Exon loss accounts for differential sorting of Na-K-Cl cotransporters in polarized epithelial cells

The renal Na-K-Cl cotransporter (NKCC2) is selectively expressed in the apical membranes of cells of the mammalian kidney, where it is the target of the clinically important loop diuretics. In contrast, the “secretory” NKCC1 cotransporter is localized in the basolateral membranes of many epithelia. To identify the sorting signal(s) that direct trafficking of NKCCs, we generated chimeras between the two isoforms and expressed these constructs in polarized renal epithelial cell lines. This analysis revealed an amino acid stretch in NKCC2 containing apical sorting information. The NKCC1 C terminus contains a dileucine motif that constitutes the smallest essential component of its basolateral sorting signal. NKCC1 lacking this motif behaves as an apical protein. Examination of the NKCC gene structure reveals that this dileucine motif is encoded by an additional exon in NKCC1 absent in NKCC2. Phylogenetic analysis of this exon suggests that the evolutionary loss of this exon from the gene encoding the basolateral NKCC1 constitutes a novel mechanism that accounts for the apical sorting of the protein encoded by the NKCC2 gene.     

Functional comparison of the K+-Cl- cotransporters KCC1 and KCC4

The K(+)-Cl(-) cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K(+), with much higher inhibition in 50 mm K(+) versus 2 mm K(+). KCC4 is also uniquely sensitive to 10 mm barium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K(+) (K(m) values of approximately 25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl(-), they differ in the anion selectivity of K(+) transport (Cl(-) > SCN(-) = Br(-) > PO(4)(-3) > I(-) for KCC1 and Cl(-) > Br(-) > PO(4)(-3) = I(-) > SCN(-) for KCC4). Both KCCs express minimal K(+)-Cl(-) cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agent N-ethylmaleimide activates K(+)-Cl(-) cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K(+)-Cl(-) cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.     

3H]bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters

Vascular endothelial cells previously have been shown to possess a prominent Na-K-Cl cotransport system which mediates a K+ influx of approximately 20 mumols/g of protein/min. Endothelial cell cotransport has also been shown to be regulated by a variety of vasoactive agents and their second messengers, suggesting that the transport system may have an important role in endothelial cell function. In the present study we investigated the possibility that the high level of cotransport in these cells is due to a large number of Na-K-Cl cotransporters in the plasma membrane. This was done by evaluating specific saturable binding of [3H]bumetanide to cultured bovine aortic endothelial cells. We found a maximal [3H]bumetanide binding of 0.83 pmol/mg protein with a dissociation constant of 0.13 microM. From these data, the number of [3H]bumetanide binding sites/endothelial cell was determined to be approximately 230,000, and the turnover number for cotransport activity was calculated to be 300 K+ ions/site/s. These findings indicate that endothelial cells do indeed exhibit a large number of Na-K-Cl cotransporters/cell relative to other cell types. We also investigated the effects on [3H]bumetanide binding of agents known to modulate Na-K-Cl cotransport activity. Saturable binding of [3H]bumetanide was found to be reduced significantly by treatment of the cells with 8-bromo-cyclic AMP, 8-bromo-cyclic GMP, phorbol esters, norepinephrine, or rat atriopeptin III, all of which have been shown to inhibit Na-K-Cl cotransport-mediated K+ influx.     

Phylogenetic,Structural and Functional Characteristics of the Na-K-Cl Cotransporter Family

Summary Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane α-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggests that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom. Received: 27 July 1995/Revised: 8 November 1995     

Molecular evidence for two renal Na+/glucose cotransporters.

Previous studies have shown that two kinetically and genetically distinct Na+/glucose cotransporters exist in mammalian kidney. We have recently cloned and sequenced one of the rabbit renal Na+/glucose cotransporters (SGLT1) and have found that it is identical in sequence to the intestinal Na+/glucose cotransporter. Northern blots showed that SGLT1 mRNA was found predominantly in the outer medulla of rabbit kidney. Injection of mRNA from outer medulla and outer cortex into Xenopus oocytes resulted in equal expression of Na(+)-dependent sugar uptake, indicating that the outer cortex sample contained mRNA encoding both SGLT1 and a second Na+/glucose cotransporter. Western blots using antipeptide antibodies against SGLT1 showed that the SGLT1 protein is more abundant in outer medulla than outer cortex. However, brush border membrane vesicles prepared from outer cortex had a greater capacity for Na(+)-dependent glucose transport, indicating the presence of a second transporter in the vesicles from outer cortex. It appears that the cloned renal Na+/glucose cotransporter, SGLT1, is the 'high affinity, low capacity' transporter found predominantly in outer medulla. There is evidence that a second transporter, the 'low affinity, high capacity' transporter, is in outer cortex. Finally, the cDNA and protein sequences of the two renal Na+/glucose cotransporters are predicted to differ by more than 20%.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

Detection of distant evolutionary relationships between protein families using theory of sequence profile-profile comparison

Background  

Detection of common evolutionary origin (homology) is a primary means of inferring protein structure and function. At present, comparison of protein families represented as sequence profiles is arguably the most effective homology detection strategy. However, finding the best way to represent evolutionary information of a protein sequence family in the profile, to compare profiles and to estimate the biological significance of such comparisons, remains an active area of research.     

A comparison of programmed cell death between species

Key components of the programmed cell death pathway are conserved between Caenorhabditis elegans, Drosophila melanogaster and humans. The search for additional homologs has been facilitated by the availability of the entire genomic sequence for each of these organisms.     

Horizontal transfer of Wolbachia between phylogenetically distant insect species by a naturally occurring mechanism

Wolbachia is a genus of alpha-proteobacteria found in obligate intracellular association with a wide variety of arthropods, including an estimated 10-20% of all insect species [1]. Wolbachia represents one of a number of recently identified 'reproductive parasites' [2] which manipulate the reproduction of their hosts in ways that enhance their own transmission [3] [4] [5] [6] [7] [8] [9]. The influence of Wolbachia infection on the dynamics of host populations has focused considerable interest on its possible role in speciation through reproductive isolation [3] [10] [11] and as an agent of biological control [2] [12] [13]. Although Wolbachia normally undergoes vertical transmission through the maternal line of its host population [14], there is compelling evidence from molecular phylogenies that extensive horizontal (intertaxon) transmission must have occurred [1] [9] [15] [16] [17]. Some of the best candidate vectors for the horizontal transmission of Wolbachia are insect parasitoids [15], which comprise around 25% of all insect species and attack arthropods from an enormous range of taxa [18]. In this study, we used both fluorescence microscopy and PCR amplification with Wolbachia-specific primers to show that Wolbachia can be transmitted to a parasitic wasp (Leptopilina boulardi) from its infected host (Drosophila simulans) and subsequently undergo diminishing vertical transmission in this novel host species. These results are, to our knowledge, the first to reveal a natural horizontal transfer route for Wolbachia between phylogenetically distant insect species.     

Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na：1K：2C1（乌贼轴突中是2Na：1K：3C1）,其活性被布美他尼（bumetanide）和呋塞米（furosemide）所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体：NKCCl和NKCC2。NKCCl存在于多个组织中,合有NKCCl的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密斑的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用（尤其是NKCCl亚基）是通过特定的激酶使该转运蛋白自身发生氧化／硝化、磷酸化／去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

Clustering orthologous proteins across phylogenetically distant species

The quality of orthologous protein clusters (OPCs) is largely dependent on the results of the reciprocal BLAST (basic local alignment search tool) hits among genomes. The BLAST algorithm is very efficient and fast, but it is very difficult to get optimal solution among phylogenetically distant species because the genomes with large evolutionary distance typically have low similarity in their protein sequences. To reduce the false positives in the OPCs, thresholding is often employed on the BLAST scores. However, the thresholding also eliminates large numbers of true positives as the orthologs from distant species likely have low BLAST scores. To rectify this problem, we introduce a new hybrid method combining the Recursive and the Markov CLuster (MCL) algorithms without using the BLAST thresholding. In the first step, we use InParanoid to produce n(n-1)/2 ortholog tables from n genomes. After combining all the tables into one, our clustering algorithm clusters ortholog pairs recursively in the table. Then, our method employs MCL algorithm to compute the clusters and refines the clusters by adjusting the inflation factor. We tested our method using six different genomes and evaluated the results by comparing against Kegg Orthology (KO) OPCs, which are generated from manually curated pathways. To quantify the accuracy of the results, we introduced a new intuitive similarity measure based on our Least-move algorithm that computes the consistency between two OPCs. We compared the resulting OPCs with the KO OPCs using this measure. We also evaluated the performance of our method using InParanoid as the baseline approach. The experimental results show that, at the inflation factor 1.3, we produced 54% more orthologs than InParanoid sacrificing a little less accuracy (1.7% less) than InParanoid, and at the factor 1.4, produced not only 15% more orthologs than InParanoid but also a higher accuracy (1.4% more) than InParanoid.     

Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na:1K:2Cl(乌贼轴突中是2Na:1 K:3Cl),其活性被布美他尼(bumetanide)和呋塞米(furosemide)所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体:NKCC1和NKCC2。NKCC1存在于多个组织中,含有NKCC1的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密班的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用(尤其是NKCC1亚基)是通过特定的激酶使该转运蛋白自身发生氧化/硝化、磷酸化/去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

A multi-environment comparison of senescence between sister species of Daphnia

Senescence is a general decline of physiological state that accompanies advancing age. It affects nearly all organisms, but patterns of senescence vary markedly, even among closely related taxa. Understanding the evolution of this diversity requires information about environmental effects on the expression of variation among taxa. I examined genetically-based variation of senescence within and between two species complexes of Daphnia in four environments. The environments were defined by large differences in food and temperature, two factors known to influence senescence. The species studied were chosen to represent sister species that likely experience divergent (D. pulex and D. pulicaria) or similar (D. mendotate and D. dentifera) selection pressures on senescence. Overall, D. pulex expressed the greatest demographic senescence, D. mendotae and D. dentifera were intermediate, and D. pulicaria expressed the least. In environments representative of typical natural conditions, D. pulex had greater senescence than D. pulicaria, regardless of how late-life performance was assessed. This shows that genetic-environment interactions do not confound the interpretation of senescence differences between these species as the result of selective differences between their habitats. Comparison of D. mendotae and D. dentifera primarily revealed similar life histories, although differences in reproductive declines occurred in some environments. The joint observation of similar mortality patterns but dissimilar fecundity declines suggests that the trade-off between survival and reproduction changes with age. This calls into question the utility of only studying mortality for understanding evolutionary change of senescence in nature.     

Glucose transport by human renal Na+/D-glucose cotransporters SGLT1 and SGLT2

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s?1) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.     

Functional morphology of bushcricket ears: comparison between two species belonging to the Phaneropterinae and Decticinae (Insecta,Ensifera)

Summary The morphology of the complex tibial organs in the forelegs of two bushcricket species belonging to the Phaneropterinae and Decticinae (Tettigoniidae) is described comparatively. In both species the tibial organs are made up of the subgenual organ, the intermediate organ and the crista acustica; the latter are parts of the tympanal organs and serve as auditory receptors. The very thin tympana in the forelegs ofPholidoptera griseoaptera (Decticinae) are protected by tympanal covers whereas inLeptophyes punctatissima (Phaneropterinae) the tympana are thicker and fully exposed. The overall auditory sensitivity ofL. punctatissima is lower and the sensitivity maximum of the hearing threshold lies at higher frequencies compared toP. griseoaptera. The number of scolopidia in the three scolopale organs and the dimensions of parts of the sound conducting system differs in the two species. In the crista acustica ofL. punctatissima a higher number of scolopidia is distributed in a smaller range than inP. griseoaptera; the scolopidia are especially concentrated in the distal part. Morphometrical analyses indicate that the dimensions of the spiracles, the acoustic trachea and the tympana determine the overall auditory sensitivity and that the arrangement of the scolopidia and the dimensions of structures in the crista acustica affect the frequency tuning of the hearing threshold.