Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

Investigation of erythrocytes from spontaneous or engineered germ-line mutant mice has been instrumental in characterizing the physiological functions of components of the red cell cytoskeleton and membrane. However, the red blood cell expresses some proteins whose germline loss-of-function is embryonic-lethal, perinatal-lethal, or confers reduced post-weaning viability. Promoter regions of erythroid-specific genes have been used to engineer erythroid-specific expression of Cre recombinase. Through breeding with mice carrying appropriately spaced insertions of loxP sequences, generation of erythroid-specific knockouts has been carried out for signaling enzymes, transcription factors, peptide hormones, and single transmembrane span signaling receptors. We report here the use of Cre recombinase expression driven by the erythropoietin receptor (EpoR) promoter to generate EpoR-Cre;Kcc3(f/f) mice, designed to express erythroid-specific knockout of the KCC3 K-Cl cotransporter encoded by Kcc3/Slc12A6. We confirm KCC3 as the predominant K-Cl cotransporter of adult mouse red cells in mice with better viability than previously exhibited by Kcc3(-/-) germline knockouts. We demonstrate roughly proportionate preservation of K-Cl stimulation by hypotonicity, staurosporine, and urea in the context of reduced, but not abrogated, K-Cl function in EpoR-Cre;Kcc3(f/f) mice. We also report functional evidence suggesting incomplete recombinase-mediated excision of the Kcc3 gene in adult erythroid tissues.

The molecular identity of Psickle, the deoxygenation-activated cation conductance of the human sickle erythrocyte, remains unknown. We observed in human sickle red cells that inhibitors of TRPA1 and TRPV1 inhibited Psickle, whereas a TRPV1 agonist activated a Psickle-like cation current. These observations prompted us to test the roles of TRPV1 and TRPA1 in Psickle in red cells of the SAD mouse model of sickle cell disease. We generated SAD mice genetically deficient in either TRPV1 or TRPA1. SAD;Trpv1-/- and SAD;Trpa1-/- mice were indistinguishable in appearance, hematological indices, and osmotic fragility from SAD mice. We found that deoxygenation-activated cation currents remained robust in SAD;Trpa1-/- and SAD;Trpv1-/- mice. In addition, 45Ca2+ influx into SAD mouse red cells during prolonged deoxygenation was not reduced in red cells from SAD;Trpa1-/- and SAD;Trpv1-/- mice. We conclude that the nonspecific cation channels TRPA1 and TRPV1 are not required for deoxygenation to stimulate Psickle-like activity in red cells of the SAD mouse model of sickle cell disease. (159).

Not available.

Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.

Distal renal tubular acidosis is a rare renal tubular disorder characterized by hyperchloremic metabolic acidosis and impaired urinary acidification. Mutations in three genes (ATP6V0A4, ATP6V1B1 and SLC4A1) constitute a monogenic causation in 58-70% of familial cases of distal renal tubular acidosis. Recently, mutations in FOXI1 have been identified as an additional cause. Therefore, we hypothesized that further monogenic causes of distal renal tubular acidosis remain to be discovered. Panel sequencing and/or whole exome sequencing was performed in a cohort of 17 families with 19 affected individuals with pediatric onset distal renal tubular acidosis. A causative mutation was detected in one of the three "classical" known distal renal tubular acidosis genes in 10 of 17 families. The seven unsolved families were then subjected to candidate whole exome sequencing analysis. Potential disease causing mutations in three genes were detected: ATP6V1C2, which encodes another kidney specific subunit of the V-type proton ATPase (1 family); WDR72 (2 families), previously implicated in VATPase trafficking in cells; and SLC4A2 (1 family), a paralog of the known distal renal tubular acidosis gene SLC4A1. Two of these mutations were assessed for deleteriousness through functional studies. Yeast growth assays for ATP6V1C2 revealed loss-of-function for the patient mutation, strongly supporting ATP6V1C2 as a novel distal renal tubular acidosis gene. Thus, we provided a molecular diagnosis in a known distal renal tubular acidosis gene in 10 of 17 families (59%) with this disease, identified mutations in ATP6V1C2 as a novel human candidate gene, and provided further evidence for phenotypic expansion in WDR72 mutations from amelogenesis imperfecta to distal renal tubular acidosis.

β-thalassemia (β-Thal) is caused by defective β-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress. We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of β-thalassemia intermedia. In the absence of these transporters, the anemia of β-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of β-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved β-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of β-thalassemia.

Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.

Supplemental Digital Content is available in the text

Supplemental Digital Content is available in the text.

Hereditary Xerocytosis (HX) is an autosomal dominant hemolytic anemia of variable penetrance associated with red cell dehydration, elevated corpuscular hemoglobin concentration mean (CHCM) and resistance to hypo-osmotic lysis. These properties are accompanied less reliably by macrocytosis and stomatocytosis of low-to-moderate frequency. The disease prevalence originally estimated at 1 per 10,000 may be considerably higher. Most HX is caused by heterozygous missense mutations in PIEZO1, a mechanosensitive, Ca-permeable cation channel polypeptide of 2521 aa in length. The mutations confer gain-of-function arising from delayed inactivation of the cation channel, leading to increased Ca entry into the red cell. Prolongation of the brief elevations in HX red cell cytosolic Ca that follow transient openings of mutant PIEZO1 channels are believed to increase activation of the erythroid Gardos channel, KCNN4, leading to red cell dehydration and MCHC elevation. Indeed, pathogenic gain-of-function KCNN4 mutations R352 (in the calmodulin-binding domain), V282M, and V282E (at the cytoplasmic face of TM6) have been reported in families with normal PIEZO1. The central role of KCNN4 activity in the pathogenesis of HX has encouraged the proposal that the KCNN4 antagonist senicapoc should be clinically tested in HX. Susceptibility to inhibition by the KCNN4 antagonist, senicapoc, is preserved by some HX-associated KCNN4 mutants, as evidenced by whole cell recording of recombinant channel activity expressed in HEK-293 cells. KCNN4 V282M retained native senicapoc affinity, whereas senicapoc inhibition of KCNN4 R352H was 10-fold more potent and KCNN4 V282E exhibited severely reduced affinity. Here, we report our results characterizing red cell KCNN4 function and senicapoc sensitivity and other red cell properties in a cohort of HX patients heterozygous for KCNN4 mutation V282M (family WO in reference 5’s Figure 2B). HX patients heterozygous for KCNN4 V282M were anemic, with hematocrit of 35.260.9% (n522) vs control value of 4561.0% (n521; P<10), and with reticulocytosis of 9.96 1.0% (n522) vs. control value of 1.460.1% (n521). HX red cells from patients heterozygous for KCNN4 V282M also exhibited a severely left-shifted osmotic fragility curve (Figure 1A), indicative of extreme resistance to osmotic lysis. This resistance to osmotic lysis corresponded to a substantial rightshift in the phthalate density profile (Figure 1B), demonstrating substantially increased density of HX red cells. HX red cells also showed reduced K content (178632 vs. 314645 mmol/kg Hb), with increased Na content (4165 vs 2465 mmol/kg Hb; n512, P<10 for both). The ektacytometry profile of red cells heterozygous for KCNN4 V282M is left-shifted to an unusually extreme degree, with reductions in Dmax as well as of Ohyp values (Figure 1C; left shift is too extreme to allow determination of Omin value). This left-shifted curve indicating reduced shear-sensitive deformability is consistent with profound red cell dehydration and contrasts with the relatively normal ektacytometry profiles reported for HX red cells from patients heterozygous for KCNN4 R352H. Whereas control red cells exhibited minimal channel activity in oncell patch recordings, red cells from patients heterozygous for KCNN4 V282M showed increased spontaneous channel activity. NPo was eightfold elevated and inhibited nearly completely by 200 nM senicapoc (Figure 1D). This increased basal channel activity (unitary chord conductance 12 pS, not shown) was reflected in increased net K efflux insensitive to ouabain plus bumetanide, but partially sensitive to 200 nM senicapoc (Figure 1E), a component of activity undetected in control red cells. Moreover, unidirectional K influx (measured as Rb) insensitive to ouabain plus bumetanide but sensitive to senicapoc was undetected in unstimulated control red cells, but present in unstimulated HX red cells from patients heterozygous for KCNN4 V282M (Figure 1F). The combined data indicate that HX red cells from anemic patients heterozygous for KCNN4 mutation V282M exhibit severe dehydration characterized by high red cell density with resistance to deformability and to osmotic lysis. Red cells from patients heterozygous for the KCNN4 mutation V282M exhibit increased basal senicapoc-sensitive channel activity, accompanied by increased senicapoc-sensitive net K efflux and increased senicapoc-sensitive unidirectional K influx. The demonstration by several methods of increased senicapoc-sensitive, basal K leak from HX red cells of patients heterozygous for the causative KCNN4 mutation V282M strengthens the proposal of senicapoc as a promising treatment for patients with this subtype of HX, as well as for HX secondary to the KCNN4 R352H mutation. The relationship between HX red cell dehydration and the anemia of HX secondary to gain-of-function KCNN4 mutations remains to be investigated. Successful senicapoc treatment of anemia in HX patients would support the intrinsic pathogenicity of increased red cell density in this context, and perhaps more generally.

Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation - a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl--sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl- uptake and stimulation of KCC3-mediated Cl- extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought "Cl-/volume-sensitive kinase" of the cation-Cl- cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.

Objectives/Hypothesis: Hearing loss and enlarged vestibular aqueduct ( EVA) can be inherited as an autosomal recessive trait caused by mutant alleles of the SLC26A4 gene. In some other families, EVA does not segregate in a typical autosomal recessive pattern. The goal of this study was to characterize the SLC26A4 genotypes and phenotypes of extended families with atypical segregation of EVA.Study Design: Prospective study of cohort of families ascertained between 1998 and 2014 at the National Institutes of Health Clinical Center.Methods: Study subjects were members of eight families segregating EVA in at least two members who were not related as siblings. Evaluations included pure-tone audiometry, temporal bone imaging, SLC26A4 nucleotide sequence analysis, SLC26A4-linked marker genotype and haplotype analysis, and pedigree analysis.Results: One family had members with EVA caused by different etiologies, and two families had pseudodominant inheritance of recessive mutations of SLC26A4. In five families, the etiology remained unknown and could include inheritance of mutant alleles at another genetic locus, nongenetic influences, or a combination of these factors.Conclusions: Familial EVA can demonstrate a variety of atypical segregation patterns. Pseudodominant inheritance of SLC26A4 mutations or recessive alleles of other hearing loss genes may be more likely to occur in families in which deaf individuals have intermarried. The etiologic basis of atypical segregation of EVA without detectable SLC26A4 mutations remains unknown. Future studies of these families may reveal novel genes for EVA.

We welcome the Comment of Faucherre et al. in response to our correspondence concerning their report of hemolytic anemia in zebrafish embryos subjected to morpholino knockdown of piezo1. We note, however, that in their Comment, Faucherre et al. neither dispute nor question our descriptions or analyses of their paper on piezo1 morpholino knockdown. Nor do they question any of our data showing the absence of detectable anemia in zebrafish red cells from piezo1 germline knockout embryos and adults, a phenotype quite different from that reported in their morpholino knockdown studies. The rationale of Faucherre et al. for contributing their Comment seems rather to be based on the following contentions. 1. Faucherre et al. state that an article by Rossi et al. "has added a new angle to this debate" [concerning phenotypic differences (between morpholino knockdowns and germline knockouts in zebrafish)], suggesting we did not discuss it. However, we note that this very paper was, indeed, discussed and cited in our correspondence. 2. Faucherre et al. state that this same new angle "may have led to an oversight by Shmukler et al.," but Faucherre et al. do not elaborate on the nature of the oversight. They note only that our correspondence "do[es] not explain why there appears to be such a large discrepancy between these [mouse and zebrafish] phenotypes". Indeed, neither we nor Faucherre et al. can further explain this difference in a brief exchange of correspondence. As was true for Rossi et al. and for Kok et al., the explanation requires directed experimentation for each individual gene for which knockdown and knockout phenotypes differ (a situation describing as many as 80% of zebrafish genes). 3. Faucherre et al. further state that "the lack of any phenotype in the piezo1 KO zebrafish line has possibly led the authors to misinterpret [the data of] Cahalan et al." We cited the data of Cahalan et al. as they were published. We pointed out that the mild hemolytic anemia of the mouse with red blood cells deficient in PIEZO1 was fully compensated, and that the normal hematocrit and red cell number thus resembled the normal hematologic phenotype of piezo1 zebrafish. In their Comment, Faucherre et al. choose to emphasize the qualitative similarity between the mild hemolysis and red cell volume increase of the mouse and the apparently severe hemolysis and red cell volume increase in the morpholino knockdown zebrafish. Each interpretation, although divergent, is legitimate; neither is a "misinterpretation". 4. Faucherre et al. contend that "the piezo1KO zebrafish line is a global KO of piezo1 and should first be compared to the mouse global KO of piezo1 not the erythrocyte-specific [conditional] KO line." We respond without qualification that phenotypic comparison of zebrafish morpholino knockdowns with the corresponding germline knockouts needs no justification. Faucherre et al. further note in their Comment that the "zebrafish piezo1 KO line .... appears to all intents and purposes to have no observable phenotype"[in contrast to the embryonic lethal vascular phenotype in KO mice]. We agree, and note in addition that recently described patients with autosomal recessive lymphatic dysplasia secondary to homozygous loss-of-function mutations in human PIEZO1 are remarkable for normal vasculature and an asymptomatic, fully compensated, very mild hemolytic state of incomplete penetrance. The concern of Faucherre et al. about these contrasting phenotypes associated with PIEZO1 loss-of-function only serves to highlight their curious choice not to report the vascular phenotype of their piezo1 morpholino knockdown fish, despite their commendable use of the Lmo2:dsRed reporter line (delineating vasculature as well as erythrocytes). Figure 3 from the original report by Faucherre et al. presents images of dsRed-expressing erythrocytes from morpholino-injected Lmo2:dsRed reporter line fish (which of the two morpholinos used was not described but seems to be MO1, assuming conditions of their Online Supplementary Figure S5 applied also to Figures 3 and 4). The absence of comment about a vascular phenotype in the dsRed-expressing blood vessels of this reporter line suggests the likelihood of normal vasculature, as was indeed observed in germline piezo1 fish by Kok et al. If so, the difference in vascular phenotype between the global knockout mouse and the PIEZO1-deficient fish would seem to be independent of whether the knockout mouse is compared to a morpholino knockdown fish or to a germline knockout fish.

Distal renal tubular acidosis caused by missense mutations in kidney isoform of anion exchanger 1 (kAE1/SLC4A1), the basolateral membrane Cl-/HCO3- exchanger of renal alpha-intercalated cells, has been extensively investigated in heterologous expression systems but rarely in human kidneys. The preferential apical localization of distal renal tubular acidosis (dRTA)-associated kAE1 mutants R901X, G609R and M909T in cultured epithelial monolayers has not been examined in human kidney. Here, we present kidney tissues from dRTA-affected siblings heterozygous for kAE1 G609R, characterized by predominant absence rather than mistargeting of kAE1 in intercalated cells. Thus, studies of heterologous recombinant expression of mutant proteins should be, whenever possible, interpreted in comparison to affected patient tissues.

J. F. Heneghan, D. H. Vandorpe, B. E. Shmukler, J. A. Giovinazzo, J. Raper, D. J. Friedman, M. R. Pollak, and S. L. Alper Renal Division, Beth Israel Deaconess Medical Center, Boston, Massachusetts; Vascular Biology Research Center, Beth Israel Deaconess Medical Center, Boston, Massachusetts; Department of Medicine, Harvard Medical School, Boston, Massachusetts; and The Graduate Center and Department of Biological Sciences, Hunter College of the City University of New York, New York, New York

Ischemia-mediated activation of Na-K-Cl cotransporter (NKCC1) leads to Na+ overload, cytotoxic edema, and cell death. However, the molecular mechanisms underlying these processes are not understood. Here we investigated the role of WNK (with no lysine = K)-SPAK/OSR1 kinase signaling pathway in regulating the function of NKCC1 in cerebral ischemia. We show that genetic deletion of WNK3, a WNK family member highly expressed in brain, or siRNA-mediated knockdown of SPAK or OSR1, protects against ischemic neuroglial death triggered by oxygen-glucose deprivation/reoxygenation. Using phospho specific antibodies, we detected robust up regulation of p-NKCC1 as well as p-SPAK/p-OSR1expression in peri-infarct cortex, striatum and corpus callosum in wild type mice subjected to transient middle cerebral artery occlusion. In WNK3 knockout mice, ischemia-induced hyper-phosphorylations of both the SPAK/OSR1 catalytic T-loop and NKCC1 are abolished, infarct volume and axonal demyelination are reduced, and neurobehavioral recovery is accelerated. These data provide fresh insight into the roles of ion transporters and their regulatory kinases in ischemic neuroglial injury, and identify the WNK3-SPAK/OSR1 kinase complex as a compelling target for novel neuroprotective strategies following stroke. Support: NIH R01NS38118