PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1)

Although the phosphorylation-dependent activation of the Na-K-Cl cotransporter (NKCC1) has been previously well documented, the identity of the kinase(s) responsible for this regulation has proven elusive. Recently, Piechotta et al. (Piechotta, K., Lu, J., and Delpire, E. (2002) J. Biol. Chem. 277, 50812-50819) reported the binding of PASK (also referred as SPAK (STE20/SPS1-related proline-alanine-rich kinase)) and OSR1 (oxidative stress response kinase) to cation-chloride cotransporters KCC3, NKCC1, and NKCC2. In this report, we show that overexpression of a kinase inactive, dominant negative (DN) PASK mutant drastically reduces both shark (60 +/- 5%) and human (80 +/- 3%) NKCC1 activation. Overexpression of wild type PASK causes a small (sNKCC1 22 +/- 8% p < 0.05, hNKCC1 12 +/- 3% p < 0.01) but significant increase in shark and human cotransporter activity in HEK cells. Importantly, DNPASK also inhibits the phosphorylation of two threonines, contained in the previously described N-terminal regulatory domain. We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional cotransporter expression. Coimmunoprecipitation assays confirm PASK binding to NKCC1 in transfected HEK cells and further suggest that this binding is not a regulated event; neither PASK nor NKCC1 activity affects the association. In cells preloaded with 32Pi, the phosphorylation of PASK, but not DNPASK, coincides with that of NKCC1 and increases 5.5 +/- 0.36-fold in low [Cl]e. These data conclusively link PASK with the phosphorylation and activation of NKCC1.     

Regulatory activation is accompanied by movement in the C terminus of the Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC1) is expressed in most vertebrate cells and is crucial in the regulation of cell volume and intracellular chloride concentration. To study the structure and function of NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins at two sites within the C terminus and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Both singly and doubly tagged NKCC1s were appropriately produced, trafficked to the plasma membrane, and exhibited (86)Rb transport activity. When both fluorescent probes were placed within the same C terminus of an NKCC1 transporter, we recorded an 11% FRET decrease upon activation of the transporter. This result clearly demonstrates movement of the C terminus during the regulatory response to phosphorylation of the N terminus. When we introduced CFP and YFP separately in different NKCC1 constructs and cotransfected these in HEK cells, we observed FRET between dimer pairs, and the fractional FRET decrease upon transporter activation was 46%. Quantitatively, this indicates that the largest FRET-signaled movement is between dimer pairs, an observation supported by further experiments in which the doubly tagged construct was cotransfectionally diluted with untagged NKCC1. Our results demonstrate that regulation of NKCC1 is accompanied by a large movement between two positions in the C termini of a dimeric cotransporter. We suggest that the NKCC1 C terminus is involved in transport regulation and that dimerization may play a key structural role in the regulatory process. It is anticipated that when combined with structural information, our findings will provide a model for understanding the conformational changes that bring about NKCC1 regulation.     

Substrates induce conformational changes in human anion exchanger 1 (hAE1) as observed by fluorescence resonance energy transfer

The one-for-one exchange of Cl(-) and HCO(3)(-) ions is catalyzed by human erythrocyte anion exchanger 1 (hAE1) through a ping-pong mechanism whereby the protein exists in two main conformations, with the single anion-binding site exposed at either the cytoplasmic (inner) side (E(i)) or the extracellular side (E(o)), with interconversion between the two states being possible only after anion binding. Steady-state and time-resolved resonance energy transfer (FRET) techniques were used to determine the distance of the binding site for diTBA (bis-(1,3-diethylthiobarbituric acid)trimethine oxonol), a high affinity fluorescent oxonol inhibitor of hAE1, from a benchmark site (probably Lys-430) labeled by external fluorescein maleimide (FM). Using red cell ghost membranes, energy transfer distances were measured in media containing different anions between FM as the donor, covalently attached to one monomer, and diTBA as the acceptor, reversibly bound to the adjacent monomer of a hAE1 dimer. Energy transfer increased significantly in chloride or bicarbonate buffers relative to conditions where no transportable anions were present, that is, in citrate buffer. These differences in transfer efficiencies were interpreted in light of the conformational distributions of hAE1 in various buffers and the possible effects of diTBA itself on the distribution. The analysis indicates that the diTBA binding site comes closer to the FM site by approximately 7 A in chloride buffer as compared to that in citrate (or equivalent changes in diTBA orientation occur) because of the effects of anion binding. This provides the first direct physical evidence for structural changes in hAE1 induced by substrates.     

Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Fo?rster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.     

Loop diuretic and ion-binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of Na-K-Cl cotransporter (NKCC1)

The Na-K-Cl cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition.     

Rare mutations in the human Na-K-Cl cotransporter (NKCC2) associated with lower blood pressure exhibit impaired processing and transport function

The Na-K-Cl cotransporter (NKCC2) is the major salt transport pathway in the thick ascending limb of Henle's loop and is part of the molecular mechanism for blood pressure regulation. Recent screening of ～3,000 members of the Framingham Heart Study identified nine rare independent mutations in the gene encoding NKCC2 (SLC12A1) associated with clinically reduced blood pressure and protection from hypertension (Ji WZ, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State M, Levy D, Lifton RP. Nat Genet 40: 592-599, 2008). To investigate their functional consequences, we introduced the nine mutations in human NKCC2A and examined protein function, expression, localization, regulation, and ion transport kinetics using heterologous expression in Xenopus laevis oocytes and HEK-293 cells. When expressed in oocytes, four of the mutants (T235M, R302W, L505V, and P569H) exhibited reduced transport function compared with wild-type. In HEK-293 cells, the same four mutants exhibited reduced function, and in addition N399S and P1083A had significantly lower activity than wild-type. The two most functionally impaired mutants (R302W and L505V) exhibited dramatically diminished production of complex-glycosylated protein and a decrease in or absence of plasma membrane localization, indicative of a processing defect. All of the functional human (h) NKCC2A variants were regulated by changes in oocyte volume and intracellular chloride in HEK cells, but P254A and N399S exhibited a lower constitutive activity in HEK cells. The P569H mutant exhibited a 50% reduction in sodium affinity compared with wild-type, predicting lower transport activity at lower intratubular salt concentrations, while the P254A mutant exhibited a 35% increase in rubidium affinity. We conclude that defects in NKCC2 processing, transport turnover rate, regulation, and ion affinity contribute to impaired transport function in six of the nine identified mutants, providing support for the predictive approach of Ji et al. to identify functionally important residues by sequence conservation. Such mutations in hNKCC2A are likely to reduce renal salt reabsorption, providing a mechanism for lower blood pressure.     

Intramolecular fluorescence resonance energy transfer (FRET) sensors of the orexin OX1 and OX2 receptors identify slow kinetics of agonist activation

Intramolecular fluorescence resonance energy transfer (FRET) sensors able to detect changes in distance or orientation between the 3rd intracellular loop and C-terminal tail of the human orexin OX(1) and OX(2) G protein-coupled receptors following binding of agonist ligands were produced and expressed stably. These were directed to the plasma membrane and, despite the substantial sequence alterations introduced, in each case were able to elevate [Ca(2+)](i), promote phosphorylation of the ERK1/2 MAP kinases and become internalized effectively upon addition of the native orexin peptides. Detailed characterization of the OX(1) sensor demonstrated that it was activated with rank order of potency orexin A > orexin B > orexin A 16-33, that it bound antagonist ligands with affinity similar to the wild-type receptor, and that mutation of a single residue, D203A, greatly reduced the binding and function of orexin A but not antagonist ligands. Addition of orexin A to individual cells expressing an OX(1) sensor resulted in a time- and concentration-dependent reduction in FRET signal consistent with mass-action and potency/affinity estimates for the peptide. Compared with the response kinetics of a muscarinic M(3) acetylcholine receptor sensor upon addition of agonist, response of the OX(1) and OX(2) sensors to orexin A was slow, consistent with a multistep binding and activation process. Such sensors provide means to assess the kinetics of receptor activation and how this may be altered by mutation and sequence variation of the receptors.     

Mouse mammary epithelial cells express the Na-K-Cl cotransporter,NKCC1: characterization,localization, and involvement in ductal development and morphogenesis

Despite the fact that physiological evidence points to the existence of a functional Na-K-Cl cotransporter in the mammary gland, the molecular identity of this transport process remains unknown. We now show that the Na-K-Cl cotransporter isoform, NKCC1, is expressed in mammary tissue. Developmental profiling revealed that the level of NKCC1 protein was significantly influenced by the stage of mammary gland development, and immunolocalization studies demonstrated that NKCC1 was present on the basolateral membrane of mammary epithelial cells. To examine whether functional NKCC1 is required for mammary epithelial cell development, we used NKCC1 -/- mice. We demonstrate that NKCC1 -/- mammary epithelium exhibited a significant delay in ductal outgrowth and an increase in branching morphogenesis during virgin development. These effects were autonomous to the epithelium as assessed by mammary gland transplantation. Although the absence of NKCC1 had no apparent effect on gross mammary epithelial cell morphology during lactation, pups born to NKCC1 -/- mice failed to thrive. Finally, analysis of NKCC1 protein in mouse models that exhibit defects in mammary gland development demonstrate that high levels of NKCC1 protein are indicative of ductal epithelial cells, and the presence of NKCC1 protein is characteristic of mammary epithelial cell identity.     

DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.

Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET The approaches used in the design of new DNA FRET probes are discussed.     

Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer

The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically regulated by the enzymatic generation of lipid second messengers following phagocyte activation. In the present study, a Cyt b-specific monoclonal antibody (mAb 44.1) was labeled with Cascade Blue (CCB) and used in resonance energy transfer (RET) studies probing the effects of a panel of lipid species on the structure of Cyt b. The binding of CCB-mAb 44.1 to immunoaffinity-purified Cyt b was both highly specific and resulted in significant quenching of the steady state donor fluorescence. Titration of the CCB-mAb 44.1:Cyt b complex with the anionic amphiphile lithium dodecyl sulfate (LDS) resulted in a saturable relaxation of fluorescence quenching due to conformational changes in Cyt b at concentrations of the amphiphile required for maximum rates of superoxide production by Cyt b in cell-free assays. Similar results were observed for the anionic amphiphile arachidonic acid (AA), although no relaxation of fluorescence quenching was observed for arachidonate methyl ester (AA-ME). Saturable relaxation of fluorescence quenching was also observed with the anionic, 18:1 phospholipids phosphatidic acid (DOPA) and phosphatidylserine (DOPS), while no relaxation was observed upon addition of the neutral 18:1 lipids phosphatidylcholine (DOPC), phosphatidylethanolamine (DOPE) or diacylglycerol (DAG) at similar levels. Further examination of a variety of phosphatidic acid (PA) species demonstrated DOPA to both potently induce conformational changes in Cyt b and to cause more dramatic conformational changes than PA species with shorter, saturated acyl chains. The data presented in this study support the hypothesis that second messenger lipids, such as AA and PA, directly bind to flavocytochrome b and modulate conformational states relevant to the activation of superoxide production.     

Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA)

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

HU binding to bent DNA: a fluorescence resonance energy transfer and anisotropy study

HU, an architectural DNA-binding protein, either stabilizes DNA in a bent conformation or induces a bend upon binding to give other proteins access to the DNA. In this study, HU binding affinity for a bent DNA sequence relative to a linear sequence was investigated using fluorescence anisotropy measurements. A static bend was achieved by the introduction of two phased A4T4 tracts in a 20 bp duplex. Binding affinity for 20 bp duplexes containing two phased A-tracts in either a 5'-3' or 3'-5' orientation was found to be almost 10-fold higher than HU binding to a random sequence 20 bp duplex (6.1 vs 0.68 microM(-1)). The fluorescence technique of resonance energy transfer was used to quantitatively determine the static bend of the DNA duplexes and the HU-induced bend. DNA molecules were 5'-end labeled with fluorescein as the donor or rhodamine as the acceptor. From the efficiency of energy transfer, the end-to-end distance of the DNA duplexes was calculated. The end-to-end distance relative to DNA contour length (R/R(C)) yields a bend angle for the A-tract duplex of 45 +/- 7 degrees in the absence of HU and 70 +/- 3 degrees in the presence of HU. The bend angle calculated for the T4A4 tract duplex was 62 +/- 4 degrees after binding two HU dimers. Fluorescence anisotropy measurements reveal that HU binds in a 1:1 stoichiometry to the A4T4 tract duplex but a 2:1 stoichiometry to the T4A4 tract and random sequence duplex. These findings suggest that HU binding and recognition of DNA may be governed by a structural mechanism.     

Structure and conformational changes in the C-terminal domain of the beta2-adrenoceptor: insights from fluorescence resonance energy transfer studies

The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.     

Ligand-mediated conformational changes and positioning of tryptophans in reconstituted human sodium/D-glucose cotransporter1 (hSGLT1) probed by tryptophan fluorescence

Recombinant purified human sodium/D-glucose cotransporter1 (hSGLT1) was reconstituted in a functional form into phospholipid vesicles and its conformational states in the absence and presence of ligands and inhibitors were probed by intrinsic tryptophan fluorescence. In the presence of sodium, sugars increase intrinsic fluorescence (maximum 17%) in a saturable manner in the following order alpha-MDG >D-Glu approximately D-Gal > D-Man >D-All, with no effect of L-Glu. Apparent affinities ranging from 0.65 to 10.4 mM were observed. In addition, D-Glu increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary, the transport inhibitor phlorizin decreased Trps fluorescence in a sodium-dependent manner by 50% with a red shift of 4-6 nm and decreased quencher accessibility, these effects were saturable with a high affinity of 5 microM. Furthermore, the positioning of the tryptophans in the reconstituted transporter was investigated. hSGLT1 Trps fluorescence was reduced by N-bromosuccinimide treatment maximally 25% in membranes and 65% in solution. The fluorescence was also significantly but differently quenched by the lipid-soluble spin labeled probes 5-Doxyl-phosphatidylcholine (40%) and 12-Doxyl-phosphatidylcholine (26%). Depth-calculation using the parallax method suggested a location of Trps at an average depth of 10 angstrom from the center of the bilayer. These studies demonstrate the existence of different conformational states of the membrane-embedded transporter in its glucose-free form, as sodium-glucose-carrier complex and as sodium-phlorizin-carrier complex. They further indicate that most of the Trp residues in hSGLT1 are located in hydrophobic regions of the protein or in contact with the lipid bilayer of the membrane. There, they are located close to the membrane-water interface contributing to the vectorial nature of the transporter.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

Allophycocyanin 1 as a near-infrared fluorescent tracer: isolation, characterization, chemical modification, and use in a homogeneous fluorescence resonance energy transfer system

Allophycocyanin 1 (APC1), isolated from Mastigocladus laminosus, retains the same (alpha-beta)(3) trimeric structure as allophycocyanin (APC), but incorporates a peptide linker in its core leading to a 28% increase in its fluorescence quantum yield compared to APC. Moreover, APC1 exhibits an unexpectedly good stability at very low concentrations, at extreme pHs, or diluted in a low ionic strength medium whereas, under the same conditions, APC dissociates into an (alpha-beta) monomer, indicating that the peptide linker acts as a stabilizer of its trimeric structure. APC1 crosslinking experiments performed using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide gave a high reaction yield (95%) and showed a similar crosslinking process as previously described for APC. Fluorescence quantum yields of crosslinked APC1 or APC decrease by 20% after labeling on antibody or streptavidin. However, quantum yields of the crosslinked APC1 conjugates remain 25% higher than those of crosslinked APC conjugates. Associated with a europium trisbipyridine cryptate as donor, crosslinked APC1 was compared with crosslinked APC as acceptor in homogeneous time resolved fluorescence technology based on a fluorescence resonance energy transfer process. Using crosslinked APC1, assay performances were increased by 20%, showing that APC1 could be considered as a very promising near infrared fluorescent probe to replace APC in its biological applications.     

The residues determining differences in ion affinities among the alternative splice variants F, A, and B of the mammalian renal Na-K-Cl cotransporter (NKCC2)

Three alternatively spliced variants of the renal Na-K-Cl cotransporter (NKCC2) are found in distinct regions of the thick ascending limb of the mammalian kidney; these variants mediate Na(+)K(+)2Cl(-) transport with different ion affinities. Here, we examine the specific residues involved in the variant-specific affinity differences, utilizing a mutagenic approach to change the NKCC2B variant into the A or F variant, with functional expression in Xenopus oocytes. The splice region contains the second transmembrane domain (TM2) and the putative intracellular loop (ICL1) connecting TM2 and TM3. It is found that the B variant is functionally changed to the F variant by replacement of six residues, half of the effect brought about by three TM2 residues and half by three ICL1 residues. The involvement of the ICL1 residues strongly suggests that this region of ICL1 may actually be part of a membrane-embedded domain. Changing six residues is also sufficient to bring about the smaller functional change from the B to the A variant; three residues in TM2 appear to be primarily responsible, two of which correspond to residues involved in the B-to-F changes. A B-variant mutation reported in a mild case of Bartter disease was found to render the cotransporter inactive. These results identify the combination of amino acid variations responsible for the differences among the three splice variants of NKCC2, and they support a model in which a reentrant loop following TM2 contributes to the chloride binding and translocation domains.