EAST/SeSAME Syndrome and Beyond: The Spectrum of Kir4.1- and Kir5.1-Associated Channelopathies.

In 2009, two groups independently linked human mutations in the inwardly rectifying K+ channel Kir4.1 (gene name KCNJ10) to a syndrome affecting the central nervous system (CNS), hearing, and renal tubular salt reabsorption. The autosomal recessive syndrome has been named EAST (epilepsy, ataxia, sensorineural deafness, and renal tubulopathy) or SeSAME syndrome (seizures, sensorineural deafness, ataxia, intellectual disability, and electrolyte imbalance), accordingly. Renal dysfunction in EAST/SeSAME patients results in loss of Na+, K+, and Mg2+ with urine, activation of the renin-angiotensin-aldosterone system, and hypokalemic metabolic alkalosis. Kir4.1 is highly expressed in affected organs: the CNS, inner ear, and kidney. In the kidney, it mostly forms heteromeric channels with Kir5.1 (KCNJ16). Biallelic loss-of-function mutations of Kir5.1 can also have disease significance, but the clinical symptoms differ substantially from those of EAST/SeSAME syndrome: although sensorineural hearing loss and hypokalemia are replicated, there is no alkalosis, but rather acidosis of variable severity; in contrast to EAST/SeSAME syndrome, the CNS is unaffected. This review provides a framework for understanding some of these differences and will guide the reader through the growing literature on Kir4.1 and Kir5.1, discussing the complex disease mechanisms and the variable expression of disease symptoms from a molecular and systems physiology perspective. Knowledge of the pathophysiology of these diseases and their multifaceted clinical spectrum is an important prerequisite for making the correct diagnosis and forms the basis for personalized therapies.

Genetic variants in eleven central and peripheral chemoreceptor genes in sudden infant death syndrome.

BACKGROUND: Sudden infant death syndrome (SIDS) is still one of the leading causes of postnatal infant death in developed countries. The occurrence of SIDS is described by a multifactorial etiology that involves the respiratory control system including chemoreception. It is still unclear whether genetic variants in genes involved in respiratory chemoreception might play a role in SIDS. METHODS: The exome data of 155 SIDS cases were screened for variants within 11 genes described in chemoreception. Pathogenicity of variants was assigned based on the assessment of variant types and in silico protein predictions according to the current recommendations of the American College of Medical Genetics and Genomics. RESULTS: Potential pathogenic variants in genes encoding proteins involved in respiratory chemoreception could be identified in 5 (3%) SIDS cases. Two of the variants (R137S/A188S) were found in the KNCJ16 gene, which encodes for the potassium channel Kir5.1, presumably involved in central chemoreception. Electrophysiologic analysis of these KCNJ16 variants revealed a loss-of-function for the R137S variant but no obvious impairment for the A188S variant. CONCLUSIONS: Genetic variants in genes involved in respiratory chemoreception may be a risk factor in a fraction of SIDS cases and may thereby contribute to the multifactorial etiology of SIDS. IMPACT: What is the key message of your article? Gene variants encoding proteins involved in respiratory chemoreception may play a role in a minority of SIDS cases. What does it add to the existing literature? Although impaired respiratory chemoreception has been suggested as an important risk factor for SIDS, genetic variants in single genes seem to play a minor role. What is the impact? This study supports previous findings, which indicate that genetic variants in single genes involved in respiratory control do not have a dominant role in SIDS.

A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Distinct Mitochondrial Pathologies Caused by Mutations of the Proximal Tubular Enzymes EHHADH and GATM.

The mitochondria of the proximal tubule are essential for providing energy in this nephron segment, whose ATP generation is almost exclusively oxygen dependent. In addition, mitochondria are involved in a variety of metabolic processes and complex signaling networks. Proximal tubular mitochondrial dysfunction can therefore affect renal function in very different ways. Two autosomal dominantly inherited forms of renal Fanconi syndrome illustrate how multifaceted mitochondrial pathology can be: Mutation of EHHADH, an enzyme in fatty acid metabolism, results in decreased ATP synthesis and a consecutive transport defect. In contrast, mutations of GATM, an enzyme in the creatine biosynthetic pathway, leave ATP synthesis unaffected but do lead to mitochondrial protein aggregates, inflammasome activation, and renal fibrosis with progressive renal failure. In this review article, the distinct pathophysiological mechanisms of these two diseases are presented, which are examples of the spectrum of proximal tubular mitochondrial diseases.

Defects in KCNJ16 Cause a Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness.

BACKGROUND: The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. METHODS: A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. RESULTS: We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. CONCLUSIONS: Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption.

Collecting system-specific deletion of Kcnj10 predisposes for thiazide- and low-potassium diet-induced hypokalemia.

The basolateral potassium channel KCNJ10 (Kir4.1), is expressed in the renal distal convoluted tubule and controls the activity of the thiazide-sensitive sodium chloride cotransporter. Loss-of-function mutations of KCNJ10 cause EAST/SeSAME syndrome with salt wasting and severe hypokalemia. KCNJ10 is also expressed in the principal cells of the collecting system. However, its pathophysiological role in this segment has not been studied in detail. To address this, we generated the mouse model AQP2cre:Kcnj10flox/flox with a deletion of Kcnj10 specifically in the collecting system (collecting system-Kcnj10-knockout). Collecting system-Kcnj10-knockout mice responded normally to standard and high potassium diet. However, this knockout exhibited a higher kaliuresis and lower plasma potassium than control mice when treated with thiazide diuretics. Likewise, collecting systemKcnj10-knockout displayed an inadequately high kaliuresis and renal sodium retention upon dietary potassium restriction. In this condition, these knockout mice became hypokalemic due to insufficient downregulation of the epithelial sodium channel (ENaC) and the renal outer medullary potassium channel (ROMK) in the collecting system. Consistently, the phenotype of collecting system-Kcnj10-knockout was fully abrogated by ENaC inhibition with amiloride and ameliorated by genetic inactivation of ROMK in the collecting system. Thus, KCNJ10 in the collecting system contributes to the renal control of potassium homeostasis by regulating ENaC and ROMK. Hence, impaired KCNJ10 function in the collecting system predisposes for thiazide and low potassium diet-induced hypokalemia and likely contributes to the pathophysiology of renal potassium loss in EAST/SeSAME syndrome.

Novel mutations in the KCNJ10 gene associated to a distinctive ataxia, sensorineural hearing loss and spasticity clinical phenotype.

KCNJ10 encodes the inward-rectifying potassium channel (Kir4.1) that is expressed in the brain, inner ear, and kidney. Loss-of-function mutations in KCNJ10 gene cause a complex syndrome consisting of epilepsy, ataxia, intellectual disability, sensorineural deafness, and tubulopathy (EAST/SeSAME syndrome). Patients with EAST/SeSAME syndrome display renal salt wasting and electrolyte imbalance that resemble the clinical features of impaired distal tubular salt transport in Gitelman's syndrome. A key distinguishing feature between these two conditions is the additional neurological (extrarenal) manifestations found in EAST/SeSAME syndrome. Recent reports have further expanded the clinical and mutational spectrum of KCNJ10-related disorders including non-syndromic early-onset cerebellar ataxia. Here, we describe a kindred of three affected siblings with early-onset ataxia, deafness, and progressive spasticity without other prominent clinical features. By using targeted next-generation sequencing, we have identified two novel missense variants, c.488G>A (p.G163D) and c.512G>A (p.R171Q), in the KCNJ10 gene that, in compound heterozygosis, cause this distinctive EAST/SeSAME phenotype in our family. Electrophysiological characterization of these two variants confirmed their pathogenicity. When expressed in CHO cells, the R171Q mutation resulted in 50% reduction of currents compared to wild-type KCNJ10 and G163D showed a complete loss of function. Co-expression of G163D and R171Q had a more pronounced effect on currents and membrane potential than R171Q alone but less severe than single expression of G163D. Moreover, the effect of the mutations seemed less pronounced in the presence of Kir5.1 (encoded by KCNJ16), with whom the renal Kir4.1 channels form heteromers. This partial functional rescue by co-expression with Kir5.1 might explain the lack of renal symptoms in the patients. This report illustrates that a spectrum of disorders with distinct clinical symptoms may result from mutations in different parts of KCNJ10, a gene initially associated only with the EAST/SeSAME syndrome.

Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol.

The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the Gq/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+/H+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.

Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization.

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes.

Novel role of mechanosensitive AT1B receptors in myogenic vasoconstriction.

Myogenic vasoconstriction is an inherent property of vascular smooth muscle cells (VSMCs) of resistance arteries harboring ill-defined mechanosensing and mechanotransducing elements. G protein-coupled receptors (GPCRs) are discussed as mechanosensors in VSMCs. In this study, we sought to identify and characterize the role and impact of GPCRs on myogenic vasoconstriction. Thus, we analyzed mRNA expression levels of GPCRs in resistance versus preceding conduit arteries revealing a significant enrichment of several GPCRs in resistance vessels. Selective pharmacological blockade of the highly expressed GPCRs in isolated murine mesenteric arteries ex vivo decreased myogenic vasoconstriction. In particular, candesartan and losartan most prominently suppressed myogenic tone, suggesting that AT1 receptors play a central role in myogenic vasoconstriction. Analyzing angiotensinogen(-/-) mice, a relevant contribution of locally produced angiotensin II to myogenic tone could be excluded. Investigation of AT1A (-/-) and AT1B (-/-) murine mesenteric arteries revealed that AT1B, but not AT1A, receptors are key components of myogenic regulation. This notion was supported by examining fura-2-loaded isolated AT1A (-/-) and AT1B (-/-) VSMCs. Our results indicate that in VSMCs, AT1B receptors are more mechanosensitive than AT1A receptors even at comparable receptor expression levels. Furthermore, we demonstrate that the mechanosensitivity of GPCRs is agonist-independent and positively correlates with receptor expression levels.

Transient receptor potential channel 1 (TRPC1) reduces calcium permeability in heteromeric channel complexes.

Specific biological roles of the classical transient receptor potential channel 1 (TRPC1) are still largely elusive. To investigate the function of TRPC1 proteins in cell physiology, we studied heterologously expressed TRPC1 channels and found that recombinant TRPC1 subunits do not form functional homomeric channels. Instead, by electrophysiological analysis TRPC1 was shown to form functional heteromeric, receptor-operated channel complexes with TRPC3, -4, -5, -6, and -7 indicating that TRPC1 proteins can co-assemble with all members of the TRPC subfamily. In all TRPC1-containing heteromers, TRPC1 subunits significantly decreased calcium permeation. The exchange of select amino acids in the putative pore-forming region of TRPC1 further reduced calcium permeability, suggesting that TRPC1 subunits contribute to the channel pore. In immortalized immature gonadotropin-releasing hormone neurons endogenously expressing TRPC1, -2, -5, and -6, down-regulation of TRPC1 resulted in increased calcium permeability and elevated basal cytosolic calcium concentrations. We did not observe any involvement of TRPC1 in store-operated cation influx. Notably, TRPC1 suppressed the migration of gonadotropin-releasing hormone neurons without affecting cell proliferation. Conversely, in TRPC1 knockdown neurons, specific migratory properties like distance covered, locomotion speed, and directionality were increased. These findings suggest a novel regulatory mechanism relying on the expression of TRPC1 and the subsequent formation of heteromeric TRPC channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration.

CD2AP in mouse and human podocytes controls a proteolytic program that regulates cytoskeletal structure and cellular survival.

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-ß1-dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-ß1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-ß response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.

Enzymatic disease of the podocyte.

Proteinuria is an early sign of kidney disease and has gained increasing attention over the past decade because of its close association with cardio-vascular and renal morbidity and mortality. Podocytes have emerged as the cell type that is critical in maintaining proper functioning of the kidney filter. A few genes have been identified that explain genetic glomerular failure and recent insights shed light on the pathogenesis of acquired proteinuric diseases. This review highlights the unique role of the cysteine protease cathepsin L as a regulatory rather than a digestive protease and its action on podocyte structure and function. We provide arguments why many glomerular diseases can be regarded as podocyte enzymatic disorders.

Functional analysis of the Kv1.1 N255D mutation associated with autosomal dominant hypomagnesemia.

Mutations in the voltage-gated K(+) channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936-942). A conserved asparagine at position 255 in the third transmembrane segment was converted into an aspartic acid, resulting in a non-functional channel. In this study, we explored the functional consequence of this conserved residue by substitution with other hydrophobic, polar, or charged amino acids (N255E, N255Q, N255A, N255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, and N255H) did not prevent ion conduction, and these mutant channels activated at more negative potentials when compared with wild-type channels, -41.5 +/- 1.6, -45.5 +/- 2.0, -50.5 +/- 1.9, and -33.8 +/- 1.3 mV to -29.4 +/- 1.1 mV, respectively. The time constant of activation was significantly faster for the two most hydrophobic mutations, N255A (6.2 +/- 0.2 ms) and N255V (5.2 +/- 0.3 ms), and the hydrophilic mutant N255T (9.8 +/- 0.4 ms) in comparison with wild type (13.0 +/- 0.9 ms). Furthermore, the voltage dependence of inactivation was shifted approximately 13 mV to more negative potentials in all mutant channels except for N255H. Taken together, our data showed that an asparagine at position 255 in Kv1.1 is required for normal voltage dependence and kinetics of channel gating.