Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis.

BACKGROUND: In physiological and pathological conditions activated protein kinace C (PKC) has been observed in the erythrocytes. Externalization of ankyrin followed by Arg-Gly-Asp (RGD)/integrin recognition also triggers erythrophagocytosis. In the present study, to test whether activated PKC is associated with ankyrin exposure in erythrophagocytosis. METHODS: Phorbol 12-myristate-13-acetate (PMA)-induced PKC activation and ankyrin phosphorylation were tested, and under different treatment conditions the subpopulation of erythrocytes with ankyrin exposure and the levels of intracellular calcium were analyzed by flow cytometry. RESULTS: Results showed that treatment of erythrocytes with PMA in a calcium-containing buffer led to ankyrin exposure. In the absence of extracellular calcium, no ankyrin exposure was observed. PKC inhibition with calphostin C, a blocker of the PMA binding site, completely prevented the calcium entry, protein phosphorylation and ankyrin exposure. PKC inhibition with chelerythrine chloride, an inhibitor of the active site, diminished the level of ankyrin-exposing cells and ankyrin phosphorylation; however it even led to a higher percentage of cells with increased levels of calcium than with PMA treatment alone. Although PKC was activated and ankyrin phosphorylation occurred, no ankyrin exposure was observed in the absence of extracellular calcium. CONCLUSION: Analyses of results suggested that PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes and the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.

AnkG hemizygous mice present cognitive impairment and elevated anxiety/depressive-like traits associated with decreased expression of GABA receptors and postsynaptic density protein.

Recent genome-wide association studies (GWAS) of patient populations and genetic linkage assessments have demonstrated that the ankyrin-G (AnkG) gene is involved in neuropsychiatric disorders, including bipolar disorder, schizophrenia, and Alzheimer's disease, but it remains unclear how the genetic variants of AnkG contribute to neuropsychiatric disorders. Here, we generated AnkG hemizygous mice using the gene trapping approach. Homozygous AnkG was embryonically lethal. Western blotting and real-time polymerase chain reaction (qPCR) assessments of wild type (WT) and AnkG +/- mutant mice demonstrated a 50% reduction of ANKG levels, at the gene and protein levels, in AnkG hemizygous mice. In behavioral tests, AnkG hemizygous mice exhibited elevated anxiety- and depression-like traits, as well as cognitive impairment. Moreover, the expression levels of cognitive-related proteins (including metabotropic glutamate receptor subtype-1, brain-derived neurotrophic factor, postsynaptic density-95, GABA-B receptor, and GABA-A receptor alpha-1) were significantly decreased (P < 0.05), suggesting a possible role for AnkG in cognition. It is possible that the loss of AnkG in the brain disrupts the excitation/inhibition balance of neurotransmitters, hindering the synaptic plasticity of neurons, and consequently leading to abnormal behavioral symptoms. Therefore, AnkG possibly contributes to neuroprotection and normal brain function, and may constitute a new target for treating neuropsychiatric diseases, especially cognitive dysfunction.

Selective filtering defect at the axon initial segment in Alzheimer's disease mouse models.

Axon pathology has been widely reported in Alzheimer's disease (AD) patients and AD mouse models. Herein we report that increased miR-342-5p down-regulates the expression of ankyrin G (AnkG), a protein known to play a critical role in establishing selective filtering machinery at the axon initial segment (AIS). Diminished AnkG expression leads to defective AIS filtering in cultured hippocampal neurons from AD mouse models, as monitored by selective exclusion of large macromolecules from the axons. Furthermore, AnkG-deficiency impairs AIS localization of Nav 1.6 channels and confines NR2B to the somatodendritic compartments. The expression of exogenous AnkG improved the cognitive performance of 12-mo-old APP/PS1 mice; thus, our data suggest that AnkG and impairment of AIS filtering may play important roles in AD pathology.

Dysfunction in the ßII spectrin-dependent cytoskeleton underlies human arrhythmia.

BACKGROUND: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. METHODS AND RESULTS: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that ßII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. ßII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/ßII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac ßII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, ßII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in ßII spectrin-deficient mice. CONCLUSIONS: Our findings identify ßII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

The ANK3 gene and facial affect processing: An ERP study.

ANK3 is one of the most promising candidate genes for bipolar disorder (BD). A polymorphism (rs10994336) within the ANK3 gene has been associated with BD in at least three genome-wide association studies of BD [McGuffin et al., 2003; Kieseppä, 2004; Edvardsen et al., 2008]. Because facial affect processing is disrupted in patients with BD, the current study aimed to explore whether the BD risk alleles are associated with the N170, an early event-related potential (ERP) component related to facial affect processing. We collected data from two independent samples of healthy individuals (Ns = 83 and 82, respectively) to test the association between rs10994336 and an early event-related potential (ERP) component (N170) that is sensitive to facial affect processing. Repeated-measures analysis of covariance in both samples consistently revealed significant main effects of rs10994336 genotype (Sample I: F (1, 72) = 7.24, P = 0.009; Sample II: F (1, 69) = 11.81, P = 0.001), but no significant interaction of genotype × electrodes (Ps > 0.05) or genotype × emotional conditions (Ps > 0.05). These results suggested that rs10994336 was linked to early ERP component reflecting facial structural encoding during facial affect processing. These results shed new light on the brain mechanism of this risk SNP and associated disorders such as BD. © 2016 Wiley Periodicals, Inc.

Rapid nutritional remodeling of the host cell upon attachment of Legionella pneumophila.

Upon entry of Legionella pneumophila into amoebas and macrophages, host-mediated farnesylation of the AnkB effector enables its anchoring to the Legionella-containing vacuole (LCV) membrane. On the LCV, AnkB triggers docking of K(48)-linked polyubiquitinated proteins that are degraded by the host proteasomes to elevate cellular levels of amino acids needed for intracellular proliferation. Interference with AnkB function triggers L. pneumophila to exhibit a starvation response and differentiate into the nonreplicative phase in response to the basal levels of cellular amino acids that are not sufficient to power intracellular proliferation of L. pneumophila. Therefore, we have determined whether the biological function of AnkB is temporally and spatially triggered upon bacterial attachment to the host cell to circumvent a counterproductive bacterial differentiation into the nonreplicative phase upon bacterial entry. Here, we show that upon attachment of L. pneumophila to human monocyte-derived macrophages (hMDMs), the host farnesylation and ubiquitination machineries are recruited by the Dot/Icm system to the plasma membrane exclusively beneath sites of bacterial attachment. Transcription and injection of ankB is triggered by attached extracellular bacteria followed by rapid farnesylation and anchoring of AnkB to the cytosolic side of the plasma membrane beneath bacterial attachment, where K(48)-linked polyubiquitinated proteins are assembled and degraded by the proteasomes, leading to a rapid rise in the cellular levels of amino acids. Our data represent a novel strategy by an intracellular pathogen that triggers rapid nutritional remodeling of the host cell upon attachment to the plasma membrane, and as a result, a gratuitous surplus of cellular amino acids is generated to support proliferation of the incoming pathogen.

An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier.

Neurons are highly polarized cells with functionally distinct axonal and somatodendritic compartments. Voltage-gated sodium channels Na(v)1.2 and Na(v)1.6 are highly enriched at axon initial segments (AISs) and nodes of Ranvier, where they are necessary for generation and propagation of action potentials. Previous studies using reporter proteins in unmyelinated cultured neurons suggest that an ankyrinG-binding motif within intracellular loop 2 (L2) of sodium channels is sufficient for targeting these channels to the AIS, but mechanisms of channel targeting to nodes remain poorly understood. Using a CD4-Na(v)1.2/L2 reporter protein in rat dorsal root ganglion neuron-Schwann cell myelinating cocultures, we show that the ankyrinG-binding motif is sufficient for protein targeting to nodes of Ranvier. However, reporter proteins cannot capture the complexity of full-length channels. To determine how native, full-length sodium channels are clustered in axons, and to show the feasibility of studying these channels in vivo, we constructed fluorescently tagged and functional mouse Na(v)1.6 channels for in vivo analysis using in utero brain electroporation. We show here that wild-type tagged-Na(v)1.6 channels are efficiently clustered at nodes and AISs in vivo. Furthermore, we show that mutation of a single invariant glutamic acid residue (E1100) within the ankyrinG-binding motif blocked Na(v)1.6 targeting in neurons both in vitro and in vivo. Additionally, we show that caseine kinase phosphorylation sites within this motif, while not essential for targeting, can modulate clustering at the AIS. Thus, the ankyrinG-binding motif is both necessary and sufficient for the clustering of sodium channels at nodes of Ranvier and the AIS.

Nodes of Ranvier and axon initial segments are ankyrin G-dependent domains that assemble by distinct mechanisms.

Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and betaIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.

AnkyrinG is required to maintain axo-dendritic polarity in vivo.

Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG(-/-) mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG(-/-) mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.

From global to local: a new understanding of cardiac electromechanical coupling.

BACKGROUND: The coupling between depolarisation and the heart's contraction is fundamental to the physiology and pathophysiology of the heart. This paper describes how the coupling depends on the interaction between proteins in "microdomains" in the heart muscle cells. METHOD: The paper is based on the authors'' own research and on a discretionary selection of articles found by means of a literature search in PubMed. RESULTS: Essential aspects of the physiology and pathophysiology of the heart must be understood through the interaction between proteins in delimited parts of the cells. The significance of the binding protein ankyrin-B and the Ca2+ channel IP3R (inositol 1,4,5-triphosphate receptor) is best understood in this context. Abnormal function of ankyrin-B and IP3R is involved in congenital diseases with increased risk of arrhythmia and in weakened contractility and arrhythmia in connection with heart failure. The pathophysiological mechanism involves a change in Ca2+ homeostasis locally in the heart muscle cells. INTERPRETATION: Normal cardiac electromechanical coupling depends on control of ionic homeostasis in intracellular microdomains. Insight into the interaction between proteins in these "local neighbourhoods" provides new explanations for the pathophysiology of heart disease and paves the way for further research on arrhythmia mechanisms in hereditary diseases such as ankyrin-B syndrome.

Activation characteristics of transient receptor potential ankyrin 1 and its role in nociception.

Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.

Transient receptor potential A1 channel contributes to activation of the muscle reflex.

This study was undertaken to elucidate the role played by transient receptor potential A1 channels (TRPA1) in activating the muscle reflex, a sympathoexcitatory drive originating in contracting muscle. First, we tested the hypothesis that stimulation of the TRPA1 located on muscle afferents reflexly increases sympathetic nerve activity. In decerebrate rats, allyl isothiocyanate, a TRPA1 agonist, was injected intra-arterially into the hindlimb muscle circulation. This led to a 33% increase in renal sympathetic nerve activity (RSNA). The effect of allyl isothiocyanate was a reflex because the response was prevented by sectioning the sciatic nerve. Second, we tested the hypothesis that blockade of TRPA1 reduces RSNA response to contraction. Thirty-second continuous static contraction of the hindlimb muscles, induced by electrical stimulation of the peripheral cut ends of L(4) and L(5) ventral roots, increased RSNA and blood pressure. The integrated RSNA during contraction was reduced by HC-030031, a TRPA1 antagonist, injected intra-arterially (163 ± 24 vs. 95 ± 21 arbitrary units, before vs. after HC-030031, P < 0.05). Third, we attempted to identify potential endogenous stimulants of TRPA1, responsible for activating the muscle reflex. Increases in RSNA in response to injection into the muscle circulation of arachidonic acid, bradykinin, and diprotonated phosphate, which are metabolic by-products of contraction and stimulants of muscle afferents during contraction, were reduced by HC-030031. These observations suggest that the TRPA1 located on muscle afferents is part of the muscle reflex and further support the notion that arachidonic acid metabolites, bradykinin, and diprotonated phosphate are candidates for endogenous agonists of TRPA1.

Novel roles for erythroid Ankyrin-1 revealed through an ENU-induced null mouse mutant.

Insights into the role of ankyrin-1 (ANK-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an N-ethyl-N-nitrosourea (ENU)-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero and most die perinatally, indicating that Ank-1 plays a nonredundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein and a marked reduction in beta-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture and severely altered hemorheologic properties. Heterozygous mutant mice, which have wild-type levels of ANK-1 and spectrin in their RBC membranes and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.

Ankyrins and cellular targeting of diverse membrane proteins to physiological sites.

Ankyrins are spectrin-binding proteins that associate via ANK repeats with a variety of ion channels/pumps, calcium release channels and cell adhesion molecules. Recent studies in mice indicate that ankyrins have a physiological role in restricting voltage-gated sodium channels and members of the L1 CAM family of cell adhesion molecules to excitable membranes in the central nervous system and in targeting calcium-release channels to the calcium homeostasis compartment of striated muscle.

Neurovascular aspects of skin neurogenic inflammation.

Neurogenic inflammation is involved in skin inflammation. It is hypothesized that it is involved in the pathogenesis of the common chronic cutaneous vascular disorder rosacea, but the exact mechanism of action is currently unknown. Transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) are widely expressed on primary sensory neuron endings and non-neuronal cells such as keratinocytes. Here we describe the potential for TRPV1 and TRPA1 receptors to be involved in the pathophysiology of rosacea due to their polymodal activation, including cold and hot temperature, pungent products from vegetable and spices, reactive oxygen species, and mechanical stimuli. We discuss the role of both receptors and the sensory neuropeptides that they release in inflammation and pain sensation and evidence suggesting that both TRPV1 and TRPA1 receptors may be promising therapeutic targets for the treatment of the inflammatory symptoms of rosacea.

Perisomatic GABAergic innervation in prefrontal cortex is regulated by ankyrin interaction with the L1 cell adhesion molecule.

The L1 adhesion molecule functions in axon growth and guidance, but a role in synaptic development of cortical inhibitory interneurons is largely unexplored. L1 mediates adhesion by engaging the actin cytoskeleton through binding the actin/spectrin adapter protein ankyrin. Loss of L1-ankyrin interaction impaired process elaboration/branching by GABAergic interneurons, including basket cells, and reduced the number of perisomatic synapses in the cingulate cortex as shown in L1 mutant mice (L1YH) with a mutation in the ankyrin-binding site, either alone or intercrossed with GAD67-enhanced green fluorescence protein reporter mice. Electron microscopy revealed that perisomatic inhibitory synapses but not excitatory synapses in the neuropil were specifically affected. In wild-type cingulate cortex, L1 colocalized with perisomatic synaptic markers, whereas L1 phosphorylation on Tyr(1229) decreased postnatally, correlating with increased ankyrin binding and synaptic development. These results suggest a novel role for L1 engagement with the actin cytoskeleton in development of inhibitory connectivity within the cingulate cortex.

Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease.

The identification of nearly a dozen ion channel genes involved in the genesis of human atrial and ventricular arrhythmias has been critical for the diagnosis and treatment of fatal cardiovascular diseases. In contrast, very little is known about the genetic and molecular mechanisms underlying human sinus node dysfunction (SND). Here, we report a genetic and molecular mechanism for human SND. We mapped two families with highly penetrant and severe SND to the human ANK2 (ankyrin-B/AnkB) locus. Mice heterozygous for AnkB phenocopy human SND displayed severe bradycardia and rate variability. AnkB is essential for normal membrane organization of sinoatrial node cell channels and transporters, and AnkB is required for physiological cardiac pacing. Finally, dysfunction in AnkB-based trafficking pathways causes abnormal sinoatrial node (SAN) electrical activity and SND. Together, our findings associate abnormal channel targeting with human SND and highlight the critical role of local membrane organization for sinoatrial node excitability.

Morphological properties of the axon initial segment-like process of AII amacrine cells in the rat retina.

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage-gated Na+ (Nav ) channel-dependent action potentials. The AII, a narrow-field, bistratified axon-less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Nav channels colocalized with the cytoskeletal protein ankyrin-G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS-like process could provide information relevant for its functional importance, including potential pre- and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin-G. Subsequently, this enabled us to reliably identify AII-type processes among ankyrin-G-labeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS-like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS-like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors.

Transient receptor potential ankyrin 1 channel: An evolutionarily tuned thermosensor.

The discovery of the role of the transient receptor potential ankyrin 1 (TRPA1) channel as a polymodal detector of cold and pain-producing stimuli almost two decades ago catalyzed the consequent identification of various vertebrate and invertebrate orthologues. In different species, the role of TRPA1 has been implicated in numerous physiological functions, indicating that the molecular structure of the channel exhibits evolutionary flexibility. Until very recently, information about the critical elements of the temperature-sensing molecular machinery of thermosensitive ion channels such as TRPA1 had lagged far behind information obtained from mutational and functional analysis. Current developments in single-particle cryo-electron microscopy are revealing precisely how the thermosensitive channels operate, how they might be targeted with drugs, and at which sites they can be critically regulated by membrane lipids. This means that it is now possible to resolve a huge number of very important pharmacological, biophysical and physiological questions in a way we have never had before. In this review, we aim at providing some of the recent knowledge on the molecular mechanisms underlying the temperature sensitivity of TRPA1. We also demonstrate how the search for differences in temperature and chemical sensitivity between human and mouse TRPA1 orthologues can be a useful approach to identifying important domains with a key role in channel activation.

Mechanisms and consequences of casein kinase II and ankyrin-3 regulation of the epithelial Na+ channel.

Activity of the Epithelial Na+ Channel (ENaC) in the distal nephron fine-tunes renal sodium excretion. Appropriate sodium excretion is a key factor in the regulation of blood pressure. Consequently, abnormalities in ENaC function can cause hypertension. Casein Kinase II (CKII) phosphorylates ENaC. The CKII phosphorylation site in ENaC resides within a canonical "anchor" ankyrin binding motif. CKII-dependent phosphorylation of ENaC is necessary and sufficient to increase channel activity and is thought to influence channel trafficking in a manner that increases activity. We test here the hypothesis that phosphorylation of ENaC by CKII within an anchor motif is necessary for ankyrin-3 (Ank-3) regulation of the channel, which is required for normal channel locale and function, and the proper regulation of renal sodium excretion. This was addressed using a fluorescence imaging strategy combining total internal reflection fluorescence (TIRF) microscopy with fluorescence recovery after photobleaching (FRAP) to quantify ENaC expression in the plasma membrane in living cells; and electrophysiology to quantify ENaC activity in split-open collecting ducts from principal cell-specific Ank-3 knockout mice. Sodium excretion studies also were performed in parallel in this knockout mouse. In addition, we substituted a key serine residue in the consensus CKII site in ß-ENaC with alanine to abrogate phosphorylation and disrupt the anchor motif. Findings show that disrupting CKII signaling decreases ENaC activity by decreasing expression in the plasma membrane. In the principal cell-specific Ank-3 KO mouse, ENaC activity and sodium excretion were significantly decreased and increased, respectively. These results are consistent with CKII phosphorylation of ENaC functioning as a "switch" that favors Ank-3 binding to increase channel activity.