Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.

Through their capacity to sense danger signals and to generate active interleukin-1β (IL-1β), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1β, little is known about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1β was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.

Effect of a temperature increase in the non-noxious range on proton-evoked ASIC and TRPV1 activity.

Acid-sensing ion channels (ASICs) are neuronal H(+)-gated cation channels, and the transient receptor potential vanilloid 1 channel (TRPV1) is a multimodal cation channel activated by low pH, noxious heat, capsaicin, and voltage. ASICs and TRPV1 are present in sensory neurons. It has been shown that raising the temperature increases TRPV1 and decreases ASIC H(+)-gated current amplitudes. To understand the underlying mechanisms, we have analyzed ASIC and TRPV1 function in a recombinant expression system and in dorsal root ganglion (DRG) neurons at room and physiological temperature. We show that temperature in the range studied does not affect the pH dependence of ASIC and TRPV1 activation. A temperature increase induces, however, a small alkaline shift of the pH dependence of steady-state inactivation of ASIC1a, ASIC1b, and ASIC2a. The decrease in ASIC peak current amplitudes at higher temperatures is likely in part due to the observed accelerated open channel inactivation kinetics and for some ASIC types to the changed pH dependence of steady-state inactivation. The increase in H(+)-activated TRPV1 current at the higher temperature is at least in part due to a hyperpolarizing shift in its voltage dependence. The contribution of TRPV1 relative to ASICs to H(+)-gated currents in DRG neurons increases with higher temperature and acidity. Still, ASICs remain the principal pH sensors of DRG neurons at 35°C in the pH range ≥6.

Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a heterogeneous retinal disorder characterized by visual impairment under low light conditions. This disorder is due to a signal transmission defect from rod photoreceptors to adjacent bipolar cells in the retina. Two forms can be distinguished clinically, complete CSNB (cCSNB) or incomplete CSNB; the two forms are distinguished on the basis of the affected signaling pathway. Mutations in NYX, GRM6, and TRPM1, expressed in the outer plexiform layer (OPL) lead to disruption of the ON-bipolar cell response and have been seen in patients with cCSNB. Whole-exome sequencing in cCSNB patients lacking mutations in the known genes led to the identification of a homozygous missense mutation (c.1807C>T [p.His603Tyr]) in one consanguineous autosomal-recessive cCSNB family and a homozygous frameshift mutation in GPR179 (c.278delC [p.Pro93Glnfs(∗)57]) in a simplex male cCSNB patient. Additional screening with Sanger sequencing of 40 patients identified three other cCSNB patients harboring additional allelic mutations in GPR179. Although, immunhistological studies revealed Gpr179 in the OPL in wild-type mouse retina, Gpr179 did not colocalize with specific ON-bipolar markers. Interestingly, Gpr179 was highly concentrated in horizontal cells and Müller cell endfeet. The involvement of these cells in cCSNB and the specific function of GPR179 remain to be elucidated.

MAP/ERK kinase kinase 1 (MEKK1) mediates transcriptional repression by interacting with polycystic kidney disease-1 (PKD1) promoter-bound p53 tumor suppressor protein.

Mitogen-activated protein kinase (MAPK) cascades regulate a wide variety of cellular processes that ultimately depend on changes in gene expression. We have found a novel mechanism whereby one of the key MAP3 kinases, Mekk1, regulates transcriptional activity through an interaction with p53. The tumor suppressor protein p53 down-regulates a number of genes, including the gene most frequently mutated in autosomal dominant polycystic kidney disease (PKD1). We have discovered that Mekk1 translocates to the nucleus and acts as a co-repressor with p53 to down-regulate PKD1 transcriptional activity. This repression does not require Mekk1 kinase activity, excluding the need for an Mekk1 phosphorylation cascade. However, this PKD1 repression can also be induced by the stress-pathway stimuli, including TNFα, suggesting that Mekk1 activation induces both JNK-dependent and JNK-independent pathways that target the PKD1 gene. An Mekk1-p53 interaction at the PKD1 promoter suggests a new mechanism by which abnormally elevated stress-pathway stimuli might directly down-regulate the PKD1 gene, possibly causing haploinsufficiency and cyst formation.

Conditional gene targeting of the ENaC subunit genes Scnn1b and Scnn1g.

Epithelial sodium channels (ENaC) are members of the degenerin/ENaC superfamily of non-voltage-gated, highly amiloride-sensitive cation channels that are composed of three subunits (alpha-, beta-, and gamma-ENaC). Since complete gene inactivation of the beta- and gamma-ENaC subunit genes (Scnn1b and Scnn1g) leads to early postnatal death, we generated conditional alleles and obtained mice harboring floxed and null alleles for both gene loci. Using quantitative RT-PCR analysis, we showed that the introduction of the loxP sites did not interfere with the mRNA transcript expression level of the Scnn1b and Scnn1g gene locus, respectively. Upon a regular and salt-deficient diet, both beta- and gamma-ENaC floxed mice showed no difference in their mRNA transcript expression levels, plasma electrolytes, and aldosterone concentrations as well as weight changes compared with control animals. These mice can now be utilized to dissect the role of ENaC function in classical and nonclassic target organs/tissues.

Store-operated Ca2+ Entry Mediated by Orai1 and TRPC1 Participates to Insulin Secretion in Rat β-Cells.

Store-operated Ca(2+) channels (SOCs) are voltage-independent Ca(2+) channels activated upon depletion of the endoplasmic reticulum Ca(2+) stores. Early studies suggest the contribution of such channels to Ca(2+) homeostasis in insulin-secreting pancreatic β-cells. However, their composition and contribution to glucose-stimulated insulin secretion (GSIS) remains unclear. In this study, endoplasmic reticulum Ca(2+) depletion triggered by acetylcholine (ACh) or thapsigargin stimulated the formation of a ternary complex composed of Orai1, TRPC1, and STIM1, the key proteins involved in the formation of SOCs. Ca(2+) imaging further revealed that Orai1 and TRPC1 are required to form functional SOCs and that these channels are activated by STIM1 in response to thapsigargin or ACh. Pharmacological SOCs inhibition or dominant negative blockade of Orai1 or TRPC1 using the specific pore mutants Orai1-E106D and TRPC1-F562A impaired GSIS in rat β-cells and fully blocked the potentiating effect of ACh on secretion. In contrast, pharmacological or dominant negative blockade of TRPC3 had no effect on extracellular Ca(2+) entry and GSIS. Finally, we observed that prolonged exposure to supraphysiological glucose concentration impaired SOCs function without altering the expression levels of STIM1, Orai1, and TRPC1. We conclude that Orai1 and TRPC1, which form SOCs regulated by STIM1, play a key role in the effect of ACh on GSIS, a process that may be impaired in type 2 diabetes.

Physiological analysis of central and peripheral insect circadian pacemaker neurons

Alle bisher untersuchten Lebewesen besitzen (circadiane) innere Uhren, die eine endogene Perioden-länge von ungefähr 24 Stunden generieren. Eine innere Uhr kann über Zeitgeber mit der Umwelt synchronisiert werden und ermöglicht dem Organismus, rhythmische Umweltveränderungen vorweg zu nehmen. Neben einem zentralen Schrittmacher, der Physiologie und Verhalten des Organismus steuert, gibt es in unterschiedlichen Organen auch periphere Uhren, die die zeitlichen Abläufe in der spezifischen Funktion dieser Organe steuern. In dieser Arbeit sollten zentrale und periphere Schrittmacherneurone von Insekten physiologisch untersucht und verglichen werden. Die Neurone der akzessorischen Medulla (AME) von Rhyparobia maderae dienten als Modellsystem für zentrale Schrittmacher, während olfaktorische Rezeptorneurone (ORNs) von Manduca sexta als Modellsystem für periphere Schrittmacher dienten. Die zentralen Schrittmacherneurone wurden in extrazellulären Ableitungen an der isolierten AME (Netzwerkebene) und in Patch-Clamp Experimenten an primären AME Zellkulturen (Einzelzellebene) untersucht. Auf Netzwerkebene zeigten sich zwei charakteristische Aktivitätsmuster: regelmäßige Aktivität und Wechsel zwischen hoher und niedriger Aktivität (Oszillationen). Es wurde gezeigt, dass Glutamat ein Neurotransmitter der weitverbreiteten inhibitorischen Synapsen der AME ist, und dass in geringem Maße auch exzitatorische Synapsen vorkommen. Das Neuropeptid pigment-dispersing factor (PDF), das von nur wenigen AME Neuronen exprimiert wird und ein wichtiger Kopplungsfaktor im circadianen System ist, führte zu Hemmungen, Aktivierungen oder Oszillationen. Die Effekte waren transient oder langanhaltend und wurden wahrscheinlich durch den sekundären Botenstoff cAMP vermittelt. Ein Zielmolekül von cAMP war vermutlich exchange protein directly activated by cAMP (EPAC). Auf Einzelzellebene wurde gezeigt, dass die meisten AME Neurone depolarisiert waren und deshalb nicht feuerten. Die Analyse von Strom-Spannungs-Kennlinien und pharmakologische Experimente ergaben, dass unterschiedliche Ionenkanäle vorhanden waren (Ca2+, Cl-, K+, Na+ Kanäle sowie nicht-spezifische Kationenkanäle). Starke, bei hohen Spannungen aktivierende Ca2+ Ströme (ICa) könnten eine wichtige Rolle bei Ca2+-abhängiger Neurotransmitter-Ausschüttung, Oszillationen, und Aktionspotentialen spielen. PDF hemmte unterschiedliche Ströme (ICa, IK und INa) und aktivierte nicht-spezifische Kationenströme (Ih). Es wurde angenommen, dass simultane PDF-abhängige Hyper- und Depolarisationen rhythmische Membranpotential-Oszillationen verursachen. Dieser Mechanismus könnte eine Rolle bei PDF-abhängigen Synchronisationen spielen. Die Analyse peripherer Schrittmacherneurone konzentrierte sich auf die Charakterisierung des olfaktorischen Corezeptors von M. sexta (MsexORCO). In anderen Insekten ist ORCO für die Membran-Insertion von olfaktorischen Rezeptoren (ORs) erforderlich. ORCO bildet Komplexe mit den ORs, die in heterologen Expressionssystemen als Ionenkanäle fungieren und Duft-Antworten vermitteln. Es wurde die Hypothese aufgestellt, dass MsexORCO in pheromonsensitiven ORNs in vivo nicht als Teil eines ionotropen Rezeptors sondern als Schrittmacherkanal fungiert, der unterschwellige Membranpotential-Oszillationen generiert. MsexORCO wurde mit vermeintlichen Pheromonrezeptoren in human embryonic kidney (HEK 293) Zellen coexprimiert. Immuncytochemie und Ca2+ Imaging Experimente zeigten sehr schwache Expressionsraten. Trotzdem war es möglich zu zeigen, dass MsexORCO wahrscheinlich ein spontan-aktiver, Ca2+-permeabler Ionenkanal ist, der durch den ORCO-Agonisten VUAA1 und cyclische Nucleotide aktiviert wird. Außerdem wiesen die Experimente darauf hin, dass MsexOR-1 offensichtlich der Bombykal-Rezeptor ist. Eine weitere Charakterisierung von MsexORCO in primären M. sexta ORN Zellkulturen konnte nicht vollendet werden, weil die ORNs nicht signifikant auf ORCO-Agonisten oder -Antagonisten reagierten.

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+](cyt)) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+] cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+] cyt, and may function as peroxidases in vitro.

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca2+-dependent and Ca2+-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

Actions of octocoral and tobacco cembranoids on nicotinic receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies. (C) 2009 Elsevier Ltd. All rights reserved.

Availability of a rich source of sodium during the perinatal period programs the fluid balance restoration pattern in adult offspring

Osmoregulatory mechanisms can be vulnerable to electrolyte and/or endocrine environmental changes during the perinatal period, differentially programming the developing offspring and affecting them even in adulthood. The aim of this study was to evaluate whether availability of hypertonic sodium solution during the perinatal period may induce a differential programming in adult offspring osmoregulatory mechanisms. With this aim, we studied water and sodium intake after Furosemide-sodium depletion in adult offspring exposed to hypertonic sodium solution from 1 week before mating until postnatal day 28 of the offspring, used as a perinatal manipulation model [PM-Na group]. In these animals, we also identified the cell population groups in brain nuclei activated by Furosemide-sodium depletion treatment, analyzing the spatial patterns of Fos and Fos-vasopressin immunoreactivity. In sodium depleted rats, sodium and water intake were significantly lower in the PM-Na group vs. animals without access to hypertonic sodium solution [PM-Ctrol group]. Interestingly, when comparing the volumes consumed of both solutions in each PM group, our data show the expected significant differences between both solutions ingested in the PM-Ctrol group, which makes an isotonic cocktail: however, in the PM-Na group there were no significant differences in the volumes of both solutions consumed after Furosemide-sodium depletion, and therefore the sodium concentration of total fluid ingested by this group was significantly higher than that in the PM-Ctrol group. With regard to brain Fos immunoreactivity, we observed that Furosemide-sodium depletion in the PM-Na group induced a higher number of activated cells in the subfornical organ, ventral subdivision of the paraventricular nucleus and vasopressinergic neurons of the supraoptic nucleus than in the PM-Ctrol animals. Moreover, along the brainstem, we found a decreased number of sodium depletion-activated cells within the nucleus of the solitary tract of the PM-Na group. Our data indicate that early sodium availability induces a long-term effect on fluid drinking and on the cell activity of brain nuclei involved in the control of hydromineral balance. These results also suggest that availability of a rich source of sodium during the perinatal period may provoke a larger anticipatory response in the offspring, activating the vasopressinergic system and reducing thirst after water and sodium depletion, as a result of central osmosensitive mechanism alterations. (C) 2011 Elsevier Inc. All rights reserved.

Investigation of the inhibitory effects of the benzodiazepine derivative, 5-BDBD on P2X4 purinergic receptors by two complementary methods

BACKGROUND/AIMS ATP-gated P2X4 purinergic receptors (P2X4Rs) are cation channels with important roles in diverse cell types. To date, lack of specific inhibitors has hampered investigations on P2X4Rs. Recently, the benzodiazepine derivative, 5-BDBD has been proposed to selectively inhibit P2X4Rs. However, limited evidences are currently available on its inhibitory properties. Thus, we aimed to characterize the inhibitory effects of 5-BDBD on recombinant human P2X4Rs. METHODS We investigated ATP-induced intracellular Ca(2+) signals and whole cell ion currents in HEK 293 cells that were either transiently or stably transfected with hP2X4Rs. RESULTS Our data show that ATP (< 1 μM) stimulates P2X4R-mediated Ca(2+) influx while endogenously expressed P2Y receptors are not activated to any significant extent. Both 5-BDBD and TNP-ATP inhibit ATP-induced Ca(2+) signals and inward ion currents in a concentration-dependent manner. Application of two different concentrations of 5-BDBD causes a rightward shift in ATP dose-response curve. Since the magnitude of maximal stimulation does not change, these data suggest that 5-BDBD may competitively inhibit the P2X4Rs. CONCLUSIONS Our results demonstrate that application of submicromolar ATP concentrations allows reliable assessment of recombinant P2XR functions in HEK 293 cells. Furthermore, 5-BDBD and TNP-ATP have similar inhibitory potencies on the P2X4Rs although their mechanisms of actions are different.

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Homology Cloning, Heterologous Expression and Characterization of a New Channelrhodopsin

Channelrhodopsins are phototaxis receptors in the plasma membranes of motile unicellular algae. They function as light-gated cation channels and this channel activity has been exploited to trigger action potentials in neurons with light to control neural circuits (“optogenetics"). Four channelrhodopsins were identified in two algal species, Chlamydomonas reinhardtii and Volvox carteri, with known genome sequences; each species contains 2 channelrhodopsins, one absorbing at longer wavelengths and one at shorter wavelengths, named CrChR1 and CrChR2, respectively. Our goals are to expand knowledge of channelrhodopsin mechanisms and also to identify new channelrhodopsins from various algal species with improved properties for optogenetic use. For these aims we are targeting algae from extreme environments to establish the natural diversity of their properties. We cloned a new channelrhodopsin from the psychrophilic (cold-loving) alga, Chlamydomonas augustae, with degenerate primers based on the 4 known homologs. The new protein is 48% and 52% identical to CrChR1 and CrChR2, respectively. We expressed the channelrhodopsin in HEK293 cells and measured light-induced currents to assess their kinetics and action spectrum. Based on the primary structure, kinetics of light-induced photocurrents in HEK293 cells, and action spectrum maximum of 520 nm near that of the two previously found CrChR1, we named the new channelrhodopsin CaChR1. The properties of robust channel activity at physiological pH, fast on-and-off kinetics, and greatly red-shifted action spectrum maximum from that of CrChR2, make CaChR1 advantageous as an optogenetic tool. To know this new channelrhodopsin better, we expressed His-tagged CaChR1 in Pichia pastoris and the yield is about 6 mg/L. The purified His-tagged CaChR1 exhibited an absorption spectrum identical to the action spectrum of CaChR1-generated photocurrents. The future work will be measurement of the photocycles of CaChR1 by flash photolysis, crystallization of CaChR1 for the structure and mutagenesis of CaChR1 to find the critical amino acids accounting for red-shifted spectra, slow inactivation and rapid on-and-off kinetics. Seven new channelrhodopsins including CaChR1 from different algal species have been cloned in our lab at this time, bringing the total known to 13. The work of cloning of these new channelrhodopsins along with the expression of CaChR1 was published in Photochemistry and Photobiology in January 2012