Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells

Carmosino, Monica; Gíménez, Ignacio; Caplan, Michael J.; Forbush, Bliss

doi:10.1091/mbc.e08-05-0478

Cited by 75 publications

(82 citation statements)

References 42 publications

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“…This variant is also present in mouse, in which both transcripts are expressed in the brain and the longer transcript also in the cochlea (Dixon et al, 1999;Randall et al, 1997), and in human, in which the ratio of the two transcripts varies between expressing tissues (Vibat et al, 2001). In the mammalian gene, the sequence encoded by exon 21 has been proposed to include a conserved basolateral sorting motif that might direct the correct subcellular localisation of the protein in vitro (Carmosino et al, 2008). We are unable to confirm whether this is the case in zebrafish, as we cannot discern which spliceoform is expressed in the ear.…”

Section: Zebrafish Nkcc1 Splice Variants Correspond To Those In the Mmentioning

confidence: 81%

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Abbas

Whitfield

2009

Development

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Endolymph is the specialised extracellular fluid present inside the inner ear. In mammals, disruptions to endolymph homeostasis can result in either collapse or distension of the endolymphatic compartment in the cochlea, with concomitant hearing loss. The zebrafish little ears (lte) mutant shows a collapse of the otic vesicle in the larva, apparently owing to a loss of endolymphatic fluid in the ear, together with an over-inflation of the swim bladder. Mutant larvae display signs of abnormal vestibular function by circling and swimming upside down. The two available alleles of lte are homozygous lethal: mutant larvae fail to thrive beyond 6 days postfertilisation. Patterning of the otic vesicle is apparently normal. However, the expression of several genes thought to play a role in endolymph production is downregulated, including the sodium-potassium-chloride cotransporter gene nkcc1 (slc12a2) and several Na + /K + -ATPase channel subunit genes. We show here that lte mutations correspond to lesions in nkcc1. Each allele has a point mutation that disrupts splicing, leading to frame shifts in the coding region that predict the generation of truncated products. Endolymph collapse in the lte/nkcc1 mutant shows distinct parallels to that seen in mouse Nkcc1 mutants, validating zebrafish as a model for the study of endolymph disorders. The collapse in ear volume can be ameliorated in the to27d allele of lte by injection of a morpholino that blocks splicing at an ectopic site introduced by the mutation. This exemplifies the use of morpholinos as potential therapeutic agents for genetic disease.

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Section: Zebrafish Nkcc1 Splice Variants Correspond To Those In the Mmentioning

confidence: 81%

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Abbas

Whitfield

2009

Development

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“…SPAK and oxidative stress response kinase were found to interact with the phosphoacceptor site of NKCC isoforms (7,27,32,34). Apical trafficking and surface expression of NKCC2 have further been established upon application of AVP (13,25,44), and regulatory domains of NKCC2 have been identified (4,47).…”

Section: Discussionmentioning

confidence: 99%

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

Mutig

Saritas

Uchida

et al. 2010

American Journal of Physiology-Renal Physiology

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Vasopressin influences salt and water transport in renal epithelia. This is coordinated by the combined action of V 2 receptor-mediated effects along distinct nephron segments. Modulation of NaCl reabsorption by vasopressin has been established in the loop of Henle, but its role in the distal convoluted tubule (DCT), an effective site for fine regulation of urinary electrolyte composition and the target for thiazide diuretics, is largely unknown. The Na ϩ -Cl Ϫ cotransporter (NCC) of DCT is activated by luminal trafficking and phosphorylation at conserved NH 2-terminal residues. Here, we demonstrate the effects of short-term vasopressin administration (30 min) on NCC activation in Brattleboro rats with central diabetes insipidus (DI) using the V 2 receptor agonist desmopressin (dDAVP). The fraction of NCC abundance in the luminal plasma membrane was significantly increased upon dDAVP as shown by confocal microscopy, immunogold cytochemistry, and Western blot, suggesting increased apical trafficking of the transporter. Changes were paralleled by augmented phosphorylation of NCC as detected by antibodies against phospho-threonine and phospho-serine residues (2.5-fold increase at Thr53 and 1.4-fold increase at Ser71). dDAVP-induced phosphorylation of NCC, studied in tubular suspensions in the absence of systemic effects, was enhanced as well (1.7-fold increase at Ser71), which points to the direct mode of action of vasopressin in DCT. Changes were more pronounced in early (DCT1) than in late DCT as distinguished by the distribution of 11␤-hydroxysteroid dehydrogenase 2 in DCT2. These results suggest that the vasopressin-V 2 receptor-NCC signaling cascade is a novel effector system to adjust transepithelial NaCl reabsorption in DCT. antidiuretic hormone; distal convoluted tubule; phosphorylation; sodium-chloride cotransporter trafficking ANTIDIURETIC HORMONE [arginine vasopressin (AVP)] serves to control extracellular fluid homeostasis. The principal effect of AVP is found in the collecting duct where AVP increases water reabsorption. The prerequisite to ensure this function is the creation of a hypertonic interstitium via action of the thick ascending limb (TAL), which is also promoted by AVP. These epithelial effects of AVP are mediated by type 2 AVP receptors (V 2 R) (2, 29), whereas type 1a receptor signaling rather serves to limit the antidiuretic effects of AVP (31). Mapping receptorspecific probes to the tubular segments has revealed subtypeselective distribution along TAL, macula densa, distal convolutions, and collecting ducts (3, 28). In TAL, strong V 2 R expression has been observed, which agreed with increased abundance and phosphorylation of the furosemide-sensitive NaϪ cotransporter (NKCC2) and enhanced NaCl reabsorption in TAL in response to AVP (8, 13, 28). V 2 R signaling also activates epithelial Na ϩ channel (ENaC)-dependent Na ϩ reabsorption in collecting ducts (16); AVP-induced antinatriuresis in this segment has therefore been considered as a causal element in the context of salt-sensitive hype...

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“…A very recent study conducted by Carmosino et al (52) reported that the region corresponding to amino acids 807-884 contains the sorting signal that directs apical trafficking of the NKCC2 protein. The results of the present work showed evidence that this region is not sufficient for NKCC2 exit from the ER.…”

Section: Discussionmentioning

confidence: 99%

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

Zaarour

Demaretz

Defontaine

et al. 2009

Journal of Biological Chemistry

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Mutations in the apically located Na؉ -K ؉ -2Cl ؊ co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet the mechanisms underlying the regulation of mutated NKCC2 proteins in renal cells have not been investigated. Here, we identified a trihydrophobic motif in the distal COOH terminus of NKCC2 that was required for endoplasmic reticulum (ER) exit and surface expression of the cotransporter. Indeed, microscopic confocal imaging showed that a naturally occurring mutation depriving NKCC2 of its distal COOH-terminal region results in the absence of cell surface expression. Biotinylation assays revealed that lack of cell surface expression was associated with abolition of mature complexglycosylated NKCC2. Pulse-chase analysis demonstrated that the absence of mature protein was not caused by reduced synthesis or increased rates of degradation of mutant co-transporters. Co-immunolocalization experiments revealed that these mutants co-localized with the ER marker protein-disulfide isomerase, demonstrating that they are retained in the ER. Cell treatment with proteasome or lysosome inhibitors failed to restore the loss of complex-glycosylated NKCC2, further eliminating the possibility that mutant co-transporters were processed by the Golgi apparatus. Serial truncation of the NKCC2 COOH terminus, followed by site-directed mutagenesis, identified hydrophobic residues 1081 LLV 1083 as an ER exit signal necessary for maturation of NKCC2. Mutation of 1081 LLV 1083 to AAA within the context of the full-length protein prevented NKCC2 ER exit independently of the expression system. This trihydrophobic motif is highly conserved in the COOH-terminal tails of all members of the cation-chloride co-transporter family, and thus may function as a common motif mediating their transport from the ER to the cell surface. Taken together, these data are consistent with a model whereby naturally occurring premature terminations that interfere with the LLV motif compromise co-transporter surface delivery through defective trafficking.The Na-K-2Cl co-transporter, NKCC2, provides the major route for sodium/chloride transport across the apical plasma membrane of the thick ascending limb (TAL) 3 of the kidney (1). This co-transporter is critical for salt reabsorption, acid-base regulation, and divalent mineral cation metabolism (2). The prominent importance of NKCC2 in renal functions is evidenced by the effect of loop diuretics, which as pharmacologic inhibitors of NKCC2, are extensively used in the treatment of edematous states (2). Even more impressive, inactivating mutations of the NKCC2 gene in humans causes Bartter syndrome type 1 (BS1), a life-threatening renal tubular disorder for which the diagnosis is usually made in the antenatal-neonatal period, due to the presence of polyhydramnios, premature delivery, salt loss, hypokalemia, metabolic alkalosis, hypercalciuria, and nephrocalcinosis (3). Without appropriate treatment, patients with BS1 will not survive the early neonatal period (4). In congruen...

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Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells

Cited by 75 publications

References 42 publications

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

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Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells

Cited by 75 publications

References 42 publications

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Nkcc1 (Slc12a2) is required for the regulation of endolymph volume in the otic vesicle and swim bladder volume in the zebrafish larva

Short-term stimulation of the thiazide-sensitive Na+-Cl−cotransporter by vasopressin involves phosphorylation and membrane translocation

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

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Short-term stimulation of the thiazide-sensitive Na⁺-Cl⁻cotransporter by vasopressin involves phosphorylation and membrane translocation