Functional Significance of K+ Channel β-Subunit KCNE3 in Auditory Neurons

The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall functional relevance of the single transmembrane domain peptide on the pore-forming K⁺ channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3^−/− mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K⁺ currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.     

KCNE2 and the K+ channel: The tail wagging the dog

KCNE2, originally designated MinK-related peptide 1 (MiRP1), belongs to a five-strong family of potassium channel ancillary (β) subunits that, despite the diminutive size of the family and its members, has loomed large in the field of ion channel physiology. KCNE2 dictates K⁺ channel gating, conductance, α subunit composition, trafficking and pharmacology, and also modifies functional properties of monovalent cation-nonselective HCN channels. The Kcne2^−/− mouse exhibits cardiac arrhythmia and hypertrophy, achlorhydria, gastric neoplasia, hypothyroidism, alopecia, stunted growth and choroid plexus epithelial dysfunction, illustrating the breadth and depth of the influence of KCNE2, mutations which are also associated with human cardiac arrhythmias. Here, the modus operandi and physiological roles of this potent regulator of membrane excitability and ion secretion are reviewed with particular emphasis on the ability of KCNE2 to shape the electrophysiological landscape of both excitable and non-excitable cells.     

N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes

Background and Objective

The slow delayed rectifier current (I_Ks) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I_Ks and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I_Ks modulation is poorly understood and was subject of this study.

Methods

We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy.

Results

All KCNE1 constructs physically interacted with Kv7.1. I_Ks resulting from co-expression of Kv7.1 with non-atrial fibrillation ‘38S’ was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions (‘38G-3xA’) were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct (‘Δ1-38’). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct.

Conclusions

The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I_Ks. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.     

HCN1 and HCN2 in Rat DRG Neurons: Levels in Nociceptors and Non-Nociceptors,NT3-Dependence and Influence of CFA-Induced Skin Inflammation on HCN2 and NT3 Expression

I_h, which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie I_h were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aβ-nociceptors and Aα/β-LTMs. High HCN1 and HCN2 levels in Aα/β-neurons may, via I_h, influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high I_h in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable I_h. In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported I_h magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie I_h in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.     

Hyperpolarization-Activated Current (Ih) Is Reduced in Hippocampal Neurons from Gabra5?/? Mice

Changes in the expression of γ-aminobutyric acid type A (GABA_A) receptors can either drive or mediate homeostatic alterations in neuronal excitability. A homeostatic relationship between α5 subunit-containing GABA_A (α5GABA_A) receptors that generate a tonic inhibitory conductance, and HCN channels that generate a hyperpolarization-activated cation current (I_h) was recently described for cortical neurons, where a reduction in I_h was accompanied by a reciprocal increase in the expression of α5GABA_A receptors resulting in the preservation of dendritosomatic synaptic function. Here, we report that in mice that lack the α5 subunit gene (Gabra5−/−), cultured embryonic hippocampal pyramidal neurons and ex vivo CA1 hippocampal neurons unexpectedly exhibited a decrease in I_h current density (by 40% and 28%, respectively), compared with neurons from wild-type (WT) mice. The resting membrane potential and membrane hyperpolarization induced by blockade of I_h with ZD-7288 were similar in cultured WT and Gabra5−/− neurons. In contrast, membrane hyperpolarization measured after a train of action potentials was lower in Gabra5−/− neurons than in WT neurons. Also, membrane impedance measured in response to low frequency stimulation was greater in cultured Gabra5−/− neurons. Finally, the expression of HCN1 protein that generates I_h was reduced by 41% in the hippocampus of Gabra5−/− mice. These data indicate that loss of a tonic GABAergic inhibitory conductance was followed by a compensatory reduction in I_h. The results further suggest that the maintenance of resting membrane potential is preferentially maintained in mature and immature hippocampal neurons through the homeostatic co-regulation of structurally and biophysically distinct cation and anion channels.     

HCN1 subunits contribute to the kinetics of Ih in neonatal cortical plate neurons

The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel constituting the molecular substrate of hyperpolarization‐activated current (I_h). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of I_h in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole‐cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1^?/? and control HCN1^+/+ mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN^+/+ mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1^?/? mice other isoforms (particularly HCN4) might be compensatory up‐regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of I_h expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I_h expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I_h properties in individual cortical plate neurons. These properties are significantly changed in HCN1^?/?, either due to the lack of HCN1 itself or due to compensatory mechanisms. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 785–797, 2013     

Targeted Deletion of Kcne2 Causes Gastritis Cystica Profunda and Gastric Neoplasia

Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 α subunit and the KCNE2 β subunit provide a K⁺ efflux current to facilitate gastric acid secretion by the apical H⁺K⁺ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2 ^−/− mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2 ^−/−mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia.     

Cellular mechanisms underlying the laxative effect of flavonol naringenin on rat constipation model

Background & Aims

Symptoms of constipation are extremely common, especially in the elderly. The present study aim to identify an efficacious treatment strategy for constipation by evaluating the secretion-promoting and laxative effect of a herbal compound, naringenin, on intestinal epithelial anion secretion and a rat constipation model, respectively.

Methods/Principal Findings

In isolated rat colonic crypts, mucosal addition of naringenin (100 µM) elicited a concentration-dependent and sustained increase in the short-circuit current (I_SC), which could be inhibited in Cl⁻ free solution or by bumetanide and DPC (diphenylamine-2-carboxylic acid), but not by DIDS (4, 4′- diisothiocyanatostilbene-2, 2′-disulfonic acid). Naringenin could increase intracellular cAMP content and PKA activity, consisted with that MDL-12330A (N-(Cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride) pretreatment reduced the naringenin-induced I_SC. In addition, significant inhibition of the naringenin-induced I_SC by quinidine indicated that basolateral K⁺ channels were involved in maintaining this cAMP-dependent Cl⁻ secretion. Naringenin-evoked whole cell current which exhibited a linear I–V relationship and time-and voltage- independent characteristics was inhibited by DPC, indicating that the cAMP activated Cl⁻ conductance most likely CFTR (cystic fibrosis transmembrane conductance regulator) was involved. In rat constipation model, administration of naringenin restored the level of fecal output, water content and mucus secretion compared to loperamide-administrated group.

Conclusions

Taken together, our data suggest that naringenin could stimulate Cl⁻ secretion in colonic epithelium via a signaling pathway involving cAMP and PKA, hence provide an osmotic force for subsequent colonic fluid secretion by which the laxative effect observed in the rat constipation model. Naringenin appears to be a novel alternative treatment strategy for constipation.     

Calcium-dependent Binding of HCN1 Channel Protein to Hair Cell Stereociliary Tip Link Protein Protocadherin 15 CD3

The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca²⁺. In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K_D = 2.39 × 10^-7 m. Ca²⁺ at 26.5-68.0 μm promoted binding, with K_D = 5.26 × 10^-8 m (at 61 μm Ca²⁺). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank™ accession number {"type":"entrez-protein","attrs":{"text":"XP_238200","term_id":"293356762","term_text":"XP_238200"}}XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca²⁺-dependent, although the binding was skewed toward a lower effective maximum [Ca²⁺] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 μm Ca²⁺. Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells.HCN1² is the primary full-length HCN isoform underlying I_h (hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation channel current) in a model hair cell preparation from the trout sacccule (1). cAMP-gated I_h, possibly in addition to the mechanosensory-transduction current, sets the membrane potential for a subpopulation of saccular hair cells (2, 3). The membrane potential in the saccular hair cell subpopulation is sufficiently depolarized to activate voltage-gated calcium channels, permitting influx of calcium and secretion of hair cell transmitter (2). Given that saccular hair cells expressing I_K1 in addition to I_h are more hyperpolarized, not supporting activation of the voltage-gated calcium channels, we predicted that spontaneous release of transmitter from the subpopulation of hair cells would constitute hair cell-generated spontaneous activity for the saccule (1). However, little has been previously reported on the morphological localization of the HCN1 isoform in hair cells or possible links to structural proteins that mechanistically would localize HCN1 in hair cells (for preliminary report, see Ref. 4). In general, little is known about protein-protein interactions for the HCN isoforms that would modulate I_h and/or the associated instantaneous current (5).Protocadherin 15 is a proposed tip link protein involved in connecting shorter stereocilia to adjacent taller stereocilia in the stereociliary array of inner ear hair cells, facilitating the opening of the mechanosensory transduction channel in response to auditory and vestibular stimuli. The active tip link protein in Danio rerio is protocadherin 15a (6), characterized by splice variants in its carboxyl terminus. In the mammal, protocadherin 15 CD3 is hypothesized to be a tip link protein at insertion sites in the tips of the shorter stereocilia of the stereociliary array (7, 8).     

Processing of retinal signals in normal and HCN deficient mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1^−/− mice or during a pharmacological blockade of I_h show that, contrary to previous reports, I_kx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I_h and I_kx to the rods'' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.     

Disruption of the K+ Channel β-Subunit KCNE3 Reveals an Important Role in Intestinal and Tracheal Cl? Transport

The KCNE3 β-subunit constitutively opens outwardly rectifying KCNQ1 (Kv7.1) K⁺ channels by abolishing their voltage-dependent gating. The resulting KCNQ1/KCNE3 heteromers display enhanced sensitivity to K⁺ channel inhibitors like chromanol 293B. KCNE3 was also suggested to modify biophysical properties of several other K⁺ channels, and a mutation in KCNE3 was proposed to underlie forms of human periodic paralysis. To investigate physiological roles of KCNE3, we now disrupted its gene in mice. kcne3^−/− mice were viable and fertile and displayed neither periodic paralysis nor other obvious skeletal muscle abnormalities. KCNQ1/KCNE3 heteromers are present in basolateral membranes of intestinal and tracheal epithelial cells where they might facilitate transepithelial Cl⁻ secretion through basolateral recycling of K⁺ ions and by increasing the electrochemical driving force for apical Cl⁻ exit. Indeed, cAMP-stimulated electrogenic Cl⁻ secretion across tracheal and intestinal epithelia was drastically reduced in kcne3^−/− mice. Because the abundance and subcellular localization of KCNQ1 was unchanged in kcne3^−/− mice, the modification of biophysical properties of KCNQ1 by KCNE3 is essential for its role in intestinal and tracheal transport. Further, these results suggest KCNE3 as a potential modifier gene in cystic fibrosis.     

A host defense mechanism involving CFTR-mediated bicarbonate secretion in bacterial prostatitis

Background

Prostatitis is associated with a characteristic increase in prostatic fluid pH; however, the underlying mechanism and its physiological significance have not been elucidated.

Methodology/Principal Findings

In this study a primary culture of rat prostatic epithelial cells and a rat prostatitis model were used. Here we reported the involvement of CFTR, a cAMP-activated anion channel conducting both Cl⁻ and HCO₃ ⁻, in mediating prostate HCO₃ ⁻ secretion and its possible role in bacterial killing. Upon Escherichia coli (E coli)-LPS challenge, the expression of CFTR and carbonic anhydrase II (CA II), along with several pro-inflammatory cytokines was up-regulated in the primary culture of rat prostate epithelial cells. Inhibiting CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO₃ ⁻ content (>50 mM), rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO₃ ⁻ on bacterial killing was confirmed by its ability to increase cAMP production and suppress bacterial initiation factors in E coli. The relevance of the CFTR-mediated HCO₃ ⁻ secretion in humans was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues.

Conclusions/Significance

The CFTR and its mediated HCO₃ ⁻ secretion may be up-regulated in prostatitis as a host defense mechanism.     

Characterization of na(+) and ca(2+) channels in zebrafish dorsal root ganglion neurons

Background

Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.

Methodology/Principal Findings

We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na⁺ currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I_Na were preferentially expressed in relatively large neurons, while slowly-inactivating I_Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I_Na components. Voltage-gated Ca²⁺ currents (I_Ca) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca_V2.2 (N-type) Ca²⁺ channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I_Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.

Conclusions/Significance

Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.     

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels

Background and Aims

Experimental evidence in the literature suggests that O₂^•− produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O₂^•−.

Methods

Stress treatments were imposed using 150 mm NaCl or 300 mm sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O₂^•− was determined using nitro blue tetrazolium, and H₂O₂ was determined using 2′, 7′-dichlorofluorescin.

Key Results

In non-stressed plants, the distribution of accelerating growth and highest O₂^•− levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O₂^•− levels increased in sorbitol-treated roots and decreased in NaCl-treated roots.

Conclusions

The lack of association between apoplastic O₂^•− levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O₂^•− may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.Key words: Root tip growth, Zea mays, salt stress, reactive oxygen species, ROS     

Ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation

Background

Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models.

Methods

We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1^−/− T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25^hi, CD62L^lo). Additionally, Ceacam1^−/− CD8 T cells had greater expression of the gut-trafficking integrin α₄β₇, though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1^−/− recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1^−/− mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1⁺ lymphoma model was improved in animals receiving Ceacam1^−/− vs. control T cells.

Conclusions

We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.     

Neuronal deletion of caspase 8 protects against brain injury in mouse models of controlled cortical impact and kainic acid-induced excitotoxicity

Background

Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8 ^−/−), in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system.

Methodology/Principal Findings

Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml) or TRAIL (250 ng/mL) plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI) model of traumatic brain injury (TBI) and seizure-induced brain injury caused by kainic acid (KA). Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8 ^−/− mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.

Conclusions

Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma.     

Osteopenia due to enhanced cathepsin K release by BK channel ablation in osteoclasts

Background

The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified.

Methodology/Principal Findings

We found, that in juvenile bone the large conductance, voltage and Ca²⁺-activated (BK) K⁺ channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K⁺ outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK^−/−) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK^−/− vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca²⁺ and triiodthyronine as well as osteoclastogenesis were not altered in BK^−/− females.

Conclusion/Significance

Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK^−/− mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity.     

Filamin A Promotes Dynamin-dependent Internalization of Hyperpolarization-activated Cyclic Nucleotide-gated Type 1 (HCN1) Channels and Restricts Ih in Hippocampal Neurons

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct I_h, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced I_h density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous I_h, and enhances the rebound-response (“voltage-sag”) of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.