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1.  The N-terminal cytoplasmic region of NCBE displays features of an intrinsic disordered structure and represents a novel target for specific drug screening 
The sodium dependent bicarbonate transporter NCBE/NBCn2 is predominantly expressed in the central nervous system (CNS). The highest protein concentrations are found in the choroid plexus. The primary function of this integral plasma membrane transport protein is to regulate intracellular neuronal pH and also probably to maintain the pH homeostasis across the blood-cerebrospinal fluid barrier. NCBE is predicted to contain at least 10 transmembrane helices. The N- and C- termini are both cytoplasmic, with a large N-terminal domain (Nt-NCBE) and a relatively small C-terminal domain (Ct-NCBE). The Nt-NCBE is likely to be involved in bicarbonate recognition and transport and contains key areas of regulation involving pH sensing and protein-protein interactions. Intrinsic disordered protein regions (IDPRs) are defined as protein regions having no rigid three-dimensional structure under physiological conditions. They are believed to be involved in signaling networks in which specific, low affinity, protein-protein interactions play an important role. We predict that NCBE and other SoLute Carrier 4 (SLC4) family members have a high level of intrinsic disorder in their cytoplasmic regions. To provide biophysical evidence for the IDPRs predicted in Nt-NCBE, we produced pure (>99%), recombinant Nt-NCBE using E. coli as the expression host. The protein was used to perform differential scanning fluorescence spectroscopy (DSF), in order to search for small molecules that would induce secondary or tertiary structure in the IDPRs. We expect this to assist the development of selective pharmaceutical compounds against individual SLC4 family members. We have also determined a low resolution (4 Å) X-ray crystal structure of the N-terminal core domain. The N-terminal cytoplasmic domain (cdb3) of anion exchanger 1 (AE1) shares a similar fold with the N-terminal core domain of NCBE. Crystallization conditions for the full-length N-terminal domain have been sought, but only the core domain yields diffracting crystals.
doi:10.3389/fphys.2013.00320
PMCID: PMC3819638  PMID: 24223558
SLC4; intrinsic disorder; drug screen; NCBE; IDP; bicarbonate
2.  Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain 
Neuroscience  2007;151(2):374-385.
NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO3− transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting—both of purified peptides representing the initial ~120aa of the transporters and of full-length transporters expressed in Xenopus oocytes—demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of E18 fetal and adult choroid plexus. NCBE protein is present by western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (~150 kDa). PNGase F reduces the MW of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.
doi:10.1016/j.neuroscience.2007.10.015
PMCID: PMC2905792  PMID: 18061361
slc4a10; central nervous system; brain; oocytes; glycosylation
3.  Na+ dependent acid-base transporters in the choroid plexus; insights from slc4 and slc9 gene deletion studies 
The choroid plexus epithelium (CPE) is located in the ventricular system of the brain, where it secretes the majority of the cerebrospinal fluid (CSF) that fills the ventricular system and surrounds the central nervous system. The CPE is a highly vascularized single layer of cuboidal cells with an unsurpassed transepithelial water and solute transport rate. Several members of the slc4a family of bicarbonate transporters are expressed in the CPE. In the basolateral membrane the electroneutral Na+ dependent Cl−/HCO3− exchanger, NCBE (slc4a10) is expressed. In the luminal membrane, the electrogenic Na+:HCO3− cotransporter, NBCe2 (slc4a5) is expressed. The electroneutral Na+:HCO3− cotransporter, NBCn1 (slc4a7), has been located in both membranes. In addition to the bicarbonate transporters, the Na+/H+ exchanger, NHE1 (slc9a1), is located in the luminal membrane of the CPE. Genetically modified mice targeting slc4a2, slc4a5, slc4a7, slc4a10, and slc9a1 have been generated. Deletion of slc4a5, 7 or 10, or slc9a1 has numerous impacts on CP function and structure in these mice. Removal of the transporters affects brain ventricle size (slc4a5 and slc4a10) and intracellular pH regulation (slc4a7 and slc4a10). In some instances, removal of the proteins from the CPE (slc4a5, 7, and 10) causes changes in abundance and localization of non-target transporters known to be involved in pH regulation and CSF secretion. The focus of this review is to combine the insights gathered from these knockout mice to highlight the impact of slc4 gene deletion on the CSF production and intracellular pH regulation resulting from the deletion of slc4a5, 7 and 10, and slc9a1. Furthermore, the review contains a comparison of the described human mutations of these genes to the findings in the knockout studies. Finally, the future perspective of utilizing these proteins as potential targets for the treatment of CSF disorders will be discussed.
doi:10.3389/fphys.2013.00304
PMCID: PMC3804831  PMID: 24155723
cerebrospinal fluid; brain pH; knockout mice; membrane transporters; epithelial physiology
4.  PSD-95 Interacts with NBCn1 and Enhances Channel-like Activity without Affecting Na/HCO3 Cotransport 
Background/Aims
The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction.
Methods: Results
In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1.
Conclusion
Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed.
doi:10.1159/000343332
PMCID: PMC3705566  PMID: 23183381
Bicarbonate transport; Acid-base; pH; Electrophysiology; Neuron; Xenopus
5.  Polarization of membrane associated proteins in the choroid plexus epithelium from normal and slc4a10 knockout mice 
The choroid plexus epithelium (CPE) has served as a model-epithelium for cell polarization and transport studies and plays a crucial role for cerebrospinal fluid (CSF) production. The normal luminal membrane expression of Na+,K+-ATPase, aquaporin-1 and Na+/H+ exchanger 1 in the choroid plexus is severely affected by deletion of the slc4a10 gene that encodes the bicarbonate transporting protein Ncbe/NBCn2. The causes for these deviations from normal epithelial polarization and redistribution following specific gene knockout are unknown, but may be significant for basic epithelial cell biology. Therefore, a more comprehensive analysis of cell polarization in the choroid plexus is warranted. We find that the cytoskeleton in the choroid plexus contains αI-, αII-, βI-, and βII-spectrin isoforms along with the anchoring protein ankyrin-3, most of which are mainly localized in the luminal membrane domain. Furthermore, we find α-adducin localized near the plasma membranes globally, but with only faint expression in the luminal membrane domain. In slc4a10 knockout mice, the abundance of β1 Na+,K+-ATPase subunits in the luminal membrane is markedly reduced. Anion exchanger 2 abundance is increased in slc4a10 knockout and its anchor protein, α-adducin is almost exclusively found near the basolateral domain. The αI- and βI-spectrin abundances are also decreased in the slc4a10 knockout, where the basolateral domain expression of αI-spectrin is exchanged for a strictly luminal domain localization. E-cadherin expression is unchanged in the slc4a10 knockout, while small decreases in abundance are observed for its probable adaptor proteins, the catenins. Interestingly, the abundance of the tight junction protein claudin-2 is significantly reduced in the slc4a10 knockouts, which may critically affect paracellular transport in this epithelium. The observations allow the generation of new hypotheses on basic cell biological paradigms that can be tested experimentally in future studies.
doi:10.3389/fphys.2013.00344
PMCID: PMC3842056  PMID: 24348423
sodium hydrogen exchanger; sodium bicarbonate cotransporter; epithelial polarization; cytoskeleton; choroid plexus; cerebrospinal fluid
6.  Cell-type Specific Distribution of Chloride Transporters in the Rat Suprachiasmatic Nucleus 
Neuroscience  2009;165(4):1519.
The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use γ-aminobutyric acid (GABA) as a neurotransmitter, the principal inhibitory neurotransmitter in the adult central nervous system. Acting via the ionotropic GABAA receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl− influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (for chloride entry) and KCC1-4 (for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cationchloride cotransporters. Previously we reported that the K+/Cl− cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K+/Cl− cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl− levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.
doi:10.1016/j.neuroscience.2009.11.040
PMCID: PMC2815043  PMID: 19932740
circadian rhythms; GABA; KCC2; KCC3; KCC4; NKCC1; WNK3
7.  Exploring the retinal connectome 
Molecular Vision  2011;17:355-379.
Purpose
A connectome is a comprehensive description of synaptic connectivity for a neural domain. Our goal was to produce a connectome data set for the inner plexiform layer of the mammalian retina. This paper describes our first retinal connectome, validates the method, and provides key initial findings.
Methods
We acquired and assembled a 16.5 terabyte connectome data set RC1 for the rabbit retina at ≈2 nm resolution using automated transmission electron microscope imaging, automated mosaicking, and automated volume registration. RC1 represents a column of tissue 0.25 mm in diameter, spanning the inner nuclear, inner plexiform, and ganglion cell layers. To enhance ultrastructural tracing, we included molecular markers for 4-aminobutyrate (GABA), glutamate, glycine, taurine, glutamine, and the in vivo activity marker, 1-amino-4-guanidobutane. This enabled us to distinguish GABAergic and glycinergic amacrine cells; to identify ON bipolar cells coupled to glycinergic cells; and to discriminate different kinds of bipolar, amacrine, and ganglion cells based on their molecular signatures and activity. The data set was explored and annotated with Viking, our multiuser navigation tool. Annotations were exported to additional applications to render cells, visualize network graphs, and query the database.
Results
Exploration of RC1 showed that the 2 nm resolution readily recapitulated well known connections and revealed several new features of retinal organization: (1) The well known AII amacrine cell pathway displayed more complexity than previously reported, with no less than 17 distinct signaling modes, including ribbon synapse inputs from OFF bipolar cells, wide-field ON cone bipolar cells and rod bipolar cells, and extensive input from cone-pathway amacrine cells. (2) The axons of most cone bipolar cells formed a distinct signal integration compartment, with ON cone bipolar cell axonal synapses targeting diverse cell types. Both ON and OFF bipolar cells receive axonal veto synapses. (3) Chains of conventional synapses were very common, with intercalated glycinergic-GABAergic chains and very long chains associated with starburst amacrine cells. Glycinergic amacrine cells clearly play a major role in ON-OFF crossover inhibition. (4) Molecular and excitation mapping clearly segregates ultrastructurally defined bipolar cell groups into different response clusters. (5) Finally, low-resolution electron or optical imaging cannot reliably map synaptic connections by process geometry, as adjacency without synaptic contact is abundant in the retina. Only direct visualization of synapses and gap junctions suffices.
Conclusions
Connectome assembly and analysis using conventional transmission electron microscopy is now practical for network discovery. Our surveys of volume RC1 demonstrate that previously studied systems such as the AII amacrine cell network involve more network motifs than previously known. The AII network, primarily considered a scotopic pathway, clearly derives ribbon synapse input from photopic ON and OFF cone bipolar cell networks and extensive photopic GABAergic amacrine cell inputs. Further, bipolar cells show extensive inputs and outputs along their axons, similar to multistratified nonmammalian bipolar cells. Physiologic evidence of significant ON-OFF channel crossover is strongly supported by our anatomic data, showing alternating glycine-to-GABA paths. Long chains of amacrine cell networks likely arise from homocellular GABAergic synapses between starburst amacrine cells. Deeper analysis of RC1 offers the opportunity for more complete descriptions of specific networks.
PMCID: PMC3036568  PMID: 21311605
8.  Celsr3 Is Required for Normal Development of GABA Circuits in the Inner Retina 
PLoS Genetics  2011;7(8):e1002239.
The identity of the specific molecules required for the process of retinal circuitry formation is largely unknown. Here we report a newly identified zebrafish mutant in which the absence of the atypical cadherin, Celsr3, leads to a specific defect in the development of GABAergic signaling in the inner retina. This mutant lacks an optokinetic response (OKR), the ability to visually track rotating illuminated stripes, and develops a super-normal b-wave in the electroretinogram (ERG). We find that celsr3 mRNA is abundant in the amacrine and ganglion cells of the retina, however its loss does not affect synaptic lamination within the inner plexiform layer (IPL) or amacrine cell number. We localize the ERG defect pharmacologically to a late-stage disruption in GABAergic modulation of ON-bipolar cell pathway and find that the DNQX-sensitive fast b1 component of the ERG is specifically affected in this mutant. Consistently, we find an increase in GABA receptors on mutant ON-bipolar terminals, providing a direct link between the observed physiological changes and alterations in GABA signaling components. Finally, using blastula transplantation, we show that the lack of an OKR is due, at least partially, to Celsr3-mediated defects within the brain. These findings support the previously postulated inner retina origin for the b1 component and reveal a new role for Celsr3 in the normal development of ON visual pathway circuitry in the inner retina.
Author Summary
Visual information is transmitted through the retina from photoreceptors to bipolars to ganglion cells, the output neurons connecting to the brain. This vertical transmission of information is modulated by inhibitory lateral interneurons. Normal vision requires the proper transmission and processing of these neuronal signals. In the inner retina, amacrine cells are the main class of inhibitory interneurons. They modulate the information from bipolar to ganglion cells and are functionally responsible for adjusting image brightness and for detecting motion. Physiological studies have revealed important aspects of the mechanisms of inhibitory modulation, and anatomical studies have identified the many amacrine subclasses and their non-random arrangement within the retina. Although cell–cell interactions are thought to be critical for establishing the important physiological and morphological features of this cell class, the precise molecules and their functions are mostly unknown. In this paper we report the discovery of a mutant that identifies the atypical cell adhesion molecule, Celsr3, as critical for proper development of GABA-signaling pathways in the inner retina.
doi:10.1371/journal.pgen.1002239
PMCID: PMC3154962  PMID: 21852962
9.  Highly conductive carbon nanotube matrix accelerates developmental chloride extrusion in central nervous system neurons by increased expression of chloride transporter, KCC2** 
Small (Weinheim an der Bergstrasse, Germany)  2012;9(7):10.1002/smll.201201994.
Exceptional mechanical and electrical properties of carbon nanotubes (CNT) have attracted neuroscientists and neural tissue engineers aiming to develop novel devices that interface with nervous tissues. In the central nervous system (CNS), the perinatal chloride shift represents a dynamic change that forms the basis for physiological actions of γ-aminobutyric acid (GABA) as inhibitory neurotransmitter, a process of fundamental relevance for normal functioning of the CNS. Low intra-neuronal chloride concentrations are maintained by chloride-extruding transporter, potassium chloride cotransporter 2 (KCC2). KCC2's increasing developmental expression underlies the chloride shift. In neural injury, repressed KCC2 expression plays a co-contributory role by corrupting inhibitory neurotransmission. Mechanisms of Kcc2 up-regulation are thus pertinent because of their medical relevance, yet they remain elusive. Here we show that primary CNS neurons originating from the cerebral cortex, cultured on highly-conductive few-walled-CNT (fwCNT) have a strikingly accelerated chloride shift caused by increased KCC2 expression. KCC2 upregulation is dependent on neuronal voltage-gated calcium channels (VGCC), furthermore on calcium/calmodulin-dependent kinase II, which is linked to VGCC-mediated calcium-influx. We also demonstrate accelerated Kcc2 transcription in brain-slices prepared from genetically-engineered reporter mice, in which Kcc2 promoter drives luciferase, when the cerebral cortex of these mice is exposed to fwCNT-coated devices. Based on these findings, we can now address whether fwCNT can enhance neural engineering devices for the benefit of neural injury conditions associated with elevated neuronal intracellular chloride concentration such as pain, epilepsy, traumatic neural injury and ischemia. Taken together, our novel insights illustrate how fwCNTs can promote low neuronal chloride in individual neurons and thus inhibitory transmission in neural circuits.
doi:10.1002/smll.201201994
PMCID: PMC3822771  PMID: 23229576
few-walled; carbon; nanotube; neuronal; chloride
10.  Anion transport and GABA signaling 
Whereas activation of GABAA receptors by GABA usually results in a hyperpolarizing influx of chloride into the neuron, the reversed chloride driving force in the immature nervous system results in a depolarizing efflux of chloride. This GABAergic depolarization is deemed to be important for the maturation of the neuronal network. The concept of a developmental GABA switch has mainly been derived from in vitro experiments and reliable in vivo evidence is still missing. As GABAA receptors are permeable for both chloride and bicarbonate, the net effect of GABA also critically depends on the distribution of bicarbonate. Whereas chloride can either mediate depolarizing or hyperpolarizing currents, bicarbonate invariably mediates a depolarizing current under physiological conditions. Intracellular bicarbonate is quickly replenished by cytosolic carbonic anhydrases. Intracellular bicarbonate levels also depend on different bicarbonate transporters expressed by neurons. The expression of these proteins is not only developmentally regulated but also differs between cell types and even subcellular regions. In this review we will summarize current knowledge about the role of some of these transporters for brain development and brain function.
doi:10.3389/fncel.2013.00177
PMCID: PMC3807543  PMID: 24187533
GABA; pH; chloride; bicarbonate; ion transporter
11.  Interaction of chloride and bicarbonate transport across the basolateral membrane of rabbit proximal straight tubule. Evidence for sodium coupled chloride/bicarbonate exchange. 
Journal of Clinical Investigation  1988;81(4):1004-1011.
The existence of chloride/bicarbonate exchange across the basolateral membrane and its physiologic significance were examined in rabbit proximal tubules. S2 segments of the proximal straight tubule were perfused in vitro and changes in intracellular pH (pHi) and chloride activity (aCli) were monitored by double-barreled microelectrodes. Total peritubular chloride replacement with gluconate increased pHi by 0.8, and this change was inhibited by a pretreatment with an anion transport inhibitor, SITS. Peritubular bicarbonate reduction increased aCli, and most of this increase was lost when ambient sodium was totally removed. The reduction rates of pHi induced by a peritubular bicarbonate reduction or sodium removal were attenuated by 20% by withdrawal of ambient chloride. SITS application to the bath in the control condition quickly increased pHi, but did not change aCli. However, the aCli slightly decreased in response to SITS when the basolateral bicarbonate efflux was increased by reducing peritubular bicarbonate concentration. It is concluded that sodium coupled chloride/bicarbonate exchange is present in parallel with sodium-bicarbonate cotransport in the basolateral membrane of the rabbit proximal tubule, and it contributes to the basolateral bicarbonate and chloride transport.
PMCID: PMC329624  PMID: 2450891
12.  Functional role of ambient GABA in refining neuronal circuits early in postnatal development 
Early in development, γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the mature brain, depolarizes and excites targeted neurons by an outwardly directed flux of chloride, resulting from the peculiar balance between the cation-chloride importer NKCC1 and the extruder KCC2. The low expression of KCC2 at birth leads to accumulation of chloride inside the cell and to the equilibrium potential for chloride positive respect to the resting membrane potential. GABA exerts its action via synaptic and extrasynaptic GABAA receptors mediating phasic and tonic inhibition, respectively. Here, recent data on the contribution of “ambient” GABA to the refinement of neuronal circuits in the immature brain have been reviewed. In particular, we focus on the hippocampus, where, prior to the formation of conventional synapses, GABA released from growth cones and astrocytes in a calcium- and SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor)-independent way, diffuses away to activate in a paracrine fashion extrasynaptic receptors localized on distal neurons. The transient increase in intracellular calcium following the depolarizing action of GABA leads to inhibition of DNA synthesis and cell proliferation. Tonic GABA exerts also a chemotropic action on cell migration. Later on, when synapses are formed, GABA spilled out from neighboring synapses, acting mainly on extrasynaptic α5, β2, β3, and γ containing GABAA receptor subunits, provides the membrane depolarization necessary for principal cells to reach the window where intrinsic bursts are generated. These are instrumental in triggering calcium transients associated with network-driven giant depolarizing potentials which act as coincident detector signals to enhance synaptic efficacy at emerging GABAergic and glutamatergic synapses.
doi:10.3389/fncir.2013.00136
PMCID: PMC3741556  PMID: 23964205
development; hippocampus; tonic GABAA conductance; network activity; extrasynaptic GABAA receptor
13.  Mice with a Targeted Disruption of the Cl−/HCO3− Exchanger AE3 Display a Reduced Seizure Threshold 
Molecular and Cellular Biology  2006;26(1):182-191.
Neuronal activity results in significant pH shifts in neurons, glia, and interstitial space. Several transport mechanisms are involved in the fine-tuning and regulation of extra- and intracellular pH. The sodium-independent electroneutral anion exchangers (AEs) exchange intracellular bicarbonate for extracellular chloride and thereby lower the intracellular pH. Recently, a significant association was found with the variant Ala867Asp of the anion exchanger AE3, which is predominantly expressed in brain and heart, in a large cohort of patients with idiopathic generalized epilepsy. To analyze a possible involvement of AE3 dysfunction in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy, and neither displayed gross histological and behavioral abnormalities nor spontaneous seizures or spike wave complexes in electrocorticograms. However, the seizure threshold of AE3-knockout mice exposed to bicuculline, pentylenetetrazole, or pilocarpine was reduced, and seizure-induced mortality was significantly increased compared to wild-type littermates. In the pyramidal cell layer of the hippocampal CA3 region, where AE3 is strongly expressed, disruption of AE3 abolished sodium-independent chloride-bicarbonate exchange. These findings strongly support the hypothesis that AE3 modulates seizure susceptibility and, therefore, are of significance for understanding the role of intracellular pH in epilepsy.
doi:10.1128/MCB.26.1.182-191.2006
PMCID: PMC1317631  PMID: 16354689
14.  The Sodium-Driven Chloride/Bicarbonate Exchanger in Presynaptic Terminals 
The Journal of comparative neurology  2012;520(7):10.1002/cne.22806.
The sodium-driven chloride/bicarbonate exchanger (NDCBE), a member of the SLC4 family of bicarbonate transporters, was recently found to modulate excitatory neurotransmission in hippocampus. By using light and electron microscopic immunohistochemistry, we demonstrate here that NDCBE is expressed throughout the adult rat brain, and selectively concentrates in presynaptic terminals, where it is closely associated with synaptic vesicles. NDCBE is in most glutamatergic axon terminals, and is also present in the terminals of parvalbumin-positive γ-aminobutyric acid (GABA)ergic cells. These findings suggest that NDCBE can regulate glutamatergic transmission throughout the brain, and point to a role for NDCBE as a possible regulator of GABAergic neurotransmission.
doi:10.1002/cne.22806
PMCID: PMC3856893  PMID: 22102085
synaptic vesicle; glutamate; GABA; pH; VGLUT; VGAT; SLC4A8
15.  Excitatory Synaptic Inputs to Mouse On-Off Direction-Selective Retinal Ganglion Cells Lack Direction Tuning 
The Journal of Neuroscience  2014;34(11):3976-3981.
Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.
doi:10.1523/JNEUROSCI.5017-13.2014
PMCID: PMC3951696  PMID: 24623775
direction selectivity; glutamate sensor; mouse retina; retinal ganglion cell; synaptic mechanism; two-photon imaging
16.  Neural reprogramming in retinal degenerations 
Purpose
Early visual defects in degenerative diseases such as retinitis pigmentosa (RP) may arise from phased remodeling of the neural retina. We sought to explore the functional expression of ionotropic (iGluR) and group III, type 6 metabotropic (mGluR6) glutamate receptors in late-stage photoreceptor degenerations.
Methods
Excitation mapping with organic cations and computational molecular phenotyping were used to determine whether retinal neurons displayed functional glutamate receptor signaling in rodent models of retinal degenerations and a sample of human RP.
Results
After photoreceptor loss in rodent models of RP, bipolar cells lose mGluR6 and iGluR glutamate-activated currents, while amacrine and ganglion cells retain iGluR-mediated responsivity. Paradoxically, amacrine and ganglion cells show spontaneous iGluR signals in vivo even though bipolar cells lack glutamate-coupled depolarization mechanisms. Cone survival can rescue iGluR expression by OFF bipolar cells. In a case of human RP with cone sparing, iGluR signaling appeared intact, but the numbers of bipolar cells expressing functional iGluRs was double that of normal retina.
Conclusions
RP triggers permanent loss of bipolar cell glutamate receptor expression, though spontaneous iGluR-mediated signaling by amacrine and ganglion cells implies that such truncated bipolar cells still release glutamate in response to some non-glutamatergic depolarization. Focal cone-sparing can preserve iGluR display by nearby bipolar cells, which may facilitate late-RP photoreceptor transplant attempts. An instance of human RP provides evidence that rod bipolar cell dendrite switching likely triggers new gene expression patterns and may impair cone pathway function.
doi:10.1167/iovs.07-0032
PMCID: PMC2408857  PMID: 17591910
17.  Gating of CFTR by the STAS domain of SLC26 transporters 
Nature cell biology  2004;6(4):343-350.
Chloride absorption and bicarbonate secretion are vital functions of epithelia1–6, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR7, which activates electrogenic chloride-bicarbonate exchange by SLC26T8. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.
doi:10.1038/ncb1115
PMCID: PMC3943213  PMID: 15048129
18.  Ion Transport Function of SLC4A11 in Corneal Endothelium 
Purpose.
Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na+ borate and Na+:OH− transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]4−), bicarbonate (HCO3−), or hydroxyl (OH−) anions coupled to Na+.
Methods.
SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na+ ([Na+]i) in response to Ringer solutions with/without B(OH)4− or HCO3− after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11.
Results.
SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)4− (2.5–10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05–0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)4−. However, the rate of B(OH)4−-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)4− did not induce significant changes in resting [Na+i] or the amplitude and rate of acidification caused by Na+ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO2/HCO3−-rich Ringer, Na+-free induced acidification, or the rate of Na+ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na+ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH4+ prepulse was decreased significantly (27%) by SLC4A11 silencing.
Conclusions.
In corneal endothelium, SLC4A11 displays robust Na+-coupled OH− transport, but does not transport B(OH)4− or HCO3−.
SLC4A11 mutations cause corneal endothelial dystrophies, although the function of this putative borate transport protein is unknown. Here we show that SLC4A11 is a Na+-linked pHi regulator that does not transport bicarbonate or borate.
doi:10.1167/iovs.13-11929
PMCID: PMC3691053  PMID: 23745003
SLC4A11; intracellular pH; intracellular Na+; bicarbonate; borate transport
19.  An Allosteric Regulator of R7-RGS Proteins Influences Light-Evoked Activity and Glutamatergic Waves in the Inner Retina 
PLoS ONE  2013;8(12):e82276.
In the outer retina, G protein-coupled receptor (GPCR) signaling mediates phototransduction and synaptic transmission between photoreceptors and ON bipolar cells. In contrast, the functions of modulatory GPCR signaling networks in the inner retina are less well understood. We addressed this question by determining the consequences of augmenting modulatory Gi/o signaling driven by endogenous transmitters. This was done by analyzing the effects of genetically ablating the R7 RGS-binding protein (R7BP), a membrane-targeting protein and positive allosteric modulator of R7-RGS (regulator of the G protein signaling 7) family that deactivates Gi/oα subunits. We found that R7BP is expressed highly in starburst amacrine cells and retinal ganglion cells (RGCs). As indicated by electroretinography and multielectrode array recordings of adult retina, ablation of R7BP preserved outer retina function, but altered the firing rate and latency of ON RGCs driven by rods and cones but not rods alone. In developing retina, R7BP ablation increased the burst duration of glutamatergic waves whereas cholinergic waves were unaffected. This effect on glutamatergic waves did not result in impaired segregation of RGC projections to eye-specific domains of the dorsal lateral geniculate nucleus. R7BP knockout mice exhibited normal spatial contrast sensitivity and visual acuity as assessed by optomotor reflexes. Taken together these findings indicate that R7BP-dependent regulation of R7-RGS proteins shapes specific aspects of light-evoked and spontaneous activity of RGCs in mature and developing retina.
doi:10.1371/journal.pone.0082276
PMCID: PMC3857278  PMID: 24349243
20.  Glycinergic Transmission in the Mammalian Retina 
Glycine and γ-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Glycinergic amacrine cells are small-field amacrine cells with vertically oriented dendrites and comprise more than 10 different morphological types. The retinal distributions of glycine receptor (GlyR) α1, α2, α3 and α4 subtypes have been mapped with subunit-specific antibodies. GlyRs were clustered at postsynaptic hot spots which showed selective distributions for the different subunits. As a rule, only one α subunit was expressed at a given postsynaptic site. The kinetic properties of GlyRs were measured by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from identified retinal neurons in wild-type, Glra1spd-ot, Glra2 and Glra3 knockout mice. From observed differences of sIPSCs in wild-type and mutant mice, the cell-type specific subunit composition of GlyRs could be defined. OFF-cone bipolar cells and A-type ganglion cells receive prominent glycinergic input with fast kinetics that is mainly mediated by α1β GlyRs (decay time constant τ ∼ 5 ms). By contrast, AII amacrine cells express α3β GlyRs with medium fast kinetics (τ ∼ 11 ms). Narrow-field (NF) and wide-field amacrine cells contain predominantly α2β GlyRs with slow kinetics (τ ∼ 27 ms). Lastly, ON-starburst, narrow-field and wide-field amacrine cells in Glra2 knockout mice express α4β GlyRs with very slow kinetics (τ ∼ 70 ms).
doi:10.3389/neuro.02.006.2009
PMCID: PMC2777502  PMID: 19924257
glycine receptors; sIPSCs; retina; synapses; Glra1spd-ot mice; Glra2−/− mice; Glra3−/− mice
21.  Amacrine cell contributions to red-green color opponency in central primate retina: A model study 
Visual Neuroscience  2007;24(4):535-547.
To investigate the contributions of amacrine cells to red-green opponency, a linear computational model of the central macaque retina was developed based on a published cone mosaic. In the model, amacrine cells of ON and OFF types received input from all neighboring midget bipolar cells of the same polarity, but OFF amacrine cells had a bias toward bipolar cells whose center responses were mediated by middle wavelength sensitive cones. This bias might arise due to activity dependent plasticity because there are midget bipolar cells driven by short wavelength sensitive cones in the OFF pathway. The model midget ganglion cells received inputs from neighboring amacrine cells of both types. As in physiological experiments, the model ganglion cells showed spatially opponent responses to achromatic stimuli, but they responded to cone isolating stimuli as though center and surround were each driven by a single cone type. Without amacrine cell input, long and middle wavelength sensitive cones contributed to both the centers and surrounds of model ganglion cell receptive fields. According to the model, the summed amacrine cell input was red-green opponent even though inputs to individual amacrine cells were unselective. A key prediction is that GABA and glycine depolarize two of the four types of central midget ganglion cells; this may reflect lower levels of the potassium chloride co-transporter in their dendrites.
doi:10.1017/S0952523807070502
PMCID: PMC3348784  PMID: 17900377
Midget ganglion cell; Color vision; Parvocellular; Primate; Parafovea
22.  Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts. 
Journal of Clinical Investigation  1989;83(1):227-233.
Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagen-degrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption. Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [Cl-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.
Images
PMCID: PMC303666  PMID: 2910910
23.  Synaptic Elements for GABAergic Feed-Forward Signaling between HII Horizontal Cells and Blue Cone Bipolar Cells Are Enriched beneath Primate S-Cones 
PLoS ONE  2014;9(2):e88963.
The functional roles and synaptic features of horizontal cells in the mammalian retina are still controversial. Evidence exists for feedback signaling from horizontal cells to cones and feed-forward signaling from horizontal cells to bipolar cells, but the details of the latter remain elusive. Here, immunohistochemistry and confocal microscopy were used to analyze the expression patterns of the SNARE protein syntaxin-4, the GABA receptor subunits α1 and ρ, and the cation-chloride cotransporters NKCC and KCC2 in the outer plexiform layer of primate retina. In macaque retina, as observed previously in other species, syntaxin-4 was expressed on dendrites and axon terminals of horizontal cells at cone pedicles and rod spherules. At cones, syntaxin-4 appeared densely clustered in two bands, at horizontal cell dendritic tips and at the level of desmosome-like junctions. Interestingly, in the lower band where horizontal cells may synapse directly onto bipolar cells, syntaxin-4 was highly enriched beneath short-wavelength sensitive (S) cones and colocalized with calbindin, a marker for HII horizontal cells. The enrichment at S-cones was not observed in either mouse or ground squirrel. Furthermore, high amounts of both GABA receptor and cation-chloride cotransporter subunits were found beneath primate S-cones. Finally, while syntaxin-4 was expressed by both HI and HII horizontal cell types, the intense clustering and colocalization with calbindin at S-cones indicated an enhanced expression in HII cells. Taken together, GABA receptors beneath cone pedicles, chloride transporters, and syntaxin-4 are putative constituents of a synaptic set of proteins which would be required for a GABA-mediated feed-forward pathway via horizontal cells carrying signals directly from cones to bipolar cells.
doi:10.1371/journal.pone.0088963
PMCID: PMC3930591  PMID: 24586460
24.  IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells 
PLoS ONE  2014;9(8):e103263.
The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.
doi:10.1371/journal.pone.0103263
PMCID: PMC4139276  PMID: 25141009
25.  Bicarbonate secretion and chloride absorption by rabbit cortical collecting ducts. Role of chloride/bicarbonate exchange. 
Journal of Clinical Investigation  1985;76(3):1123-1130.
Cortical collecting ducts (CCD) from rabbits treated with deoxycorticosterone (DOC) actively secrete bicarbonate at high rates. To investigate the mechanism of bicarbonate secretion, we measured bicarbonate and chloride transport in CCD from rabbits treated with DOC for 9-24 d. Removal of chloride (replaced with gluconate) from both perfusate and bath inhibited bicarbonate secretion without changing transepithelial voltage. Removal of chloride only from the bath increased bicarbonate secretion, while removal of chloride only from the perfusate inhibited secretion. In contrast to the effect of removing chloride, removal of sodium from both the perfusate and bath (replacement with N-methyl-D-glucamine) did not change the rate of bicarbonate secretion. The rate of bicarbonate secretion equaled the rate of chloride absorption in tubules bathed with 0.1 mM ouabain to inhibit any cation-dependent chloride transport. Under these conditions, chloride absorption occurred against an electrochemical gradient. Removal of bicarbonate from both the perfusate and bath inhibited chloride absorption. Removal of bicarbonate only from the bath inhibited chloride absorption, while removal of bicarbonate from the lumen stimulated chloride absorption. We conclude that CCD from DOC-treated rabbits actively secrete bicarbonate and actively absorb chloride by an electroneutral mechanism involving 1:1 chloride/bicarbonate exchange. The process is independent of sodium.
PMCID: PMC424003  PMID: 3930570

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