scholarly journals SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia

2009 ◽  
Vol 133 (4) ◽  
pp. 421-438 ◽  
Author(s):  
Carol A. Bertrand ◽  
Ruilin Zhang ◽  
Joseph M. Pilewski ◽  
Raymond A. Frizzell
Keyword(s):  
Current Increase ◽  
Chloride Currents ◽  
Hek 293 ◽  
Anion Secretion ◽  
Anion Conductance ◽  
Hek Cells ◽  
Δf508 Cftr ◽  
Cf Transmembrane Conductance Regulator ◽  
Apical Membranes ◽  
In Cells

Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 ± 4%, 50 µM) and exhibited a near-linear current–voltage (I-V) relation during block, while GlyH-101–inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or ΔF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral α-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 ± 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 ± 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with ΔF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A–stimulated conditions, and its activity in HBE cells requires functional CFTR.

scholarly journals Cryptdin-3 induces novel apical conductance(s) in Cl− secretory, including cystic fibrosis, epithelia

2001 ◽  
Vol 280 (2) ◽  
pp. C296-C302 ◽  
Author(s):  
Didier Merlin ◽  
Gang Yue ◽  
Wayne I. Lencer ◽  
Michael E. Selsted ◽  
James L. Madara
Keyword(s):  
Cystic Fibrosis ◽  
Ion Selectivity ◽  
Secretory Cells ◽  
T84 Cells ◽  
Anion Secretion ◽  
Anion Conductance ◽  
Mucosal Surfaces ◽  
Water Secretion ◽  
Cf Transmembrane Conductance Regulator ◽  
Apical Membranes

Opening of anion-conductive pathways in apical membranes of secretory cells lining mucosal surfaces is a critical step in salt and water secretion and, thus, hydration of sites including airway and intestine. In intestine, Paneth cells are positioned at the base of the secretory gland (crypt) and release defensin peptide, in mice termed cryptdins, into the crypt lumen. Because at least some defensins have been shown to form anion-conductive channels in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes. To directly evaluate the possibility of cryptdin-3-mediated apical anion conductance ( G ap), we have utilized amphotericin B to selectively permeabilize basolateral membranes of electrically tight monolayers of polarized human intestinal secretory epithelia (T84 cells), thus isolating the apical membrane for study. Cryptdin-3 induces G ap that is voltage independent (Δ G ap = 1.90 ± 0.60 mS/cm2) and exhibits ion selectivity contrasting to that elicited by forskolin or thapsigargin (for cryptdin-3, Cl− = gluconate; for forskolin and thapsigargin, Cl− ≫ gluconate). We cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of cation and Cl− currents. Cryptdin-3 induces a current in basolaterally permeabilized epithelial monolayers derived from airway cells harboring the ΔF508 mutation of cystic fibrosis (CF; Δ G ap = 0.80 ± 0.06 mS/cm2), demonstrating that cryptdin-3 restores anion secretion in CF cells; this occurs independently of the CF transmembrane conductance regulator channel. These results support the idea that cryptdin-3 may associate with apical membranes of Cl−-secreting epithelia and self-assemble into conducting channels capable of mediating a physiological response.

Functional Comparison of Red Cell KCl Cotransporter Isoforms, KCC1, KCC3, and KCC4.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1245-1245
Author(s):  
Clinton H. Joiner ◽  
Richard Papes ◽  
Scott Crable ◽  
Dao Pan ◽  
David B. Mount
Keyword(s):  
Western Blotting ◽  
Plasma Membranes ◽  
Isotonic Saline ◽  
Flux Rate ◽  
Kinetic Characteristics ◽  
Transport Activity ◽  
Hek 293 ◽  
Hek Cells ◽  
Cellular Dehydration ◽  
In Cells

Abstract The KCl cotransporter mediates volume reduction in normal (AA) reticulocytes, and its abnormal regulation in sickle (SS) reticulocytes contributes to cellular dehydration that facilitates Hb S sickling. mRNA for three KCC genes - KCC1, KCC3, and KCC4 - as well as a splicing isoform, KCC1ex1b, is present in reticulocytes (Exp. Hem.2005; 33:624). Western blotting has demonstrated KCC1 in human RBC membranes (Su et al, AJPhysiol.1999;277:C899) and KCC3 in sheep (Lauf et al, CompBiochemPhysiol2001;130:499); KCC4 protein was found in hRBC membranes by proteomic analysis (Pasini et al, Blood2006;108:791). We confirm here the presence of KCC3 protein in hRBC via western blotting using an antibody to an exon 3 epitope distal to known N-terminal splicing sites. We sought to characterize and compare human KCC isoforms expressed in human cells (HEK 293) and assess their similarity to KCC activity in RBC. cDNAs for human KCC1, KCC1ex1b, KCC3a, and KCC4, with N-terminal c-myc epitope tags were expressed in HEK 293 cells. Stable cell lines were selected by growth in neomycin, and expression monitored by quantitative PCR analysis of the expressed construct and other endogenous isoforms and by western blotting (anti-myc) of plasma membranes. KCC activity was measured as N-ethylmaleimide(NEM)-stimulated, Cl-dependent Rb uptake in cells grown to 75–90 % confluency. Cells were incubated at 37°C in isotonic saline media with various concentrations of Rb (Na replacement), plus 0.1 mM ouabain and 0.01 mM bumetanide. At 2 and 4 min cells were washed with iced Rb-free media, then lysed and assayed for Rb by flame emission, normalized to sample protein. Flux rates were calculated from Rb uptake at 2 and 4 min. The flux rate in Cl-free sulfamate media was subtracted from that in Cl-media to yield the Cl-dependent flux rate. Wild-type HEK cells showed no increase in Cl-dependent Rb influx when exposed to 1 mM NEM, but Cl-dependent, NEM-stimulated Rb uptake was apparent in cells expressing KCC isoforms. All isoforms were stimulated by hypotonic conditions (75 mOsm), but relative to NEM-stimulated activity, KCC3 was most responsive. Kinetic characteristics of Cl-dependent, NEM-stimulated Rb influx in HEK cell expressing human KCC1, KCC3a, and KCC4 isoforms isoforms are given below, compared to RBC KCC fluxes. A splice variant, KCC1ex1b, coding for a protein with a truncated N-terminus, exhibited 48 ± 7% of the activity of the full-length KCC1 isoform, but did not alter transport activity of coexpressed KCC3a. Thus, KCC isoforms expressed in HEK cells share some, but not all, characteristics with RBC KCC fluxes. The isoform kinetics also differ from those previously published in xenopus oocyte expression systems, suggesting that the membrane milieu in which KCC proteins are expressed may influence their functional characteristics. Likewise, KCC activity in RBC may represent an average of several transporters operating in parallel. The truncated KCC1ex1b isoform, expressed at higher levels in normal than in sickle reticulocytes, has lower activity than KCC1. KCC3 and KCC4 exhibit more robust transport activity than KCC1 and may mediate a substantial part of KCC activity in RBC. KCC Kinetic Characteristics VMax (μmol/mgprot./min) Km (ext. Rb, mM) Anion Selectivity hKCC1 28.9 ± 5.6 16.7 ± 5.4 Cl > Br > I > SCN hKCC3a 107 ± 45 11.0 ± 7.1 Cl = Br > I > SCN hKCC4 206 ± 16 15.6 ± 8.2 Cl > Br >> I = SCN RBC NA 12 - 20 Cl = Br >> I = SCN

Effect of Ag+ on isolated bullfrog gastric mucosa

1985 ◽  
Vol 248 (4) ◽  
pp. G443-G449 ◽  
Author(s):  
P. K. Rangachari ◽  
J. Matthews
Keyword(s):  
Gastric Mucosa ◽  
Potential Difference ◽  
Anion Conductance ◽  
Luminal Side ◽  
Apical Membranes ◽  
Nitrate Solutions

In nitrate solutions, Ag+ added to the luminal side had marked effects on transmucosal conductance and potential difference (PD). Conductance increased quickly (85% within 60 s, 420% by 10 min); PD increased initially (11% within 30 s) and then fell precipitously (58% decreased within 2 min, 85% decreased by 10 min). During this period, no increase in mannitol permeability was found. These changes were essentially similar in histamine-stimulated, spontaneously secreting, and metiamide-inhibited fundic mucosae. Replacement of luminal Na+ by choline had no effect on the changes observed. Similar changes occurred also in the antrum. In SO2-4 media the increases in conductance occurred more slowly (40% within 2 min, 150% after 10 min); PD increased initially for 4-6 min and then slowly declined over 60 min to 74% of control values. After Ag+ treatment, replacement of luminal SO2-4 by nitrate led to an inversion of the PD by up to 20 mV (serosa -ve). Brief exposure of the mucosa (2 min) to Ag+ did not show any obvious damage, although surface cells were damaged following more prolonged exposures. After a 4-min treatment with Ag+, electrical and secretory parameters showed substantial recoveries. Ag+ appears to increase anion conductance; these effects appear to occur on the apical membranes of tubular and/or surface cells of the fundus and antrum.

scholarly journals The expression of wild-type pendrin (SLC26A4) in human embryonic kidney (HEK 293 Phoenix) cells leads to the activation of cationic currents

2005 ◽  
Vol 153 (5) ◽  
pp. 693-699 ◽  
Author(s):  
Silvia Dossena ◽  
Antonella Maccagni ◽  
Valeria Vezzoli ◽  
Claudia Bazzini ◽  
Maria Lisa Garavaglia ◽  
...  
Keyword(s):  
Chloride Transport ◽  
Cellular Systems ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Pendred Syndrome ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Hek 293 ◽  
Prelingual Deafness ◽  
Open Question

Objective: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. Methods: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. Results: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. Conclusion: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.

Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Daniel C Villela ◽  
Anke Teichmann ◽  
Sebastian Kirsch ◽  
Maibritt Mardahl ◽  
Lisa M Münter ◽  
...  
Keyword(s):  
Cross Correlation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Expression Vectors ◽  
Physical Interaction ◽  
At2 Receptor ◽  
Hek 293 ◽  
Fret Efficiency ◽  
In Cells

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist. HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS. FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules. Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor. Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%). FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p<0.0001). Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.

scholarly journals Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Karen N. Barnard ◽  
Brian R. Wasik ◽  
Justin R. LaClair ◽  
David W. Buchholz ◽  
Wendy S. Weichert ◽  
...  
Keyword(s):  
Cell Lines ◽  
Influenza A ◽  
A549 Cells ◽  
Sialic Acids ◽  
Influenza Viruses ◽  
Obvious Effect ◽  
Hek 293 ◽  
Chemically Modified ◽  
Modified Forms ◽  
In Cells

ABSTRACT Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection. IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.

Splice variants of a ClC-2 chloride channel with differing functional characteristics

2000 ◽  
Vol 279 (4) ◽  
pp. C1198-C1210 ◽  
Author(s):  
L. Pablo Cid ◽  
María-Isabel Niemeyer ◽  
Alfredo Ramírez ◽  
Francisco V. Sepúlveda
Keyword(s):  
Splice Variants ◽  
Donor Site ◽  
Proximal Colon ◽  
Site Directed Mutagenesis ◽  
Intestinal Epithelial ◽  
Chloride Currents ◽  
Hek 293 ◽  
Internal Donor ◽  
293 Cells ◽  
Functional Consequences

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH2 terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Δ77–86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Δ77–86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Δ77–86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH2-terminal domain could have functional consequences in tissues where their relative expression levels are different.

Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells

2001 ◽  
Vol 281 (5) ◽  
pp. G1301-G1308 ◽  
Author(s):  
Tracy Greeley ◽  
Holli Shumaker ◽  
Zhaohui Wang ◽  
Clifford W. Schweinfest ◽  
Manoocher Soleimani
Keyword(s):  
Pancreatic Duct ◽  
Northern Hybridization ◽  
Transmembrane Conductance Regulator ◽  
Duct Cells ◽  
Anion Transporter ◽  
Pancreatic Duct Cells ◽  
Cf Transmembrane Conductance Regulator ◽  
Immunohistochemical Studies ◽  
Exchange Activity ◽  
In Cells

The mechanism of the pancreatic ductal HCO[Formula: see text] secretion defect in cystic fibrosis (CF) is not well defined. However, a lack of apical Cl−/HCO[Formula: see text] exchange may exist in CF. To test this hypothesis, we examined the expression of Cl−/HCO[Formula: see text] exchangers in cultured pancreatic duct epithelial cells with physiological features prototypical of CF [CFPAC-1 cells lacking a functional CF transmembrane conductance regulator (CFTR)] or normal duct cells (CFPAC-1 cells transfected with functional wild-type CFTR, CFPAC-WT). Cl−/HCO[Formula: see text] exchange activity, assayed with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in cells grown on coverslips, increased about twofold in cells transfected with functional CFTR. This correlated with increased apical 36Cl influx in cells expressing functional CFTR and grown on permeable support. Northern hybridizations indicated the induction of downregulated in adenoma (DRA) in cells expressing functional CFTR. The expression of putative anion transporter PAT1 also increased significantly in cells expressing functional CFTR. DRA was detected at high levels in native mouse pancreas by Northern hybridization and localized to the apical domain of the duct cells by immunohistochemical studies. In conclusion, CFTR upregulates DRA and PAT1 expression in cultured pancreatic duct cells. We propose that the pancreatic HCO[Formula: see text] secretion defect in CF patients is partly due to the downregulation of apical Cl−/HCO[Formula: see text] exchange activity mediated by DRA (and possibly PAT1).

scholarly journals Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

1999 ◽  
Vol 276 (2) ◽  
pp. C328-C336 ◽  
Author(s):  
Christopher M. Gillen ◽  
Bliss Forbush
Keyword(s):  
Cell Swelling ◽  
Primary Role ◽  
Functional Interaction ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Hek Cells ◽  
293 Cells ◽  
Hypotonic Cell Swelling ◽  
The Relationship ◽  
Fold Activation

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

Structural and Functional Interactions of KCl Cotransport Proteins KCC1 and KCC3 in Sickle and Normal Erythrocyte Membranes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2474-2474
Author(s):  
Mary Risinger ◽  
Jesse Rinehart ◽  
Scott Crable ◽  
Anna Ottlinger ◽  
Richard Winkelmann ◽  
...  
Keyword(s):  
Western Blot ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Erythrocyte Membranes ◽  
Western Blots ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
In Cells

Abstract The KCl cotransporter (KCC) mediates volume reduction in normal reticulocytes and exaggerated KCC activity in sickle red blood cells (SS RBC) (Joiner et al, Blood109:1728, 2007) contributes to pathological dehydration that potentiates sickling. Three separate genes (KCC1, KCC3, KCC4) are expressed in RBC (Crable et al, Exp. Hem.33:624, 2005). KCC1 and KCC3 proteins have been shown to interact in ex vivo expression systems (Simard et al, JBC282(25):18083, 2007), and co-expression of an N-terminal truncation of KCC1 reduces KCC activity mediated by full-length KCC1 or KCC3 (Casula et al. JBC276:41870, 2001), suggesting functional interaction. We show here via western blot analysis that SS RBC membranes contain more KCC1 protein (relative to KCC3) than AA RBC, independent of the reticulocytosis of sickle blood. Immunoprecipitation of solubilized SS RBC membranes with KCC3-specific antibody yielded a band at 125 kD on SDS PAGE which contained KCC1, as identified by western blotting with KCC1-specific antibody and by TOF mass spectroscopy. The effect of co-expression of KCC1 and KCC3 on KCC activity was assessed by measuring NEM-stimulated, Cl-dependent, (ouabain + bumetanide)-insensitive Rb uptake in HEK 293 cells. The Flip-In T-rex HEK 293 cell line (Invitrogen) containing a tetracycline-response promoter was transfected with a pcDNA5a plasmid containing KCC3a cDNA. Recombination of the plasmid with the integrated tet-promoter construct inserts the KCC3a gene under control of a tetracycline-responsive promoter. These cells were subsequently transduced with a retroviral vector (SF-91. Hildinger et at, Gene Ther. 5:1575, 1998) containing KCC1 cDNA linked to a GFP cassette. Control cells contained SF-91 vector lacking KCC1. Cells were selected for GFP expression and grown in the absence (un-induced, no KCC3a expression) or presence of tetracycline (induced, KCC3a expression). From this binary matrix, four types of cells were obtained: Cells with no additional KCC expression, representing endogenous KCC activity; cells with only KCC1 or KCC3a expression; cells with both KCC1 and KCC3a expression. Western blots indicated similar KCC1 expression in cells with KCC1 only and [KCC1 + KCC3] and similar KCC3 expression in cells with KCC3 only and [KCC1 + KCC3]. Thus, the expression of neither isoform was affected by the presence of the other. KCC activity in cells overexpressing KCC1 only was similar to endogenous activity in HEK 293 cells; i.e., transport activity of KCC1 alone was minimal. Cells overexpressing KCC3 only had a 5-fold increase in KCC activity over endogenous levels. When KCC1 was co-expressed with KCC3 in [KCC1 + KCC3] cells, an additional 50% increase in KCC activity was observed (p &lt; 0.05 by paired t-test, N=4), despite similar levels of KCC3 expression by western blot analysis. This synergistic effect was dependent on the cytoplasmic N-terminus of KCC1, as it was not seen when the first 39 amino acids of KCC1 were removed. Interestingly, removal of the entire cytoplasmic N-terminal domain (117 aa) produced an inhibitory effect when co-expressed with KCC3a in HEK cells, as previously reported in Xenopus oocytes (Casula et al.). These data indicate that KCC1 and KCC3 interact structurally and functionally in RBC membranes, and provide another potential mechanism for regulation of KCC activity via multimeric associations between KCC isoforms. Thus, KCC activity could be modulated not only by transcriptional mechanisms and post-translational modification (phosphorylation), but also by altering the ratios of KCC isoforms or the kinetics of their association. We speculate that higher levels of KCC1 protein relative to KCC3 in SS RBC membranes could account for higher KCC activity in these cells relative to AA RBC.

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