Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

[Ion transporters in the lungs. Use as therapeutic targets]. / Transportadores de iones en pulmón. Uso como dianas terapéuticas.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

Transportadores de iones en pulmón: Uso como dianas terapéuticas / Ion transporters in the lungs. Use as therapeutic targets

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Intercalated Cells of the Kidney Collecting Duct in Kidney Physiology.

The epithelium of the kidney collecting duct (CD) is composed mainly of two different types of cells with distinct and complementary functions. CD principal cells traditionally have been considered to have a major role in Na+ and water regulation, while intercalated cells (ICs) were thought to largely modulate acid-base homeostasis. In recent years, our understanding of IC function has improved significantly owing to new research findings. Thus, we now have a new model for CD transport that integrates mechanisms of salt and water reabsorption, K+ homeostasis, and acid-base status between principal cells and ICs. There are three main types of ICs (type A, type B, and non-A, non-B), which first appear in the late distal convoluted tubule or in the connecting segment in a species-dependent manner. ICs can be detected in CD from cortex to the initial part of the inner medulla, although some transport proteins that are key components of ICs also are present in medullary CD, cells considered inner medullary. Of the three types of ICs, each has a distinct morphology and expresses different complements of membrane transport proteins that translate into very different functions in homeostasis and contributions to CD luminal pro-urine composition. This review includes recent discoveries in IC intracellular and paracrine signaling that contributes to acid-base regulation as well as Na+, Cl-, K+, and Ca2+ homeostasis. Thus, these new findings highlight the potential role of ICs as targets for potential hypertension treatments.

Pharmacological characterization of histamine-gated chloride channels from the housefly Musca domestica.

The biogenic amine histamine (HA) is not only the neurotransmitter of photoreceptors but also has important roles in mechanosensory reception, temperature preference, sleep and olfactory processing in insects. Two cDNAs (MdhclA and MdhclB) that encode HA-gated chloride channel subunits (MdHCLA and MdHCLB) were cloned from the housefly Musca domestica. The cRNAs were injected into Xenopus laevis oocytes to examine the functions and pharmacological characteristics of MdHCLA and MdHCLB channels using a two-electrode voltage clamp method. HA was used to activate MdHCLA and MdHCLB channels to evoke inward currents with EC50s of 33.1µM and 6.28µM, respectively. 2-(3-Trifluoromethylphenyl)histamine, an HA H1 receptor agonist, was a partial agonist of MdHCLB receptors with an EC50 of 49.4µM. MdHCLB channels were also activated by γ-aminobutyric acid (GABA) and monoamines, such as octopamine, serotonin (5-HT) and dopamine (DA); 5-HT and DA also acted as competitive antagonists. GABA acted as a full agonist of MdHCLB receptors with an EC50 of 1.11mM. d-Tubocurarine, cimetidine and picrotoxinin were poor inhibitors of HA- and GABA-evoked currents in MdHCLB channels. Our data show that HCLB channels are more sensitive to agonists when compared with HCLA channels. HCLB channels are also affected by antagonists but insusceptible to known insecticides that target GABA- and glutamate-gated chloride channels.

Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels.

BACKGROUND AND PURPOSE: Docking studies predict that the insecticides, lindane and fipronil, block GABA(A) receptors by binding to 6' pore-lining residues. However, this has never been tested at any Cys-loop receptor. The neurotoxic effects of these insecticides are also thought to be mediated by GABA(A) receptors, although a recent morphological study suggested glycine receptors mediated fipronil toxicity in zebrafish. Here we investigated whether human α1, α1ß, α2 and α3 glycine receptors were sufficiently sensitive to block by either compound as to represent possible neurotoxicity targets. We also investigated the mechanisms by which lindane and fipronil inhibit α1 glycine receptors. EXPERIMENTAL APPROACH: Glycine receptors were recombinantly expressed in HEK293 cells and insecticide effects were studied using patch-clamp electrophysiology. KEY RESULTS: Both compounds completely inhibited all tested glycine receptor subtypes with IC(50) values ranging from 0.2-2 µM, similar to their potencies at vertebrate GABA(A) receptors. Consistent with molecular docking predictions, both lindane and fipronil interacted with 6' threonine residues via hydrophobic interactions and hydrogen bonds. In contrast with predictions, we found no evidence for lindane interacting at the 2' level. We present evidence for fipronil binding in a non-blocking mode in the anaesthetic binding pocket, and for lindane as an excellent pharmacological tool for identifying the presence of ß subunits in αß heteromeric glycine receptors. CONCLUSIONS AND IMPLICATIONS: This study implicates glycine receptors as novel vertebrate toxicity targets for fipronil and lindane. Furthermore, lindane interacted with pore-lining 6' threonine residues, whereas fipronil may have both pore and non-pore binding sites.

Direct and indirect control of orexin/hypocretin neurons by glycine receptors.

Hypothalamic hypocretin/orexin (hcrt/orx) neurons promote arousal and reward seeking, while reduction in their activity has been linked to narcolepsy, obesity and depression. However, the mechanisms influencing the activity of hcrt/orx networks in situ are not fully understood. Here we show that glycine, a neurotransmitter best known for its actions in the brainstem and spinal cord, elicits dose dependent postsynaptic Cl⁻ currents in hcrt/orx cells in acute mouse brain slices. The effect was blocked by the glycine receptor (GLyR) antagonist strychnine and mimicked by the GlyR agonist alanine. Postsynaptic GlyRs on hcrt/orx cells remained functional during both early postnatal and adult periods, and gramicidin-perforated patch-clamp recordings revealed that they progressively switch from excitatory to inhibitory during the first two postnatal weeks. The pharmacological profile of the glycine response suggested that developed hcrt/orx neurons contain α/ß-heteromeric GlyRs that lack α2-subunits, whereas α2-subunits, whereas α2-subunits are present in early postnatal hcrt/orx neurons. All postsynaptic currents (PSCs) in developed hcrt/orx cells were blocked by inhibitors of GABA and glutamate receptors, with no evidence of GlyR-mediated PSCs. However, the frequency but not amplitude of miniature PSCs was reduced by strychnine and increased by glycine in ~50% of hcrt/orx neurons. Together, these results provide the first evidence for functional GlyRs in identified hcrt/orx circuits and suggest that the activity of developed hcrt/orx cells is regulated by two GlyR pools: inhibitory extrasynaptic GlyRs located on all hcrt/orx cells and excitatory GlyRs located on presynaptic terminals contacting some hcrt/orx cells.

Chloride intracellular channel 5 modulates adipocyte accumulation in skeletal muscle by inhibiting preadipocyte differentiation.

Intramuscular fat, the total lipid deposited within skeletal muscle, has been regarded as a potential factor responsible for meat quality in animal production and insulin resistance in humans. The objective of present study was to identify candidate genes which control intramuscular fat accumulation through using animal models. PIC pigs (lean-type) and Rongchang pigs (obese-type) were used. By scanning the mRNA samples of longissimus dorsi muscle with Affymetrix Gene-Chip microarray technology, sus scrofa chloride intracellular channel 5 (CLIC5) was isolated, and its mRNA abundance and protein expression level were reversely related with the intramuscular fat content of pigs. Furthermore, over-expression of CLIC5 dramatically increased the proliferation of 3T3-L1 preadipocytes, while inhibited adipocytic differentiation accompanied by the down-regulation of c/EBPalpha, LPL, and PPARgamma protein. Our results suggest that CLIC5 might be a crucial regulator of adipose accumulation in skeletal muscle of pigs.

CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

CFTR mediates apoptotic volume decrease and cell death by controlling glutathione efflux and ROS production in cultured mice proximal tubules.

We have previously shown that despite the presence of mRNA encoding CFTR, renal proximal cells do not exhibit cAMP-sensitive Cl(-) conductance (Rubera I, Tauc M, Bidet M, Poujeol C, Cuiller B, Watrin A, Touret N, Poujeol P. Am J Physiol Renal Physiol 275: F651-F663, 1998). Nevertheless, in these cells, CFTR plays a crucial role in the control of the volume-sensitive outwardly rectifying (VSOR) activated Cl(-) currents during hypotonic shock. The aim of this study was to determine the role of CFTR in the regulation of apoptosis volume decrease (AVD) and the apoptosis phenomenon. For this purpose, renal cells were immortalized from primary cultures of proximal convoluted tubules from cftr(+/+) and cftr(-/-) mice. Apoptosis was induced by staurosporine (STS; 1 microM). Cell volume, Cl(-) conductance, caspase-3 activity, intracellular level of reactive oxygen species (ROS), and glutathione content (GSH/GSSG) were monitored during AVD. In cftr(+/+) cells, AVD and caspase-3 activation were strongly impaired by conventional Cl(-) channel blockers and by a specific CFTR inhibitor (CFTR(inh)-172; 5 microM). STS induced activation of CFTR conductance within 15 min, which was progressively replaced by VSOR Cl(-) currents after 60 min of exposure. In parallel, STS induced an increase in ROS content in the time course of VSOR Cl(-) current activation. This increase was impaired by CFTR(inh)-172 and was not observed in cftr(-/-) cells. Furthermore, the intracellular GSH/GSSG content decreased during STS exposure in cftr(+/+) cells only. In conclusion, CFTR could play a key role in the cascade of events leading to apoptosis. This role probably involves control of the intracellular ROS balance by some CFTR-dependent modulation of GSH concentration.

In vitro effects of propofol and volatile agents on pharmacologically induced chloride channel myotonia.

BACKGROUND: Anesthetic choice for patients with chloride channel myotonia remains under debate. The authors have, therefore, investigated the in vitro effects of various anesthetic agents on pharmacologically induced chloride channel myotonia. METHODS: Functionally viable (> 10 mN force generation) rectus abdominis muscle preparations obtained from normal swine were investigated using in vitro muscle contracture test baths. During continuous 0.1-Hz supramaximal electrical stimulation, the chloride channel blocker 9-anthracenecarboxylic acid (64 microM) was added before the addition of propofol or one of three volatile anesthetics. The concentration of propofol in either Intralipid (n = 11) or dimethyl sulfoxide (n = 10) was doubled every 10 min (from 4-512 microM). The concentration of halothane (n = 8), isoflurane (n = 8), and sevoflurane (n = 8) was doubled from 0.25 vol% up to the maximum dose according to calibrated vaporizers. Control muscle bundles were either untreated (n = 30) or exposed to 9-anthracenecarboxylic acid (n = 19). RESULTS: The myotonic reactions induced by 9-anthracenecarboxylic acid were reversed by high-dose (> 64 microM) propofol (P < 0.01). Halothane, isoflurane, or sevoflurane each enhanced the myotonic reactions at 5.4 (P < 0.001), 0.21 (P < 0.01), and 0.5 minimum alveolar concentrations (P < 0.05), respectively. CONCLUSIONS: The authors' in vitro data imply that propofol administration for general anesthesia may be better suited for patients with chloride channel myotonia versus volatile anesthetics. In isolated swine skeletal muscle bundles, propofol elicited a reversal of 9-anthracenecarboxylic acid-induced chloride channel myotonia, whereas volatile anesthetics further increased the associated myotonic reactions.

Emodin augments calcium activated chloride channel in colonic smooth muscle cells by Gi/Go protein.

Emodin is a natural anthraquinone in rhubarb. It has been identified as a prokinetic drug for gastrointestinal motility in Chinese traditional medicine. Emodin contracts smooth muscle by increasing the concentration of intracellular Ca(2+). In many smooth muscles, increasing intracellular Ca(2+) activates Ca(2+)-activated Cl(-) channels (ClCA). The study was aimed to investigate the effects of emodin on ClCA channels in colonic smooth muscle. 4 channel physiology signal acquire system was used to measure isometric contraction of smooth muscle strips. ClCA currents were recorded by EPC10 with perforated whole cell model. Emodin contracted strips and cells in colonic smooth muscle and augmented ClCA currents. Niflumic acid (NFA) and 4', 4'-diisothiostilbene-2, 2-disulfonic acid (DIDS) blocked the effects. Gi/Go protein inhibits protein kinase A (PKA) and protein kinase C (PKC), and PKA and PKC reduced ClCA currents. Pertussis toxin (PTX, a special inhibitor of Gi/Go protein), 8-bromoadenosine 38, 58-cyclic monophosphate (8-BrcAMP, a membrane-permeant protein kinase A activator) and Phorbol-12-myristate-13-acetate (PMA, a membrane-permeant protein kinase C activator) inhibited the effects on ClCA currents significantly. Our findings suggest that emodin augments ClCA channels to contract smooth muscle in colon, and the effect is induced mostly by enhancement of membrane Gi/Go protein signal transducer pathway.

Investigation of the effects of the novel anticonvulsant compound carisbamate (RWJ-333369) on rat piriform cortical neurones in vitro.

BACKGROUND AND PURPOSE: Carisbamate is being developed for adjuvant treatment of partial onset epilepsy. Carisbamate produces anticonvulsant effects in primary generalized, complex partial and absence-type seizure models, and exhibits neuroprotective and antiepileptogenic properties in rodent epilepsy models. Phase IIb clinical trials of carisbamate demonstrated efficacy against partial onset seizures; however, its mechanisms of action remain unknown. Here, we report the effects of carisbamate on membrane properties, evoked and spontaneous synaptic transmission and induced epileptiform discharges in layer II-III neurones in piriform cortical brain slices. EXPERIMENTAL APPROACH: Effects of carisbamate were investigated in rat piriform cortical neurones by using intracellular electrophysiological recordings. KEY RESULTS: Carisbamate (50-400 micromol x L(-1)) reversibly decreased amplitude, duration and rise-time of evoked action potentials and inhibited repetitive firing, consistent with use-dependent Na+ channel block; 150-400 micromol x L(-1) carisbamate reduced neuronal input resistance, without altering membrane potential. After microelectrode intracellular Cl(-) loading, carisbamate depolarized cells, an effect reversed by picrotoxin. Carisbamate (100-400 micromol x L(-1)) also selectively depressed lateral olfactory tract-afferent evoked excitatory synaptic transmission (opposed by picrotoxin), consistent with activation of a presynaptic Cl(-) conductance. Lidocaine (40-320 micromol x L(-1)) mimicked carisbamate, implying similar modes of action. Carisbamate (300-600 micromol x L(-1)) had no effect on spontaneous GABA(A) miniature inhibitory postsynaptic currents and at lower concentrations (50-200 micromol x L(-1)) inhibited Mg2+-free or 4-aminopyridine-induced seizure-like discharges. CONCLUSIONS AND IMPLICATIONS: Carisbamate blocked evoked action potentials use-dependently, consistent with a primary action on Na+ channels and increased Cl(-) conductances presynaptically and, under certain conditions, postsynaptically to selectively depress excitatory neurotransmission in piriform cortical layer Ia-afferent terminals.

Xenopus oocytes as a heterologous expression system for studying ion channels with the patch-clamp technique.

Oocytes from the Xenopus laevis represent one of the most widely used expression systems for functional characterization of ion channels. Their large size facilitates both injection of heterologous cRNA and subsequent electrophysiological recordings of ion channel currents. Furthermore, Xenopus oocytes translate cRNA very efficiently, resulting in the generation of a large number of ion channels in the plasma membrane. In this chapter, we outline methods for oocyte preparation and maintenance and describe procedures for patch-clamping of oocytes, with a special focus on the macropatch technique. We discuss some common problems associated with patch-clamping of oocytes and their use as an expression system for ion channels.

ClC-3 and IClswell are required for normal neutrophil chemotaxis and shape change.

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).

Novel chloride channel mutations leading to mild myotonia among Chinese.

We describe two Chinese families with a mild form of the myotonia congenita due to novel chloride channel (ClCN1) mutations. In one case, heterozygous I553F and H555N mutations were found. The patient shared the I553F mutation with his healthy father, and his mother had a history of mild myotonia when she was younger. In another family, autosomal dominant myotonia congenita was due to a L844F change. The physiological effects of the mutations were examined by using the two-electrode voltage-clamp technique after expression of the channels in Xenopus oocytes. All mutations drastically shifted the voltage required for half-maximal activation, more under conditions mimicking the homozygous situation, than under conditions mimicking the heterozygous situation. The larger effect was seen in the compound heterozygous situation combining the I553F and the H555N mutations. Our data suggest that myotonia congenita caused by CLCN1 mutations in Chinese have similar variable features to those found in the West.

Voltage-activated ion channels and Ca(2+)-induced Ca (2+) release shape Ca (2+) signaling in Merkel cells.

Ca(2+) signaling and neurotransmission modulate touch-evoked responses in Merkel cell-neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca(2+) signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca(2+) channels (Ca(V)2.1) and voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels. Voltage-clamp recordings revealed small Ca(2+) currents, which produced Ca(2+) transients that were amplified sevenfold by Ca(2+)-induced Ca(2+) release. Merkel cells' voltage-activated K(+) currents were carried predominantly by BK(Ca) channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK(Ca) channels. Finally, blocking K(+) channels increased response magnitude and dramatically shortened Ca(2+) transients evoked by mechanical stimulation. Together, these results demonstrate that Ca(2+) signaling in Merkel cells is governed by the interplay of plasma membrane Ca(2+) channels, store release and K(+) channels, and they identify specific signaling mechanisms that may control touch sensitivity.

Leukoencephalopathy upon disruption of the chloride channel ClC-2.

ClC-2 is a broadly expressed plasma membrane chloride channel that is modulated by voltage, cell swelling, and pH. A human mutation leading to a heterozygous loss of ClC-2 has previously been reported to be associated with epilepsy, whereas the disruption of Clcn2 in mice led to testicular and retinal degeneration. We now show that the white matter of the brain and spinal cord of ClC-2 knock-out mice developed widespread vacuolation that progressed with age. Fluid-filled spaces appeared between myelin sheaths of the central but not the peripheral nervous system. Neuronal morphology, in contrast, seemed normal. Except for the previously reported blindness, neurological deficits were mild and included a decreased conduction velocity in neurons of the central auditory pathway. The heterozygous loss of ClC-2 had no detectable functional or morphological consequences. Neither heterozygous nor homozygous ClC-2 knock-out mice had lowered seizure thresholds. Sequencing of a large collection of human DNA and electrophysiological analysis showed that several ClC-2 sequence abnormalities previously found in patients with epilepsy most likely represent innocuous polymorphisms.

Cloning and heterologous expression of a Ca2+-activated chloride channel isoform from rat brain.

We previously reported the cloning of a calcium-activated chloride channel (CLCA) from rat brain (Biochem. Biophys. Res. Commun., 334, 569-576 (2005)), which we designated rbCLCA1. We further showed that rbCLCA1 is expressed in the central nervous system and peripheral organs, and may be functionally expressed in mammalian HEK293 cells. In the present study, we report the successful cloning of a second CLCA from rat cerebrum (designated rbCLCA2), using reverse transcription-PCR (RT-PCR) with primers specific for rbCLCA1. We have begun to clone this cDNA based on the rbCLCA1-like sequence. The full-length rbCLCA2 cDNA, obtained via 5' and 3' rapid amplification of cDNA ends (RACE), is 2900 bp long and encodes a putative polypeptide of 905 amino acids having at least two major transmembrane domains and showing 85.2% identity to rbCLCA1. RT-PCR analysis revealed that, similar to rbCLCA1, rbCLCA2 was predominantly expressed in the rat cerebrum, cerebellum, kidney, stomach, spinal cord, lung and small intestine, but not in the heart, large intestine, liver, orand spleen. Whole-cell patch clamp studies in HEK293 cells transiently co-transfected with expression vectors encoding rbCLCA2 and EGFP allowed us to identify the presence of niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride currents in cells expressing rbCLCA2 but not EGFP alone. Treatment of these cells with ionomycin, a Ca2+ ionophore, significantly increased the novel currents in cells expressing rbCLCA2 and EGFP, but not those expressing EGFP alone, indicating that activation of the rbCLCA2 current is Ca2+-dependent. In sum, we herein report the cloning of a second member of the rbCLCA family from rat brain and its functional expression in vitro, thus adding to our knowledge of anion channels and facilitating future exploration of brain and other organ physiology.