Until recently, anion (Cl − ) channels have received considerably less attention than cation channels. One reason for this may be that many Cl − channels perform functions that might be considered cell biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability that typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl − channels with special emphasis on new and often surprising developments over the last 5 years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels as was previously thought, and that bestrophins, previously prime candidates for Ca 2+ -activated Cl − channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl − channel world. Keywords channelopathies; ion transport; bestrophin; TMEM16; anoctamin; ClC A Short History of Chloride in Biological SystemsNot long ago, Cl − channels were the Rodney Dangerfield of the ion channel field. Rodney Dangerfield (1921Dangerfield ( -2004) was a comedian who became famous for his joke: "I get no respect. I played hide-and-seek, and they wouldn't even look for me." After a small flurry of work on Cl − channels in the 50's and 60's, interest in Cl − channels dwindled until the 1990's. In the first edition of the "bible" on ion channels published in 1984 (1), less than 3 pages were devoted to Cl − channels, because it was thought that Cl − was usually in electrochemical equilibrium across cell membranes. This made Cl − less interesting than other ions that exhibited the potential to do some work. This mis-impression occurred because many early studies on Cl − were performed on skeletal muscle and erythrocytes where resting Cl − permeability is very high so that even if there is active Cl − transport present, Cl − is in electrochemical equilibrium. §Corresponding author: H. Criss Hartzell, Department of Cell Biology, 615 Michael St., 535 Whitehead Building, Emory, University School of Medicine, Atlanta, GA 30322, criss.hartzell@emory.edu, NIH Public Access Author ManuscriptAnnu Rev Physiol. Author manuscript; available in PMC 2011 March 17. Published in final edited form as:Annu Rev Physiol. 2010 March 17; 72: 95-121. doi:10.1146/annurev-physiol-021909-135811. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe demonstration that Cl − is actively transported in squid axons (2) and secreted (as HCl) by stomach (3) did not seem to attract much attention. Even the discovery that the inhibitory action of GABA was caused by an increased Cl − conductance did little to dispel the idea that Cl − could be out of electrochemical equilibrium. Because the reversal potentials of GABA-induced i.p.s.p.'s were very close to the resting potential, it was reasonably concluded that "normally Cl − ions are in electro-chemical equilibrium across the membrane" (4).Into the 1990's, the position of Cl − channels in cell...

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a+/−) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a+/− mice on strain 129S6/SvEvTac (129.Scn1a+/−) have no overt phenotype and normal survival compared with Scn1a+/− mice bred to C57BL/6J (F1.Scn1a+/−) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal day 21–24 (P21–24) 129.Scn1a+/− or F1.Scn1a+/− mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a+/− mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa between 129.Scn1a+/− mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a+/− and F1.Scn1a+/− mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a+/− neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a+/− animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a+/− mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21–24 correlates with age-dependent onset of lethality in F1.Scn1a+/− mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

A method has been developed for the bulk isolation of Alzheimer neurofibrillary tangles (ANT) of paired helical filaments (PHF) from histopathologically confirmed cases of Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). The fresh or frozen autopsied cerebral cortex affected with Alzheimer neurofibrillary changes is dissociated by homogenization and sieving through nylon bolting cloth and the ANT are separated by a combination of sucrose discontinuous density gradient centrifugation, glass bead column chromatography, and sodium dodecyl sulfate (SDS) treatment. The isolated ANT produce red-green birefringence when viewed through polarized light after staining with Congo red. Ultrastructurally, the isolated PHF are well preserved and have the dimensions of the PHF seen in situ. Two major Populations of ANT which exist in different proportions in AD/SDAT brains are identified on the basis of their solubility in SDS. The ANT I and the ANT II are soluble and insoluble respectively on treatment with 2% SDS at room temperature for 5 min. Solubilization of the ANT II requires several repeated extractions with a solution containing 10% each of SDS and beta-mercaptoethanol (BME) at 100 degrees C for 10 min. Sonication of the ANT II greatly facilitates their solubilization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ANT reveals the presence of two major polypeptides with molecular weights (MW) of 62,000 and 57,000, several minor polypeptides with MW below 57,000, and a significant amount of material not entering the stacking and the resolving gels. Re-electrophoresis of polypeptides extracted from various areas of the resolving gel or of the material which does not enter the gel generates the same polypeptide profile as on the first gel, suggesting that the PHF material which does not enter the gel may result from the reaggregation of the polypeptides that enter the resolving gel. None of the polypeptides that enter the resolving gel. None of the polpeptides observed in the isolated PHF comigrate in the SDS-PAGE with any of the neurofilament polypeptides, tubulin, actin, or myosin.

Objective Evidence from basic neurophysiology and molecular genetics has implicated persistent sodium current conducted by voltage-gated sodium (NaV) channels as a contributor to the pathogenesis of epilepsy. Many antiepileptic drugs target NaV channels and modulate neuronal excitability mainly by a use-dependent block of transient sodium current, although suppression of persistent current may also contribute to the efficacy of these drugs. We hypothesized that a drug or compound capable of preferential inhibition of persistent sodium current would have antiepileptic activity. Methods We examined the antiepileptic activity of two selective persistent sodium current blockers ranolazine, an FDA-approved drug for treatment of angina pectoris, and GS967, a novel compound with more potent effects on persistent current, in the epileptic Scn2aQ54 mouse model. We also examined the effect of GS967 in the maximal electroshock model and evaluated effects of the compound on neuronal excitability, propensity for hilar neuron loss, development of mossy fiber sprouting and survival of Scn2aQ54 mice. Results We found that ranolazine was capable of reducing seizure frequency by ~50% in Scn2aQ54 mice. The more potent persistent current blocker GS967 reduced seizure frequency by greater than 90% in Scn2aQ54 mice and protected against induced seizures in the maximal electroshock model. GS967 greatly attenuated abnormal spontaneous action potential firing in pyramidal neurons acutely isolated from Scn2aQ54 mice. In addition to seizure suppression in vivo, GS967 treatment greatly improved the survival of Scn2aQ54 mice, prevented hilar neuron loss, and suppressed the development of hippocampal mossy fiber sprouting. Significance Our findings indicate that the selective persistent sodium current blocker GS967 has potent antiepileptic activity and this compound could inform development of new agents.

Summary Three murine monoclonal antibodies (BC 1, BC2 and BC3) were developed against human milk fat globule membrane (HMFGM). By immunoperoxidase staining, it was found that the antigenic determinants had a predominant distribution in breast cancer tissue. In addition, the antibodies reacted preferentially with mucin derived from human milk rather than that derived from the breast cancer cell line ZR75; they also recognized polymorphic high molecular weight components (MW>23OOOO) in serum and in human milk fat globule membrane. Thus the antibodies appear to react with a component of the family of mucins found in breast cancer and human milk and it appears likely that at least part of each epitope is protein in nature. Antibodies BCI, BC2 and BC3 recognized related but not identical epitopes, and they appear to be co-expressed on the same molecules as 3EI 2-defined antigen (mammary serum antigen, MSA) which is also a member of the family of breast cancer-rctated mucin. However, the 3E1 2 epitope is distinct and non-cross-reactive with those described for BCI, BC2and BC3. The BC2 and BC3 defined epitopes were examined for their value in serum assays. Immunoassay was developed with a combination of two antibodies, using antibody BC3 for antigen capture and antibody BC2 or 3E1 2 for antigen detection and gave reasonable sensitivity (-~85%) and specificity (~95%) in such serum tests for breast cancer. In a limited study, these tests appeared to complement the MSA test in the detection of breast cancer.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was ion channel ͉ molecular evolution ͉ CFTR ͉ Type II divergence

SUMMARYPurpose: A common genetic variant (rs3812718) in a splice donor consensus sequence within the neuronal sodium channel gene SCN1A (encoding Na V 1.1) modulates the proportion of transcripts incorporating either the canonical (5A) or alternative (5N) exon 5. A pharmacogenetic association has been reported whereby increased expression of exon 5N containing Na V 1.1 transcripts correlated with lower required doses of phenytoin in epileptics. We tested the hypothesis that SCN1A alternative splicing affects the pharmacology of Na V 1.1 channels. Methods: To directly examine biophysical and pharmacologic differences between the exon 5 splice variants, we performed whole-cell patch clamp recording of tsA201 cells transiently coexpressing either Na V 1.1-5A or Na V 1.1-5N with the b1 and b2 accessory subunits. We examined tonic inhibition and use-dependent inhibition of Na V 1.1 splice isoforms by phenytoin, carbamazepine, and lamotrigine. We also examined the effects of phenytoin and lamotrigine on channel biophysical properties and determined concentration-response relationships for both splice variants. Key Findings: We observed no significant differences in voltage dependence of activation, steady-state inactivation, and recovery from inactivation between splice variants. However, Na V 1.1-5N channels exhibited enhanced tonic block by phenytoin and lamotrigine compared to Na V 1.1-5A. In addition, Na V 1.1-5N exhibited enhanced use-dependent block by phenytoin and lamotrigine across a range of stimulation frequencies and concentrations. Phenytoin and lamotrigine induced shifts in steady-state inactivation and recovery from fast inactivation for both splice isoforms. No splice isoform differences were observed for channel inhibition by carbamazepine. Significance: These results suggest Na V 1.1 channels containing exon 5N are more sensitive to the commonly used antiepileptic drugs phenytoin and lamotrigine.

Dravet syndrome, an epileptic encephalopathy affecting children, largely results from heterozygous loss-of-function mutations in the brain voltage-gated sodium channel gene SCN1A. Heterozygous Scn1a knockout (Scn1a +/−) mice recapitulate the severe epilepsy phenotype of Dravet syndrome and are an accepted animal model. Because clinical observations suggest conventional sodium channel blocking antiepileptic drugs may worsen the disease, we predicted the phenotype of Scn1a +/− mice would be exacerbated by GS967, a potent, unconventional sodium channel blocker. Unexpectedly, GS967 significantly improved survival of Scn1a +/− mice and suppressed spontaneous seizures. By contrast, lamotrigine exacerbated the seizure phenotype. Electrophysiological recordings of acutely dissociated neurons revealed that chronic GS967-treatment had no impact on evoked action potential firing frequency of interneurons, but did suppress aberrant spontaneous firing of pyramidal neurons and was associated with significantly lower sodium current density. Lamotrigine had no effects on neuronal excitability of either neuron subtype. Additionally, chronically GS967-treated Scn1a +/− mice exhibited normalized pyramidal neuron sodium current density and reduced hippocampal NaV1.6 protein levels, whereas lamotrigine treatment had no effect on either pyramidal neuron sodium current or hippocampal NaV1.6 levels. Our findings demonstrate unexpected efficacy of a novel sodium channel blocker in Dravet syndrome and suggest a potential mechanism involving a secondary change in NaV1.6.