRPM7 has been implicated in Mg(2+) homeostasis and embryonic development. The intracellular delivery of PIP2 restored TRPM7-mediated Ca(2+) influx, indicating that the observed deficits in Ca(2+) entry are due to downregulation of PIP2. Conversely, PS1 and PS2 deficiency, previously shown to upregulate PIP2 levels, potentiated TRPM7-mediated Ca(2+) influx. PS-dependent changes in Ca(2+) influx could be neutralized by a TRPM7 channel blocker. Collectively, these results indicate that TRPM7 may underlie the Ca(2+) entry deficits observed in FAD-associated PS mutants and suggest that the normal function of PS involves regulation of TRPM7 through a PIP2-dependent mechanism.     

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides

The transient receptor potential (TRP) channel TRPM3 is a calcium-permeable cation channel activated by heat and by the neurosteroid pregnenolone sulfate (PregS). TRPM3 is highly expressed in sensory neurons, where it plays a key role in heat sensing and inflammatory hyperalgesia, and in pancreatic β cells, where its activation enhances glucose-induced insulin release. However, despite its functional importance, little is known about the cellular mechanisms that regulate TRPM3 activity. Here, we provide evidence for a dynamic regulation of TRPM3 by membrane phosphatidylinositol phosphates (PIPs). Phosphatidylinositol 4,5-bisphosphate (PI[4,5]P₂) and ATP applied to the intracellular side of excised membrane patches promote recovery of TRPM3 from desensitization. The stimulatory effect of cytosolic ATP on TRPM3 reflects activation of phosphatidylinositol kinases (PI-Ks), leading to resynthesis of PIPs in the plasma membrane. Various PIPs directly enhance TRPM3 activity in cell-free inside-out patches, with a potency order PI(3,4,5)P₃ > PI(3,5)P₂ > PI(4,5)P₂ ≈ PI(3,4)P₂ >> PI(4)P_. Conversely, TRPM3 activity is rapidly and reversibly inhibited by activation of phosphatases that remove the 5-phosphate from PIPs. Finally, we show that recombinant TRPM3, as well as the endogenous TRPM3 in insuloma cells, is rapidly and reversibly inhibited by activation of phospholipase C–coupled muscarinic acetylcholine receptors. Our results reveal basic cellular mechanisms whereby membrane receptors can regulate TRPM3 activity.     

C-terminal dimerization activates the nociceptive transduction channel transient receptor potential vanilloid 1

Covalent modification of the specific cysteine residue(s) by oxidative stress robustly potentiates transient receptor potential vanilloid 1 (TRPV1) and sensitizes nociception. Here we provide biochemical evidence of dimerization of TRPV1 subunits upon exposure to phenylarsine oxide and hydrogen peroxide (H(2)O(2)), two chemical surrogates of oxidative stress. A disulfide bond formed between apposing cysteines ligates two C termini, serving as the structural basis of channel sensitization by oxidative covalent C-terminal modification. Systematic cysteine scanning of the C terminus of a cysteineless TRPV1 channel revealed a critical region within which any cysteine introduced phenylarsine oxide activation to mutant TRPV1. Oxidative sensitization persisted even when this region is substituted with a random peptide linker containing a single cysteine. So did insertion of this region to TRPV3, a homolog lacking the corresponding region and resistant to oxidative challenge. These results suggest that the non-conserved linker in the TRPV1 C terminus senses environmental oxidative stress and adjusts channel activity during cumulative oxidative damage by lowering the activation threshold of gating elements shared by TRPV channels.     

Regulation of the transient receptor potential channel TRPA1 by its N-terminal ankyrin repeat domain

The transient receptor potential channel A1 (TRPA1) is unique among ion channels of higher vertebrates in that it harbors a large ankyrin repeat domain. The TRPA1 channel is expressed in the inner ear and in nociceptive neurons. It is involved in hearing as well as in the perception of pungent and irritant chemicals. The ankyrin repeat domain has special mechanical properties, which allows it to function as a soft spring that can be extended over a large range while maintaining structural integrity. A calcium-binding site has been experimentally identified within the ankyrin repeats. We built a model of the N-terminal 17 ankyrin repeat structure, including the calcium-binding EF-hand. In our simulations we find the calcium-bound state to be rigid as compared to the calcium-free state. While the end-to-end distance can change by almost 50% in the apo form, these fluctuations are strongly reduced by calcium binding. This increase in stiffness that constraints the end-to-end distance in the holo form is predicted to affect the force acting on the gate of the TRPA1 channel, thereby changing its open probability. Simulations of the transmembrane domain of TRPA1 show that residue N855, which has been associated with familial episodic pain syndrome, forms a strong link between the S4-S5 connecting helix and S1, thereby creating a direct force link between the N-terminus and the gate. The N855S mutation weakens this interaction, thereby reducing the communication between the N-terminus and the transmembrane part of TRPA1.     

Desensitization of canonical transient receptor potential channel 5 by protein kinase C

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channel in mammalian cells. TRPC5 is desensitized rapidly after activation by G protein-coupled receptor. Herein we report our investigation into the desensitization of mTRPC5 and localization of the molecular determinants of this desensitization using mutagenesis. TRPC5 was initially activated by muscarinic stimulation using 100 microM carbachol (CCh) and then decayed rapidly even in the presence of CCh (desensitization). Increased EGTA or omission of MgATP in the pipette solution slowed the rate of this desensitization. The protein kinase C (PKC) inhibitors, 1 microM chelerythrine, 100 nM GF109203X, or PKC peptide inhibitor (19-36), inhibited this desensitization of TRPC5 activated by 100 microM CCh. When TRPC5 current was activated by intracellular GTPgammaS, PKC inhibitors prevented TRPC5 desensitization and the mutation of TRPC5 T972 to alanine slowed the desensitization process dramatically. We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and suggest that threonine at residue 972 of mouse TRPC5 might be required for its phosphorylation by PKC.     

Heteromeric heat-sensitive transient receptor potential channels exhibit distinct temperature and chemical response

TRPV1 and TRPV3 are two heat-sensitive ion channels activated at distinct temperature ranges perceived by human as hot and warm, respectively. Compounds eliciting human sensations of heat or warmth can also potently activate these channels. In rodents, TRPV3 is expressed predominantly in skin keratinocytes, whereas in humans TRPV1 and TRPV3 are co-expressed in sensory neurons of dorsal root ganglia and trigeminal ganglion and are known to form heteromeric channels with distinct single channel conductances as well as sensitivities to TRPV1 activator capsaicin and inhibitor capsazepine. However, how heteromeric TRPV1/TRPV3 channels respond to heat and other stimuli remains unknown. In this study, we examined the behavior of heteromeric TRPV1/TRPV3 channels activated by heat, capsaicin, and voltage. Our results demonstrate that the heteromeric channels exhibit distinct temperature sensitivity, activation threshold, and heat-induced sensitization. Changes in gating properties apparently originate from interactions between TRPV1 and TRPV3 subunits. Our results suggest that heteromeric TRPV1/TRPV3 channels are unique heat sensors that may contribute to the fine-tuning of sensitivity to sensory inputs.     

Regulation of activity of transient receptor potential melastatin 8 (TRPM8) channel by its short isoforms

One important mechanism of the regulation of membrane ion channels involves their nonfunctional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of the transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on the TRPM8, which functions as a cold receptor in sensory neurons but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short splice variants of TRPM8, termed short TRPM8α and short TRPM8β. Our results show that both variants are in a closed configuration with the C-terminal tail of the full-length TRPM8 channel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and activity. Our findings therefore uncover a new mode of regulation of the TRPM8 channel by its splice variants.     

A cell-based impedance assay for monitoring transient receptor potential (TRP) ion channel activity

Transient receptor potential (TRP) channels are non-selective ion channels permeable to cations including Na(+), Ca(2+) and Mg(2+). They play a unique role as cellular sensors and are involved in many Ca(2+)-mediated cell functions. Failure in channel gating can contribute to complex pathophysiological mechanisms. Dysfunctions of TRP channels cause diseases but are also involved in the progress of diseases. We present a novel method to analyse chemical compounds as potential activators or inhibitors of TRP channels to provide pharmaceutical tools to regulate channel activity for disease control. Compared to common methods such as patch clamp or Ca(2+) imaging, the presented impedance assay is automatable, experimental less demanding and not restricted to Ca(2+) flow. We have chosen TRPA1 from the TRPA ('ankyrin') family as a model channel which was stimulated by allyl isothiocyanate (AITC). HEK293 cells stably transfected with human TRPA1 cDNA were grown on microelectrode arrays. Confluent cell layers of high density were analysed. Impedance spectra of cell-covered and non-covered electrodes yielded a cell-specific signal at frequencies between 70 and 120 kHz. Therefore, 100 kHz was chosen to monitor TRPA1 activity thereupon. An average impedance decrease to about 70% of its original value was observed after application of 10 μM AITC indicating an increased conductance of the cell layer mediated by TRPA1. Transfected cells pretreated with 10 μM of inhibitor ruthenium red to prevent channel conductance, as well as control cells lacking TRPA1, showed no impedance changes upon AITC stimuli demonstrating the specificity of the novel impedance assay.     

Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation.

The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.