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1.  The SLC4 Family of Bicarbonate (HCO3−) Transporters 
Molecular aspects of medicine  2013;34(2-3):159-182.
The SLC4 family consists of ten genes (SLC4A1-5; SLC4A7-11). All encode integral membrane proteins with very similar hydropathy plots—consistent with 10 – 14 transmembrane segments. Nine SLC4 members encode proteins that transport HCO3− (or a related species, such as CO3=) across the plasma membrane. Functionally, eight of these proteins fall into two major groups: three Cl-HCO3 exchangers (AE1 – 3) and five Na+-coupled HCO3− transporters (NBCe1, NBCe2, NBCn1, NBCn2, NDCBE). Two of the Na+ - coupled transporters (NBCe1, NBCe2) are electrogenic; the other three Na+-coupled HCO3− transporters and all three AEs are electroneutral. In addition, two other SLC4 members (AE4, SLC4A9 and BTR1, SLC4A11) do not yet have a firmly established function. Most, though not all, SLC4 members are functionally inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS). SLC4 proteins play important roles many modes of acid-base homeostasis: the carriage of CO2 by erythrocytes, the transport of H+ or HCO3− by several epithelia, as well as the regulation of cell volume and intracellular pH.
PMCID: PMC3605756  PMID: 23506864
SLC4; Bicarbonate; carbonate; chloride; sodium; boron; exchanger; cotransporter
2.  Functional analysis of non-synonymous single nucleotide polymorphisms in human SLC26A9 
Human Mutation  2012;33(8):1275-1284.
Slc26 anion transporters play crucial roles in transepithelial Cl− absorption and HCO3− secretion; Slc26 protein mutations lead to several diseases. Slc26a9 functions as a Cl− channel and electrogenic Cl−-HCO3− exchanger, and can interact with CFTR. Slc26a9(−/−) mice have reduced gastric acid secretion, yet no human disease is currently associated with SLC26A9 coding mutations. Therefore, we tested the function of non-synonymous, coding, single nucleotide polymorphisms (cSNPs) of SLC26A9. Presently, eight cSNPs are NCBI-documented: Y70N, T127N, I384T, R575W, P606L, V622L, V744M and H748R. Using two-electrode voltage-clamp and anion selective electrodes, we measured the biophysical consequences of these cSNPs. Y70N (cytoplasmic N-terminus) displays higher channel activity and enhanced Cl−-HCO3− exchange. T127N (transmembrane) results in smaller halide currents but not for SCN−. V622L (STAS domain) and V744M (STAS adjacent) decreased plasma membrane expression which partially accounts for decreased whole cell currents. Nevertheless, V622L transport is reduced to ~50%. SLC26A9 polymorphisms lead to several function modifications (increased activity, decreased activity, altered protein expression) which could lead to a spectrum of pathophysiologies. Thus, knowing an individual’s SLC26A9 genetics becomes important for understanding disease potentially caused by SLC26A9 mutations or modifying diseases, e.g., cystic fibrosis. Our results also provide a framework to understand SLC26A9 transport modalities and structure-function relationships.
PMCID: PMC3399991  PMID: 22544634
SLC26A9; single nucleotide polymorphisms; voltage clamp; Cl− channel; intracellular pH; Xenopus oocytes
3.  The Thellungiella salsuginea Tonoplast Aquaporin TsTIP1;2 Functions in Protection Against Multiple Abiotic Stresses 
Plant and Cell Physiology  2013;55(1):148-161.
Examination of aquaporin (AQP) membrane channels in extremophile plants may increase our understanding of plant tolerance to high salt, drought or other conditions. Here, we cloned a tonoplast AQP gene (TsTIP1;2) from the halophyte Thellungiella salsuginea and characterized its biological functions. TsTIP1;2 transcripts accumulate to high levels in several organs, increasing in response to multiple external stimuli. Ectopic overexpression of TsTIP1;2 in Arabidopsis significantly increased plant tolerance to drought, salt and oxidative stresses. TsTIP1;2 had water channel activity when expressed in Xenopus oocytes. TsTIP1;2 was also able to conduct H2O2 molecules into yeast cells in response to oxidative stress. TsTIP1;2 was not permeable to Na+ in Xenopus oocytes, but it could facilitate the entry of Na+ ions into plant cell vacuoles by an indirect process under high-salinity conditions. Collectively, these data showed that TsTIP1;2 could mediate the conduction of both H2O and H2O2 across membranes, and may act as a multifunctional contributor to survival of T. salsuginea in highly stressful habitats.
PMCID: PMC3894706  PMID: 24214268
Aquaporin; Channeling activity; Stress tolerance; Thellungiella salsuginea
4.  Ion and solute transport by prestin in Drosophila and Anopheles 
Journal of insect physiology  2012;58(4):563-569.
The gut and Malpighian tubules of insects are the primary sites of active solute and water transport for controlling hemolymph and urine composition, pH, and osmolarity. These processes depend on ATPase (pumps), channels and solute carriers (Slc proteins). Maturation of genomic databases enables us to identify the putative molecular players for these processes. Anion transporters of the Slc4 family, AE1 and NDAE1, have been reported as HCO3− transporters, but are only part of the story. Here we report Dipteran (Drosophila melanogaster (d) and Anopheles gambiae (Ag)) anion exchangers, belonging to the Slc26 family, which are multi-functional anion exchangers. One Drosophila and two Ag homologues of mammalian Slc26a5 (prestin) and Slc26a6 (aka, PAT1, CFEX) were identified and designated dPrestin, AgPrestinA and AgPrestinB. dPrestin and AgPrestinB show electrogenic anion exchange (Cl−/nHCO3−, Cl−/SO42− and Cl−/oxalate2−) in an oocyte expression system. Since these transporters are the only Dipteran Slc26 proteins whose transport is similar to mammalian Slc26a6, we submit that Dipteran Prestin are functional and even molecular orthologues of mammalian Slc26a6. OSR1 kinase increases dPrestin ion transport, implying another set of physiological processes controlled by WNK/SPAK signaling in epithelia. All of these mRNAs are highly expressed in the gut and Malpighian tubules. Dipteran Prestin proteins appear suited for central roles in bicarbonate, sulfate and oxalate metabolism including generating the high pH conditions measured in the Dipteran midgut lumen. Finally, we present and discuss Drosophila genetic models that integrate these processes.
PMCID: PMC3482613  PMID: 22321763
Cl− transport; prestin; gut; Malpighian tubules; Slc26
5.  Primers on Molecular Pathways: Bicarbonate Transport by the Pancreas 
Pancreatology  2011;10(6):660-663.
The pancreas has both endocrine and exocrine functions. As an endocrine organ, stimulation of the pancreatic β-cells results in insulin secretion to control systemic glucose levels. The exocrine function of the pancreas and the need for alkaline pancreatic secretion (pH 8.0–8.5) have been appreciated for more than 40 years. Yet, our knowledge of the cellular mechanisms (signaling, transporters and channels) which accomplish these critical functions has evolved greatly. In the mid-1990s, basolateral Na-bicarbonate (HCO3−) uptake by NBCe1 (Slc4a4) was shown to be critical for the generation of approximately 75% of stimulated HCO3− secretion. In the last 10 years, several new HCO3− transporters in the Slc26 family and their interaction with the cystic fibrosis transmembrane conductance regulator-chloride channel have elucidated the HCO3− exit step at the ductal lumen. Most recently, both IRBIT (inositol 1,4,5-trisphosphate receptor-binding protein) and WNK [with no lysine (K)] kinase have been implicated as additional HCO3− secretory controllers.
PMCID: PMC3068561  PMID: 21242704
Cystic fibrosis transmembrane conductance regulator; SLC26 family; Pancreatic duct
6.  Regulation of Electroneutral NaCl Absorption by the Small Intestine 
Annual review of physiology  2011;73:261-281.
Na+ and Cl− movement across the intestinal epithelium occurs by several interconnected mechanisms: (1) nutrient coupled Na+ absorption; (2) electroneutral NaCl absorption; (3) electrogenic Cl− secretion by CFTR; and (4) electrogenic Na+ absorption by ENaC. All of these transport modes require a favorable electrochemical gradient maintained by the basolateral Na+-K+-ATPase, a Cl− channel and K+ channels. Electroneutral NaCl absorption is observed from the small intestine to distal colon. This transport is mediated by apical Na+/H+ (NHE2/3) and Cl−/HCO3 − (Slc26a3/a6, others) exchangers that provide the major route of NaCl absorption. Electroneutral NaCl absorption and Cl− secretion by CFTR are oppositely regulated by the autonomic nerve system, immune system, and endocrine system via PKAα, PKCα, cGKII, and/or SGK1. This integrated regulation requires the formation of macromolecular complexes, which mediated by NHERF family of scaffold proteins, and involve internalization of NHE3. Using knockout mice and human mutations, a more detailed understanding of the integrated as well as subtle regulation of electroneutral NaCl absorption by the mammalian intestine has emerged.
PMCID: PMC3988668  PMID: 21054167
NHE; SLC26; NHERF; CFTR; neuroendocrine system
7.  Slc26A9 - anion exchanger, channel and Na+ transporter 
The Journal of membrane biology  2009;228(3):125-140.
The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralog expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins – cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl--HCO3- exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO3- secretion; Na+ electrodes and uptakes reveal that Slc26a9 has a cation-dependence. Single channel measurements indicate that Slc26a9 displays discrete open and close states. These experiments show that Slc26a9 has three discrete physiological modes: nCl--HCO3- exchanger, Cl- channel, and Na+-anion cotransporter. Thus, the Slc26a9 transporter-channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.
PMCID: PMC2733867  PMID: 19365592
intracellular pH; Cl-; Na+; HCO3-; Xenopus oocyte expression; epithelial localization
8.  Drosophila Melanogaster as an Emerging Translational Model of Human Nephrolithiasis 
The Journal of urology  2013;190(5):10.1016/j.juro.2013.03.010.
The limitations imposed by human clinical studies and mammalian models of nephrolithiasis have hampered the development of effective medical treatments and preventative measures for decades. The simple but elegant Drosophila melanogaster is emerging as a powerful translational model of human disease, including nephrolithiasis and may provide important information essential to our understanding of stone formation. We present the current state of research using D. melanogaster as a model of human nephrolithiasis.
Materials and Methods
A comprehensive review of the English language literature was performed using PUBMED. When necessary, authoritative texts on relevant subtopics were consulted.
The genetic composition, anatomic structure and physiologic function of Drosophila Malpighian tubules are remarkably similar to those of the human nephron. The direct effects of dietary manipulation, environmental alteration, and genetic variation on stone formation can be observed and quantified in a matter of days. Several Drosophila models of human nephrolithiasis, including genetically linked and environmentally induced stones, have been developed. A model of calcium oxalate stone formation is among the most recent fly models of human nephrolithiasis.
The ability to readily manipulate and quantify stone formation in D. melanogaster models of human nephrolithiasis presents the urologic community with a unique opportunity to increase our understanding of this enigmatic disease.
PMCID: PMC3842186  PMID: 23500641
Drosophila melanogaster; Malpighian tubule; disease model; nephrolithiasis
9.  Slc4-like anion transporters of the larval mosquito alimentary canal 
Journal of Insect Physiology  2012;58(4):551-562.
Mosquito larvae exhibit luminal pH extremes along the axial length of their alimentary canal that range from very alkaline (pH > 10) in the anterior midgut to slightly acid in the hindgut. The principal buffer in the system is thought to be bicarbonate and/or carbonate, because the lumen is known to contain high levels of bicarbonate/carbonate and is surrounded by various epithelial cell types which express a variety of carbonic anhydrases. However, the precise mechanisms responsible for the transport of bicarbonate/carbonate into and out of the lumen are unclear. In the present study, we test the hypothesis that SLC4-like anion transporters play a role in bicarbonate/carbonate accumulation in the larval mosquito alimentary canal. Molecular, physiological and immnuohistochemical characterizations of Slc4-like transporters in the gut of larval mosquitoes (Aedes aegypti and Anopheles gambiae) demonstrate the presence of both a Na+-independent chloride/bicarbonate anion exchanger (AE) as well as a Na+-dependent anion exchanger (NDAE). Notably, immunolocalization experiments in Malpighian tubules show that the two proteins can be located in the same tissue, but to different cell types. Immunolabeling experiments in the gastric caecae show that the two proteins can be found in the same cells, but on opposite sides (basal vs. apical). In summary, our results indicate that the alimentary canal of larval mosquitoes exhibits robust expression of two SLC4-like transporters in locations that are consistent with a role in the regulation of luminal pH. The precise physiological contributions of each transporter remain to be determined.
PMCID: PMC3322255  PMID: 22251674
Anion exchangers; mosquito larvae; alimentary canal; Malpighian tubule; Gastric caeca
10.  Synthesis, biological and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase 
Journal of medicinal chemistry  2011;54(20):7150-7164.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1–3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with L-glutamate diethyl ester, followed by saponification, afforded 1–3. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1, 2 and 4-atom bridge lengths for the activity of this series.
PMCID: PMC3209708  PMID: 21879757
11.  O2-Filled Swimbladder Employs Monocarboxylate Transporters for the Generation of O2 by Lactate-Induced Root Effect Hemoglobin 
PLoS ONE  2012;7(4):e34579.
The swimbladder volume is regulated by O2 transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the “Root effect.” While O2 generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H+/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.
PMCID: PMC3319611  PMID: 22496829
12.  ATP-Sensitive Potassium (KATP) Channel Activation Decreases Intraocular Pressure in the Anterior Chamber of the Eye 
In this study, the authors found that ATP-sensitive potassium channel (KATP) openers increase outflow facility in human anterior segment culture and decrease IOP in Brown Norway rat eyes. The importance of KATP channels to outflow facility and KATP channel openers as a future therapeutic class of ocular hypotensive agents is discussed.
ATP-sensitive potassium channel (KATP) openers target key cellular events, many of which have been implicated in glaucoma. The authors sought to determine whether KATP channel openers influence outflow facility in human anterior segment culture and intraocular pressure (IOP) in vivo.
Anterior segments from human eyes were placed in perfusion organ culture and treated with the KATP channel openers diazoxide, nicorandil, and P1075 or the KATP channel closer glyburide (glibenclamide). The presence, functionality, and specificity of KATP channels were determined by RT-PCR, immunohistochemistry, and inside-out patch clamp in human trabecular meshwork (TM) tissue or primary cultures of normal human trabecular meshwork (NTM) cells. The effect of diazoxide on IOP in anesthetized Brown Norway rats was measured with a rebound tonometer.
KATP channel openers increased outflow facility in human anterior segments (0.14 ± 0.02 to 0.26 ± 0.09 μL/min/mm Hg; P < 0.001) compared with fellow control eyes (0.22 ± 0.11 to 0.21 ± 0.11 μL/min/mm Hg; P > 0.5). The effect was reversible, with outflow facility returning to baseline after drug removal. The addition of glyburide inhibited diazoxide from increasing outflow facility. Electrophysiology confirmed the presence and specificity of functional KATP channels. KATP channel subunits Kir6.1, Kir6.2, SUR2A, and SUR2B were expressed in TM and NTM cells. In vivo, diazoxide significantly lowered IOP in Brown Norway rats.
Functional KATP channels are present in the trabecular meshwork. When activated by KATP channel openers, these channels increase outflow facility through the trabecular outflow pathway in human anterior segment organ culture and decrease IOP in Brown Norway rat eyes.
PMCID: PMC3176023  PMID: 21743021
13.  The MgtC Virulence Factor of Salmonella enterica Serovar Typhimurium Activates Na+,K+-ATPase 
Journal of Bacteriology  2006;188(15):5586-5594.
The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg2+-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg2+ transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H+, Mg2+, or Ca2+, thus ruling out a previously postulated function as a Mg2+/H+ antiporter. However, addition of extracellular K+ markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na+,K+-ATPase completely prevented hyperpolarization and restored the normal K+-induced depolarization response. These results suggested that the Na+,K+-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na+,K+-ATPase. Consistent with the involvement of Na+,K+-ATPase, oocytes expressing MgtC exhibited an increased rate of 86Rb+ uptake and had increased intracellular free [K+] and decreased free [Na+] and ATP. The free concentrations of Mg2+ and Ca2+ and cytosolic pH were unchanged, although the total intracellular Ca2+ content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg2+ or another ion.
PMCID: PMC1540036  PMID: 16855249
14.  Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney  
The Journal of General Physiology  2000;115(5):533-546.
We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.
PMCID: PMC2217225  PMID: 10779312
Xenopus oocytes; intracellular pH; extracellular pH; sulfite; carbonate

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