Nitrate is frequently the major nitrogen source for plants and is generally assimilated during the day. In the absence of light, nitrate can transiently accumulate in the vacuolar lumen via tonoplast transporters. CLC-a, a member of the CLC family of anion transporters, is critically involved in this nitrate storage in the vacuole, while other CLC family members apparently have different roles in diverse cell organelles. Here, CLC-b, a close relative of CLC-a, was functionally expressed in oocytes and analyzed. CLC-b conducted strongly outwardly rectifying anionic currents that were largest in the presence of nitrate. Fluorescence ratio changes of oocytes loaded with a pH-dependent fluorescent dye suggested that NO(-)(3) transport is associated with H(+) exchange. CLC-b was localized at the tonoplast, as was CLC-c, when tagged with the green fluorescent protein. CLC-b expression was strongest in young roots, hypocotyl and cotyledons. The physiological role of CLC-b was analyzed using two independent knock-out alleles. Both lines grew as the wild type in various conditions. The total chloride and nitrate content was identical in clcb lines and the wild type, potentially suggesting that mutants were able to compensate the loss of CLC-b.

Plants need nitrate for growth and store the major part of it in the central vacuole of cells from root and shoot tissues. Based on few studies on the two model plants Arabidopsis thaliana and rice, members of the large ChLoride Channel (CLC) family have been proposed to encode anion channels/transporters involved in nitrate homeostasis. Proteins from the Arabidopsis CLC family (AtClC, comprising seven members) are present in various membrane compartments including the vacuolar membrane (AtClCa), Golgi vesicles (AtClCd and AtClCf) or chloroplast membranes (AtClCe). Through a combination of electrophysiological and genetic approaches, AtClCa was shown to function as a 2NO3-/1H+ exchanger that is able to accumulate specifically nitrate into the vacuole, in agreement with the main phenotypic trait of knockout mutant plants that accumulate 50 per cent less nitrate than their wild-type counterparts. The set-up of a functional complementation assay relying on transient expression of AtClCa cDNA in the mutant background opens the way for studies on structure-function relationships of the AtClCa nitrate transporter. Such studies will reveal whether important structural determinants identified in bacterial or mammalian CLCs are also crucial for AtClCa transport activity and regulation.

Capillary liquid chromatography (CLC) coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS were explored for identification and quantification of neuropeptides in microwave-fixed rat brain tissue. Sample was separated by gradient elution on 50-mum-inner diameter reversed-phase columns at 180 nL/min. Effluent was mixed with matrix solution and transferred to a MALDI target plate by pulsed electric field deposition, yielding sample spots with 200-300-mum diameter. Mass detection limits as low as 2 amol, corresponding to 1 pM concentration, were achieved for neuropeptides. CLC-MALDI-TOF-MS analysis of microwave-fixed rat striatum tissue yielded detection of over 400 distinctive peaks. CLC-MALDI-TOF/TOF-MS allowed identification of 10 peptides including 3 novel peptides. Quantification was evaluated using substance P as analyte and 15N3-labeled substance P as an internal standard. Quantification of substance P revealed approximately 6.8-fold higher levels than previously reported in the rat striatum. This increase is attributed to use of microwave fixation, which prevented degradation of the peptide, aggressive extraction procedures, and accounting for oxidation of substance P in the analysis. These results demonstrate that CLC-MALDI-TOF-MS is a versatile tool for neuropeptide analysis in brain tissue by allowing for detection, identification, and quantification.

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl(-) channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl(-) currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl(-) currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl(-) currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl(-) currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl(-) currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl(-) currents with half-maximal inhibition at 100 and 200-230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl(-) currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl(-) currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl(-) currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl(-) currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

1. In the present work we investigated the dependence on temperature of the ionic conductance and gating of human muscle ClC-1 chloride channels, transiently expressed in human embryonic kidney (HEK 293) cells. 2. At normal pH, ClC-1 currents deactivated at negative potentials with a double-exponential time course. The time constants of the exponential components, corresponding to the relaxations of the fast and slow gates, were temperature dependent with Q(10) values of approximately 3 and approximately 4, respectively. Current amplitude increased with increasing temperature with a Q(10) of approximately 1.6. 3. The voltage dependence of the two gating processes was shifted towards more positive potentials with increasing temperature. The half-saturation voltage (V(1/2)) of the steady-state open probability (P(o)) was shifted by approximately 23 and approximately 34 mV per 10 degrees C increase in temperature, for the fast and slow gate, respectively. 4. At low pH, the voltage dependence of ClC-1 was reversed and currents were activated by hyperpolarisation with a single-exponential time course. This type of gating in ClC-1 resembled the slow gating of the Torpedo ClC-0 homologue, but differed with respect to its kinetics and temperature dependence, with a Q(10) of gating relaxations at negative potentials of approximately 5. The Arrhenius plot of ClC-1 conductance at low pH had a clear break point at approximately 25 degrees C, with higher Q(10) values at lower temperatures. 5. The temperature sensitivity of relaxation and open probability of the slow gate, which in both ClC-0 and ClC-1 controls two pores simultaneously, implies that the slow gating of ClC-1 is mechanistically different from that of ClC-0.

BACKGROUND

Single-incision laparoscopic surgery may reduce the complications of port site and postoperative pain. The improved cosmetic result also may improve the satisfaction of patients who have undergone surgery.

METHODS

The study enrolled 108 patients who consecutively underwent laparoscopic cholecystectomy by the same surgeons and randomly divided them into single-incision laparoscopic cholecystectomy (SILC) and conventional laparoscopic cholecystectomy (CLC) groups. Demographic data and short-term operative outcomes were collected and compared.

RESULTS

A total of 57 and 51 patients received SILC and CLC, respectively, from May to August 2010 at our institution. No significant difference was found with respect to demographic data including age, sex, and body mass index between the 2 groups. Similarly, short-term operative outcomes such as postoperative complications, length of stay, and visual analog pain score did not differ between the 2 groups. However, the incision of SILC (21.6 ± 2.4) was shorter than that of CLC (30.8 ± 2.6) (P=0.032).

CONCLUSIONS

SILC seems to be a safe and feasible technique. It can be undertaken without the expense of added postoperative complication and operative time and provides patients with a minimal apparent scar.

BACKGROUND

ClC-Ka and ClC-Kb, chloride channels participating in renal tubular Cl- transport, require the coexpression of barttin to become functional. Mutations of the barttin gene lead to the Bartter's syndrome variant BSND, characterized by congenital deafness and severe renal salt wasting. Barttin bears a proline-tyrosine motif, a target structure for the ubiquitin ligase Nedd4-2, which mediates the clearance of channel proteins from the cell membrane. Nedd4-2 is, in turn, a target of the serum- and glucocorticoid-inducible kinase SGK1, which phosphorylates and, thus, inactivates the ubiquitin ligase. ClC-Ka also possesses a SGK1 consensus site in its sequence. We hypothesized that ClC-Ka/barttin is stimulated by SGK1, and down-regulated by Nedd4-2, an effect that may be reversed by SGK1 and its isoforms, SGK2 or SGK3.

METHODS

To test this hypothesis, ClC-Ka/barttin was heterologously expressed in Xenopus oocytes with or without the additional expression of Nedd4-2, SGK1, SGK2, SGK3, constitutively active S422DSGK1, or inactive K127NSGK1.

RESULTS

Expression of ClC-Ka/barttin induced a slightly inwardly rectifying current that was significantly decreased upon coexpression of Nedd4-2, but not the catalytically inactive mutant C938SNedd4-2. The coexpression of S422DSGK1, SGK1, or SGK3, but not SGK2 or K127NSGK1 significantly stimulated the current. Moreover, S422DSGK1, SGK1, and SGK3 also phosphorylated Nedd4-2 and thereby inhibited Nedd4-2 binding to its target. The down-regulation of ClC-Ka/barttin by Nedd4-2 was abolished by elimination of the PY motif in barttin.

CONCLUSIONS

ClC-Ka/barttin channels are regulated by SGK1 and SGK3, which may thus participate in the regulation of transport in kidney and inner ear.

H+/Cl- antiport behavior has recently been observed in bacterial chloride channel homologs and eukaryotic CLC-family proteins. The detailed molecular-level mechanism driving the stoichiometric exchange is unknown. In the bacterial structure, experiments and modeling studies have identified two acidic residues, E148 and E203, as key sites along the proton pathway. The E148 residue is a major component of the fast gate, and it occupies a site crucial for both H+ and Cl- transport. E203 is located on the intracellular side of the protein; it is vital for H+, but not Cl-, transport. This suggests two independent ion transit pathways for H+ and Cl- on the intracellular side of the transporter. Previously, we utilized a new pore-searching algorithm, TransPath, to predict Cl- and H+ ion pathways in the bacterial ClC channel homolog, focusing on proton access from the extracellular solution. Here we employ the TransPath method and molecular dynamics simulations to explore H+ pathways linking E148 and E203 in the presence of Cl- ions located at the experimentally observed binding sites in the pore. A conclusion is that Cl- ions are required at both the intracellular (S(int)) and central (S(cen)) binding sites in order to create an electrostatically favorable H+ pathway linking E148 and E203; this electrostatic coupling is likely related to the observed 1H+/2Cl- stoichiometry of the antiporter. In addition, we suggest that a tyrosine residue side chain (Y445), located near the Cl- ion binding site at S(cen), is involved in proton transport between E148 and E203.

ClC voltage-gated anion channels have been identified in bacteria, yeast, plants, and animals. The biophysical and structural properties of ClCs have been studied extensively, but relatively little is known about their precise physiological functions. Furthermore, virtually nothing is known about the signaling pathways and molecular mechanisms that regulate channel activity. The nematode Caenorhabditis elegans provides significant experimental advantages for characterizing ion channel function and regulation. We have shown previously that the ClC Cl- channel homologue CLH-3 is expressed in C. elegans oocytes, and that it is activated during meiotic maturation and by cell swelling. We demonstrate here that depletion of intracellular ATP or removal of Mg2+, experimental maneuvers that inhibit kinase function, constitutively activate CLH-3. Maturation- and swelling-induced channel activation are inhibited by type 1 serine/threonine phosphatase inhibitors. RNA interference studies demonstrated that the type 1 protein phosphatases CeGLC-7alpha and beta, both of which play essential regulatory roles in mitotic and meiotic cell cycle events, mediate CLH-3 activation. We have suggested previously that CLH-3 and mammalian ClC-2 are orthologues that play important roles in heterologous cell-cell interactions, intercellular communication, and regulation of cell cycle-dependent physiological processes. Consistent with this hypothesis, we show that heterologously expressed rat ClC-2 is also activated by serine/threonine dephosphorylation, suggesting that the two channels have common regulatory mechanisms.

The principal function of the colon in fluid homeostasis is the absorption of NaCl and water. Apical membrane Na(+) channels, Na(+)/H(+) and Cl(-)/HCO exchangers, have all been postulated to mediate NaCl entry into colonocytes. The identity of the basolateral exit pathway for Cl(-) is unknown. We have previously demonstrated the presence of the ClC-2 transcript in the guinea pig intestine. Now we explore in more detail, the tissue and cellular distribution of chloride channel ClC-2 in the distal colon by in situ hybridization and immunohistochemistry. The patch-clamp technique was used to characterize Cl(-) currents in isolated surface epithelial cells from guinea pig distal colon and these were compared with those mediated by recombinant guinea pig (gp)ClC-2. ClC-2 mRNA and protein were found in the surface epithelium of the distal colon. Immunolocalization revealed that, in addition to some intracellular labeling, ClC-2 was present in the basolateral membranes but absent from the apical pole of colonocytes. Isolated surface epithelial cells exhibited hyperpolarization-activated chloride currents showing a Cl(-)>> I(-) permeability and Cd(2+) sensitivity. These characteristics, as well as some details of the kinetics of activation and deactivation, were very similar to those of recombinant gpClC-2 measured in parallel experiments. The presence of active ClC-2 type currents in surface colonic epithelium, coupled to a basolateral location for ClC-2 in the distal colon, suggests a role for ClC-2 channel in mediating basolateral membrane exit of Cl(-) as an essential step in a NaCl absorption process.

ClC-K channels form a subgroup of anion channels within the ClC family of anion transport proteins. They are expressed predominantly in the kidney and in the inner ear, and are necessary for NaCl resorption in the loop of Henle and for K+ secretion by the stria vascularis. Subcellular distribution as well as the function of these channels are tightly regulated by an accessory subunit, barttin. Barttin improves the stability of ClC-K channel protein, stimulates the exit from the endoplasmic reticulum and insertion into the plasma membrane and changes its function by modifying voltage-dependent gating processes. The importance of ClC-K/barttin channels is highlighted by several genetic diseases. Dysfunctions of ClC-K channels result in Bartter syndrome, an inherited human condition characterized by impaired urinary concentration. Mutations in the gene encoding barttin, BSND, affect the urinary concentration as well as the sensory function of the inner ear. Surprisingly, there is one BSND mutation that causes deafness without affecting renal function, indicating that kidney function tolerates a reduction of anion channel activity that is not sufficient to support normal signal transduction in inner hair cells. This review summarizes recent work on molecular mechanisms, physiology, and pathophysiology of ClC-K/barttin channels.

Lubiprostone activates ClC-2 chloride channels in epithelia. It is approved for treatment of chronic idiopathic constipation in adults and constipation-predominate irritable bowel syndrome in women. We tested a hypothesis that lubiprostone can reverse the constipating action of morphine and investigated the mechanism of action. Short-circuit current (Isc) was recorded in Ussing chambers as a marker for chloride secretion during pharmacological interactions between morphine and lubiprostone. Measurements of fecal wet weight were used to obtain information on morphine-lubiprostone interactions in conscious mice. Morphine decreased basal Isc, with an IC(50) of 96.1 nM. The action of dimethylphenylpiperazinium (DMPP), a nicotinic receptor agonist that stimulates neurogenic Isc, was suppressed by morphine. Lubiprostone applied after pretreatment with morphine reversed morphine suppression of both basal Isc and DMPP-evoked chloride secretion. Electrical field stimulation (EFS) of submucosal neurons evoked biphasic increases in Isc. Morphine abolished the first phase and marginally suppressed the second phase. Lubiprostone reversed, in concentration-dependent manner, the action of morphine on the first and second phases of the EFS-evoked responses. Subcutaneous lubiprostone increased fecal wet weight and numbers of pellets expelled. Morphine significantly reduced fecal wet weight and number of pellets. Injection of lubiprostone, 30-min after morphine, reversed morphine-induced suppression of fecal wet weight. We conclude that inhibitory action of morphine on chloride secretion reflects suppression of excitability of cholinergic secretomotor neurons in the enteric nervous system. Lubiprostone, which does not directly affect enteric neurons, bypasses the neurogenic constipating effects of morphine by directly opening chloride channels in the mucosal epithelium.

ClC-3 is an intracellular chloride transport protein known to reside on endosomes and synaptic vesicles. The endogenous protein has been notoriously difficult to detect in immunohistological experiments because of the lack of reliable antibodies. Using newly generated antibodies, we now examine its expression pattern at the cellular and subcellular level. In all tissues examined, immunostaining indicated that ClC-3 is a vesicular protein, with a prominent expression in endocrine cells like adrenal chromaffin cells and pancreatic islet cells. In line with a possible function of ClC-3 in regulating vesicle trafficking or exocytosis in those secretory cells, capacitance measurements and amperometry indicated that exocytosis of large dense-core vesicles (LDCVs) was decreased in chromaffin cells from ClC-3 knock-out mice. However, immunohistochemistry complemented with subcellular fractionation showed that ClC-3 is not detectable on LDCVs of endocrine cells, but localizes to endosomes and synaptic-like microvesicles in both adrenal chromaffin and pancreatic beta cells. This observation points to an indirect influence of ClC-3 on LDCV exocytosis in chromaffin cells, possibly by affecting an intracellular trafficking step.

Abnormal renal reabsorption of sodium (Na(+)) is likely to play a role in the pathogenesis of salt-sensitivity. In the kidney, chloride channels CLC-Ka (gene CLCNKA) and CLC-Kb (gene CLCNKB) and their subunit Barttin (gene BSND) have important effects on the control of Na(+) and water homeostasis. We investigated if single nucleotide polymorphisms (SNPs) or haplotypes within CLCNKA, CLCNKB and BSND loci affect salt-sensitivity in hypertensive subjects. Associations between blood pressure (BP) change after Na(+)-load and 15 SNPs spanning the length of CLCNKA and CLCNKB and six SNPs spanning the length of BSND were studied in 314 never treated essential hypertensives who underwent an i.v. infusion of saline (300 mm NaCl in 2 l H(2)O in 120 min). Four SNPs were significantly associated with BP change after Na-load. Rs848307 (P = 0.0026) and rs1739843 (P = 0.0023) map upstream the 5' of CLCNKA. Non-coding Rs1010069 (P = 0.0006) and non-synonymous rs1805152 (Thr447Ala; P = 0.0078) map within CLCNKA. Moreover, basal plasma renin activity and heart rate (measured before Na-load) were significantly lower in patients carrying the alleles associated with the larger mean BP increase after Na-load, indicating that such alleles are associated with chronic volume expansion. This study supports the candidacy of CLCNKA as a new susceptibility gene for salt-sensitivity.

Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.

After providing a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl(-) channel ClC-0 and the discovery of the CLC gene family, I succinctly describe the general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl(-) channels and vesicular Cl(-) /H(+) -exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel.

Myotonia congenita (MC) is caused by loss-of-function mutations of the muscle ClC-1 chloride channel. Clinical manifestations include the variable association of myotonia and transitory weakness. We recently described a cohort of recessive MC patients showing, at a low rate repetitive nerves stimulation protocol, different values of compound muscle action potential (CMAP) transitory depression, which is considered the neurophysiologic counterpart of transitory weakness. From among this cohort, we studied the chloride currents generated by G190S (associated with pronounced transitory depression), F167L (little or no transitory depression), and A531V (variable transitory depression) hClC-1 mutants in transfected HEK293 cells using patch-clamp. While F167L had no effect on chloride currents, G190S dramatically shifts the voltage dependence of channel activation and A531V reduces channel expression. Such variability in molecular mechanisms observed in the hClC-1 mutants may help to explain the different clinical and neurophysiologic manifestations of each ClCN1 mutation. In addition we examined five different mutations found in compound heterozygosis with F167L, including the novel P558S, and we identified additional molecular defects. Finally, the G190S mutation appeared to impair acetazolamide effects on chloride currents in vitro.

The proteins ClC-6 and ClC-7 are expressed in the endosomal-lysosomal system. Because Clcn6-deficient mice display some features of neuronal ceroid lipofuscinosis (NCL), CLCN6 may be a candidate gene for novel forms of NCL. Using landmarks of disease progression from NCL mouse models as a guide, we examined neuropathologic alterations in the central nervous system of Clcn6(-/-), Clcn7(-/-), andgl mice. gl mice bear a mutation in Ostm1, the β-subunit critical for Clcn7 function. Severely affected Clcn7(-/-) and gl mice have remarkably similar neuropathologic phenotypes, with pronounced reactive changes and neuron loss in the thalamocortical system, similar to findings in early-onset forms of NCL. In contrast, Clcn6(-/-) mice display slowly progressive, milder neuropathologic features with very little thalamic involvement or microglial activation. These findings detail for the first time the markedly different neuropathologic consequences of mutations in these two CLC genes. Clcn7(-/-) and gl mice bear a close resemblance to the progressive neuropathologic phenotypes of early onset forms of NCL, whereas the distinct phenotype of Clcn6-deficient mice suggests that this gene could be a candidate for a later-onset form of mild neurologic dysfunction with some NCL-like features.

Membrane hyperpolarization normally activates the slow gate of the Torpedo voltage-gated chloride channel (ClC-0). To elucidate the structural basis of this process, carboxy terminus truncation mutants and chimeras were constructed, expressed in Xenopus oocytes, and evaluated using a two-microelectrode voltage clamp. Introduction of stop codons at several positions between transmembrane domains 12 and 13 (D12 and D13) showed no expression, whereas a truncation just after D13 yielded wild-type currents. A chimera (022) entailing the substitution of the carboxy-terminal cytoplasmic tail after Lys-520 with the corresponding region of ClC-2 lacked slow gating, whereas a more conservative construct (chimera 002), in which D13 was replaced with its ClC-2 analog, retained its capacity to slow gate. These findings suggest that important structures reside within the interdomain stretch (IDS) between D12 and D13. Unlike ClC-2, in which transplantation of "ball" structures could restore gating to constitutively open mutants, transplantation of the ClC-0 IDS to the amino terminus of chimera 022 did not restore gating. Surprisingly, replacement of the IDS by the analogous regions of either ClC-1 or ClC-2 showed slow voltage-activated gating, although the gating was altered. Our findings lead us to conclude that both the functional expression and the slow voltage gating of ClC-0 rely on structures at the carboxy terminus of the channel.

Idiopathic epilepsies are genetically determined diseases of the central nervous system characterized by typical epileptic seizures and EEG abnormalities but not associated with structural brain lesions. In recent years, an increasing number of mutations associated with idiopathic epilepsy syndromes were identified in genes encoding subunits of voltage- or ligand-gated ion channels. These encouraging results provide a plausible pathophysiological concept, since ion channels form the basis for neuronal excitability and are the major targets for anticonvulsive pharmacotherapy. The first epilepsy genes were identified for rare autosomal dominant syndromes within large pedigrees. Recently, a few mutations were also found for the frequent classical forms of idiopathic generalized epilepsies (IGE), for example absence or juvenile myoclonic epilepsy. The mutations can affect ion channels which on one hand have been known since several decades to be crucial for neuronal function, such as the voltage-gated sodium channel or the GABA(A) receptor, or on the other hand were newly identified within the last decade as KCNQ potassium channels or the ClC-2 chloride channel. Functional studies characterizing the molecular defects of the mutant channels point to a central role of GABAergic synaptic inhibition in the pathophysiology of IGE. Furthermore, newly discovered genes may be suitable as novel targets for pharmacotherapy such as KCNQ channels for the anticonvulsant drug retigabine. Altogether, these genetic and pathophysiological investigations will enhance our knowledge about the understanding of epileptogenesis and can help to improve anticonvulsive therapy.

The role of clathrin light chain (CLC) in clathrin-mediated endocytosis is not completely understood. Previous studies showed that the CLC N-terminus (CLC-NT) binds the Hip1/Hip1R/Sla2 family of membrane/actin-binding factors and that overexpression of the CLC-NT in yeast suppresses endocytic defects of clathrin heavy-chain mutants. To elucidate the mechanistic basis for this suppression, we performed synthetic genetic array analysis with a clathrin CLC-NT deletion mutation (clc1-Δ19-76). clc1-Δ19-76 suppressed the internalization defects of null mutations in three late endocytic factors: amphiphysins (rvs161 and rvs167) and verprolin (vrp1). In actin sedimentation assays, CLC binding to Sla2 inhibited Sla2 interaction with F-actin. Furthermore, clc1-Δ19-76 suppression of the rvs and vrp phenotypes required the Sla2 actin-binding talin-Hip1/R/Sla2 actin-tethering C-terminal homology domain, suggesting that clc1-Δ19-76 promotes internalization by prolonging actin engagement by Sla2. We propose that CLC directs endocytic progression by pruning the Sla2-actin attachments in the clathrin lattice, providing direction for membrane internalization.

Synaptic inhibition by GABA(A) receptors requires a transmembrane chloride gradient. Hyperpolarization or shunting results from outward current produced by chloride flowing down this gradient, into the cell. Chloride influx necessarily depletes the chloride gradient. Therefore, mechanisms that replenish the gradient (by reducing intracellular chloride concentration, [Cl(-)](i)) are crucial for maintaining the efficacy of GABA(A) receptor-mediated inhibition. ClC-2 is an inward-rectifying chloride channel that is thought to help extrude chloride because inward rectification should, in principle, allow ClC-2 to act as a one-way chloride exit valve. But chloride efflux via ClC-2 nevertheless requires an appropriate driving force. Using computer modeling, we reproduced voltage-clamp experiments showing chloride efflux via ClC-2, but testing the same model under physiological conditions revealed that ClC-2 normally leaks chloride into the cell. The discrepancy is explained by the driving force conditions that exist under artificial versus physiological conditions, and by the fact that ClC-2 rectification is neither complete nor instantaneous. Thus, contrary to previous assertions that ClC-2 helps maintain synaptic inhibition by lowering [Cl(-)](i), our simulations show that ClC-2 mediates chloride influx, thus producing outward current and directly reducing excitability. To test how ClC-2 functions in real neurons, we used dynamic clamp to insert virtual ClC-2 channels into rat CA1 pyramidal cells with and without native ClC-2 channels blocked. Experiments confirmed that ClC-2 reduces spiking independently of inhibitory synaptic transmission. Our results highlight the importance of considering driving force when inferring how a channel functions under physiological conditions.

* In plants, the knowledge of the molecular identity and functions of anion channels are still very limited, and are almost restricted to the large ChLoride Channel (CLC) family. In Arabidopsis thaliana, some genetic evidence has suggested a role for certain AtCLC protein members in the control of plant nitrate levels. In this context, AtClCa has been demonstrated to be involved in nitrate transport into the vacuole, thereby participating in cell nitrate homeostasis. * In this study, analyses of T-DNA insertion mutants within the AtClCa and AtClCe genes revealed common phenotypic traits: a lower endogenous nitrate content; a higher nitrite content; a reduced nitrate influx into the root; and a decreased expression of several genes encoding nitrate transporters. * This set of nitrate-related phenotypes, displayed by clca and clce mutant plants, showed interconnecting roles of AtClCa and AtClCe in nitrate homeostasis involving two different endocellular membranes. * In addition, it revealed cross-talk between two nitrate transporter families participating in nitrate assimilation pathways. The contribution to nitrate homeostasis at the cellular level of members of these different families is discussed.

The CLC 'Cl(-) channel' family consists of both Cl(-)/H(+) antiporters and Cl(-) channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl(-) permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC-mediated antiport. The crystal structure of the Escherichia coli antiporter ClC-ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate-induced conformational changes in ClC-ec1. Using mutational analysis, we show that substrate-dependent (19)F spectral changes reflect functionally relevant protein movement occurring at the ClC-ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl(-) permeation pathway and show the usefulness of (19)F NMR for studying conformational changes in membrane proteins of known structure.