Knockdown of INPP5K compromises the differentiation of N2A cells.

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Histone demethylase KDM4C is a functional dependency in JAK2-mutated neoplasms.

Mutations of the JAK2 gene are frequent aberrations in the aging hematopoietic system and in myeloid neoplasms. While JAK-inhibitors efficiently reduce hyperinflammation induced by the constitutively active mutated JAK2 kinase, the malignant clone and abundance of mutated cells remains rather unaffected. Here, we sought to assess for genetic vulnerabilities of JAK2-mutated clones. We identified lysine-specific demethylase KDM4C as a selective genetic dependency that persists upon JAK-inhibitor treatment. Genetic inactivation of KDM4C in human and murine JAK2-mutated cells resulted in loss of cell competition and reduced proliferation. These findings led to reduced disease penetrance and improved survival in xenograft models of human JAK2-mutated cells. KDM4C deleted cells showed alterations in target histone residue methylation and target gene expression, resulting in induction of cellular senescence. In summary, these data establish KDM4C as a specific dependency and therapeutic target in JAK2-mutated cells that is essential for oncogenic signaling and prevents induction of senescence.

Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms.

Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2-ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.

A regulatory pathway linking caffeine action, mood and the diurnal clock.

Depression is a leading cause of disability worldwide. Circadian abnormalities and mood changes are symptoms of depression. The psychostimulant caffeine alters wakefulness and alleviates other depression-related symptoms during chronic intake, but the underlying mechanisms are unclear. It is not known, whether and how acute caffeine administration affects mood. Molecular approaches, transgenic mouse models, pharmacological intervention and behavioral analysis were combined to uncover a regulatory pathway, which connects caffeine action with diurnal signaling via the key dopaminergic protein DARPP-32 and alters mood-related phenotypes in mice, which are often assessed in the context of antidepressant action. We observed that Thr75-DARPP-32 binds to the circadian regulator CLOCK and disrupts CLOCK:BMAL1 chromatin binding, thereby affecting gene expression. T75A-DARPP-32 mutant mice show reduced caffeine effects on CLOCK:BMAL1 and lack caffeine-induced effects on mood. This study provides a link between caffeine, diurnal signaling and mood-related behaviors, which may open new perspectives for our understanding of antidepressant mechanisms in the mouse brain.

First interchromosomal insertion in a patient with cerebral and spinal cavernous malformations.

Autosomal dominant cerebral cavernous malformations (CCM) are leaky vascular lesions that can cause epileptic seizures and stroke-like symptoms. Germline mutations in either CCM1, CCM2 or CCM3 are found in the majority of patients with multiple CCMs or a positive family history. Recently, the first copy number neutral inversion in CCM2 has been identified by whole genome sequencing in an apparently mutation-negative CCM family. We here asked the question whether further structural genomic rearrangements can be detected within NGS gene panel data of unsolved CCM cases. Hybrid capture NGS data of eight index patients without a pathogenic single nucleotide, indel or copy number variant were analyzed using two bioinformatics pipelines. In a 58-year-old male with multiple CCMs in his brain and spinal cord, we identified a 294 kb insertion within the coding sequence of CCM2. Fine mapping of the breakpoints, molecular cytogenetic studies, and multiplex ligation-dependent probe amplification verified that the structural variation was an inverted unbalanced insertion that originated from 1p12-p11.2. As this rearrangement disrupts exon 6 of CCM2 on 7p13, it was classified as pathogenic. Our study demonstrates that efforts to detect structural variations in known disease genes increase the diagnostic sensitivity of genetic analyses for well-defined Mendelian disorders.

DNA Methylation-Mediated Modulation of Endocytosis as Potential Mechanism for Synaptic Function Regulation in Murine Inhibitory Cortical Interneurons.

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.

Towards Raman spectroscopy of urine as screening tool.

For the screening purposes urine is an especially attractive biofluid, since it offers easy and noninvasive sample collection and provides a snapshot of the whole metabolic status of the organism, which may change under different pathological conditions. Raman spectroscopy (RS) has the potential to monitor these changes and utilize them for disease diagnostics. The current study utilizes mouse models aiming to compare the feasibility of the urine based RS combined with chemometrics for diagnosing kidney diseases directly influencing urine composition and respiratory tract diseases having no direct connection to urine formation. The diagnostic models for included diseases were built using principal component analysis with linear discriminant analysis and validated with a leave-one-mouse-out cross-validation approach. Considering kidney disorders, the accuracy of 100% was obtained in discrimination between sick and healthy mice, as well as between two different kidney diseases. For asthma and invasive pulmonary aspergillosis achieved accuracies were noticeably lower, being, respectively, 77.27% and 78.57%. In conclusion, our results suggest that RS of urine samples not only provides a solution for a rapid, sensitive and noninvasive diagnosis of kidney disorders, but also holds some promises for the screening of nonurinary tract diseases.

Homeostatic plasticity and synaptic scaling in the adult mouse auditory cortex.

It has been demonstrated that sensory deprivation results in homeostatic adjustments recovering neuronal activity of the deprived cortex. For example, deprived vision multiplicatively scales up mEPSC amplitudes in the primary visual cortex, commonly referred to as synaptic scaling. However, whether synaptic scaling also occurs in auditory cortex after auditory deprivation remains elusive. Using periodic intrinsic optical imaging in adult mice, we show that conductive hearing loss (CHL), initially led to a reduction of primary auditory cortex (A1) responsiveness to sounds. However, this was followed by a complete recovery of A1 activity evoked sounds above the threshold for bone conduction, 3 days after CHL. Over the same time course patch-clamp experiments in slices revealed that mEPSC amplitudes in A1 layers 2/3 pyramids scaled up multiplicatively in CHL mice. No recovery of sensory evoked A1 activation was evident in TNFα KO animals, which lack synaptic scaling. Additionally, we could show that the suppressive effect of sounds on visually evoked visual cortex activity completely recovered along with TNFα dependent A1 homeostasis in WT animals. This is the first demonstration of homeostatic multiplicative synaptic scaling in the adult A1. These findings suggest that mild hearing loss massively affects auditory processing in adult A1.

Clinical and genetic studies in patients with Lafora disease from Pakistan.

Lafora disease (LD) is progressive myoclonic epilepsy with late childhood- to teenage-onset. Mutations in two genes, EPM2A and NHLRC1, are responsible for this autosomal recessive disease in many patients Worldwide. In present study, we reported two unrelated consanguineous Pakistani families with Lafora disease (Families A and B). Affected individuals in both families presented with generalized tonic clonic seizures, intellectual disability, ataxia and cognitive decline. Diagnosis of Lafora disease was made on histo-pathological analysis of the skin biopsy, found positive for lafora bodies in periodic acid schiff stain and frequent generalized epileptiform discharges on electroencephalogram (EEG). Bi-directional sequencing in family A was performed for EPM2A and NHLRC1 genes but no mutation was found. In family B, Illumina TruSight One Sequencing Panel covering 4813 OMIM genes was carried out and we identified a novel homozygous mutation c.95G>T; p.32Trp>Leu of EPM2A gene which was found co-segregated in this family through Sanger sequencing. Structural analysis of this mutation, through different in silico approaches, predicted loss of stability and conformation in Laforin protein.

Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.

Regulation of endoplasmic reticulum turnover by selective autophagy.

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Regulation of vascular tone and arterial blood pressure: role of chloride transport in vascular smooth muscle.

Recent studies suggest that primary changes in vascular resistance can cause sustained changes in arterial blood pressure. In this review, we summarize current knowledge about Cl(-) homeostasis in vascular smooth muscle cells. Within vascular smooth muscle cells, Cl(-) is accumulated above the electrochemical equilibrium, causing Cl(-) efflux, membrane depolarization, and increased contractile force when Cl(-) channels are opened. At least two different transport mechanisms contribute to raise [Cl(-)] i in vascular smooth muscle cells, anion exchange, and cation-chloride cotransport. Recent work suggests that TMEM16A-associated Ca(2+)-activated Cl(-) currents mediate Cl(-) efflux in vascular smooth muscle cells leading to vasoconstriction. Additional proteins associated with Cl(-) flux in vascular smooth muscle are bestrophins, which modulate vasomotion, the volume-activated LRRC8, and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl(-) transporters and Cl(-) channels in vascular smooth muscle cells (VSMCs) significantly contribute to the physiological regulation of vascular tone and arterial blood pressure.

Functional mutation analysis provides evidence for a role of REEP1 in lipid droplet biology.

Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

Disruption of vascular Ca2+-activated chloride currents lowers blood pressure.

High blood pressure is the leading risk factor for death worldwide. One of the hallmarks is a rise of peripheral vascular resistance, which largely depends on arteriole tone. Ca2+-activated chloride currents (CaCCs) in vascular smooth muscle cells (VSMCs) are candidates for increasing vascular contractility. We analyzed the vascular tree and identified substantial CaCCs in VSMCs of the aorta and carotid arteries. CaCCs were small or absent in VSMCs of medium-sized vessels such as mesenteric arteries and larger retinal arterioles. In small vessels of the retina, brain, and skeletal muscle, where contractile intermediate cells or pericytes gradually replace VSMCs, CaCCs were particularly large. Targeted disruption of the calcium-activated chloride channel TMEM16A, also known as ANO1, in VSMCs, intermediate cells, and pericytes eliminated CaCCs in all vessels studied. Mice lacking vascular TMEM16A had lower systemic blood pressure and a decreased hypertensive response following vasoconstrictor treatment. There was no difference in contractility of medium-sized mesenteric arteries; however, responsiveness of the aorta and small retinal arterioles to the vasoconstriction-inducing drug U46619 was reduced. TMEM16A also was required for peripheral blood vessel contractility, as the response to U46619 was attenuated in isolated perfused hind limbs from mutant mice. Out data suggest that TMEM16A plays a general role in arteriolar and capillary blood flow and is a promising target for the treatment of hypertension.

A causative link between inner ear defects and long-term striatal dysfunction.

There is a high prevalence of behavioral disorders that feature hyperactivity in individuals with severe inner ear dysfunction. What remains unknown is whether inner ear dysfunction can alter the brain to promote pathological behavior. Using molecular and behavioral assessments of mice that carry null or tissue-specific mutations of Slc12a2, we found that inner ear dysfunction causes motor hyperactivity by increasing in the nucleus accumbens the levels of phosphorylated adenosine 3',5'-monophosphate response element-binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase (pERK), key mediators of neurotransmitter signaling and plasticity. Hyperactivity was remedied by local administration of the pERK inhibitor SL327. These findings reveal that a sensory impairment, such as inner ear dysfunction, can induce specific molecular changes in the brain that cause maladaptive behaviors, such as hyperactivity, that have been traditionally considered exclusively of cerebral origin.

Renal intercalated cells are rather energized by a proton than a sodium pump.

The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na(+) has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na(+)/K(+)-ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H(+) vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na(+)/K(+)-ATPase had no effects. Basolateral NaCl exit from ß-intercalated cells was independent of the Na(+)/K(+)-ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H(+) V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H(+) V-ATPase.

Extracellular ATP induces cytoplasmic and nuclear Ca2+ transients via P2Y2 receptor in human biliary epithelial cancer cells (Mz-Cha-1).

Extracellular nucleotides such as adenosine triphosphate (ATP) play a role in biliary epithelial cell function. Since nucleotide receptors are potential targets for various diseases related to epithelial cell dysfunction and cancer, the purpose of this study was to investigate the expression and to functionally characterize the nucleotide receptor subtypes in biliary epithelial cancer cells (Mz-Cha-1). Extracellular ATP dose-dependently resulted in an intracellular Ca(2+) increase (mean effective concentration (EC(50)) 40 µM). Uridine triphosphate (UTP) produced a similar Ca(2+) response and cross-desensitation was observed. The rank order of tested agonists was ATP=UTP>> adenosine>ADP=AMP>α,ß-methylene-ATP. This confirms the functional expression of purinoceptor P2Y2 and P2Y4 in biliary epithelial cancer cell membranes. mRNAs for P2Y1, P2Y2, P2Y4 and P2Y6 purinergic receptor subtypes were found, whereas western blot analysis suggested only the expression of P2Y2 receptors. Confocal imaging and nuclear staining was used to compartmentalize ATP-induced cytosolic and nuclear Ca(2+)-transients, indicating a role for secretory ATP in regulating nuclear function, by increasing nuclear Ca(2+) concentrations. These data define the expression profile of P2Y receptors on human biliary epithelial cancer cells and indicate P2Y2 receptors as being potential targets in new treatment strategies for biliary cancer.

K-Cl cotransporter gene expression during human and murine erythroid differentiation.

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.