Molecular mechanism of a COOH-terminal gating determinant in the ROMK channel revealed by a Bartter's disease mutation

The ROMK subtypes of inward-rectifier K⁺ channels mediate potassium secretion and regulate  NaCl  reabsorption in the kidney. Loss-of-function mutations in this pH-sensitive K⁺ channel cause Bartter's disease, a familial salt wasting nephropathy. One disease-causing mutation truncates the extreme COOH-terminus and induces a closed gating conformation. Here we identify a region within the deleted domain that plays an important role in pH-dependent gating. The domain contains a structural element that functionally interacts with the pH sensor in the cytoplasmic NH₂ - terminus to set a physiological range of pH sensitivity. Removal of the domain shifts the p K _a towards alkaline pH values, causing channel inactivation under physiological conditions. Suppressor mutations within the pH sensor rescued channel gating and trans addition of the cognate peptide restored pH sensitivity. A specific interdomain interaction was revealed in an in vitro protein-protein binding assay between the NH₂- and COOH-terminal cytoplasmic domains expressed as bacterial fusion proteins. These results provide new insights into the molecular mechanisms underlying Kir channel regulation and channel gating defects that are associated with Bartter's disease.     

The role of cyclooxygenases and prostanoid receptorsin furosemide‐like salt losing tubulopathy:the hyperprostaglandin E syndrome

Hyperprostaglandin E syndrome/antenatal Bartter syndrome is characterized by NaCl wasting and volume depletion, juxtaglomerula hypertrophy, hyper‐reninism and secondary hyperaldosteronism. Primary causes are mutations in the gene for Na‐K‐2Cl‐cotransporter, NKCC2, or for potassium channel, ROMK, responsible for medullary NaCl malabsorption. Most intriguing aspect of the syndrome is the association with a massively increased renal prostaglandin production which contributes substantially to the clinical picture of the patients. Therefore the term hyperprostaglandin E syndrome has been introduced. It is unclear how prostaglandins aggravate the NaCl transport deficiency. Aspects to prostaglandin synthesis and receptor‐mediated function within the kidney in patients suffering from hyperprostaglandin E syndrome/antenatal Bartter syndrome will be discussed.     

Heterozygous mutations of the gene for Kir 1.1 (ROMK) in antenatal Bartter syndrome presenting with transient hyperkalemia,evolving to a benign course

Bartter-like syndrome encompasses a set of inherited renal tubular disorders associated with hypokalemic metabolic alkalosis, renal salt wasting, hyperreninemic hyperaldosteronism, and normal blood pressure. Antenatal Bartter syndrome, a subtype of Bartter-like syndrome, is characterized by polyhydramnios, premature delivery, life-threatening episodes of fever and dehydration during the early weeks of life, growth retardation, hypercalciuria, and early-onset nephrocalcinosis. Mutations in the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) and ATP-sensitive inwardly rectifying potassium channel (ROMK) of the thick ascending limb of Henle's loop have been identified in the antenatal Bartter syndrome. We report the identification of two heterozygous mutations of the gene for Kir 1.1 (ROMK) from an antenatal Bartter syndrome patient who presented at birth with mild salt wasting and a biochemical findings that mimicked primary pseudohypoaldosteronism type 1, such as hyperkalemia and hyponatremia, and evolved to a relatively benign course. We have identified amino acid exchanges Arg338Stop and Met357Thr in the gene exon 5 for ROMK by PCR and direct sequencing. Both mutations alter the C-terminus of the ROMK protein, and can affect channel function.     

Pharmacotyping of hypokalaemic salt‐losing tubular disorders

Long standing confusion exists in the terminology of hypokalaemic salt‐losing tubulopathies (SLTs). SLTs are autosomal recessively transmitted and characterized by normotensive secondary hyperreninism/hyperaldosteronism with hypokalaemic metabolic alkalosis. Historically, four phenotypical variants have been described: (1) the (classic) Bartter syndrome (cBS), (2) the hypomagnesaemic hypocalciuric Gitelman syndrome (GS), (3) the hypercalciuric hyperprostaglandin‐E‐syndrome (HPS) or antenatal Bartter syndrome (aBS) and (4) the hyperprostaglandin‐E‐syndrome with sensorineural deafness (HPS + SND). The latter two syndromes are the most severe variants with antenatal manifestation with polyhydramnios and life‐threatening course of salt‐ and water‐loss. Defects in five renal membrane proteins involved in electrolyte reabsorption have been identified: In HPS‐patients mutations in (1) either the furosemide‐sensitive sodium–potassium–chloride cotransporter NKCC2, or (2) in the potassium channel ROMK have been identified, and (3) HPS + SND is caused by mutations in the β‐subunit of the chloride channels ClC‐Kb and –Ka (named barttin), all mimicking the major pharmacological effects of furosemide with minor potassium‐wasting in ROMK‐patients as seen in patients treated with simultaneous furosemide and amiloride, and minor calcium‐wasting in Barttin‐patients resembling the combination of furosemide and thiazides. (4) cBS is caused by mutations in the chloride channel ClC‐Kb with similar clinical characteristics as seen under combination of thiazides and furosemide, (5) GS is caused by mutations in the thiazide‐sensitive sodium‐chloride cotransporter NCCT resembling the effect of long‐term thiazide administration. Conclusion: The combination of pharmacology and genetics suggests a new terminology for the above described SLTs: Furosemide‐like‐SLT for HPS caused by NKCC2‐mutations, furosemide/amiloride‐like‐SLT for HPS caused by ROMK‐mutations, furosemide/thiazide‐like‐SLT for HPS + SND, thiazide/furosemide‐like‐SLT for cBS, and thiazide‐like‐SLT for GS.     

The role of cyclooxygenases and prostanoid receptorsin furosemide-like salt losing tubulopathy: the hyperprostaglandin E syndrome

Hyperprostaglandin E syndrome/antenatal Bartter syndrome is characterized by NaCl wasting and volume depletion, juxtaglomerula hypertrophy, hyperreninism and secondary hyperaldosteronism. Primary causes are mutations in the gene for Na-K-2Cl-cotransporter, NKCC2, or for potassium channel, ROMK, responsible for medullary NaCl malabsorption. Most intriguing aspect of the syndrome is the association with a massively increased renal prostaglandin production which contributes substantially to the clinical picture of the patients. Therefore the term hyperprostaglandin E syndrome has been introduced. It is unclear how prostaglandins aggravate the NaCl transport deficiency. Aspects to prostaglandin synthesis and receptor-mediated function within the kidney in patients suffering from hyperprostaglandin E syndrome/antenatal Bartter syndrome will be discussed.     

Functional implications of mutations in the human renal outer medullary potassium channel (ROMK2) identified in Bartter syndrome

Bartter syndrome is an autosomal recessive heterogeneous renal tubular disorder affecting NaCl reabsorption in the thick ascending limb of Henle's loop (TAL). The aim of this study was to elucidate the functional implications of mutations in the predominant human ROMK isoform in TAL, hROMK2, involved in Bartter syndrome type II. cRNA of flag-tagged hROMK2 and eight mutants identified in seven non-related patients was expressed in Xenopus laevis oocytes. hROMK2 activity was measured by two-electrode voltage-clamp analysis and defined as the Ba2+ -sensitive current at a holding potential of -75 mV. The subcellular localization of hROMK2 in oocytes was studied by immunocytochemistry. Injection of 25 pg hROMK2 cRNA resulted in an inwardly rectifying Ba2+ -sensitive current of 522+/-43 nA ( n=22). The mutants could be divided into three distinct groups. First, at 25 pg injection mutants W80C, V103E and T313/350X exhibited no significant currents and could only be detected intracellularly. Upon 8 ng injection, plasma membrane presence was observed as well as currents up to 60% of wild-type current. Second, mutants V53E and V296G exhibited no Ba2+ -sensitive current, but were present in the plasma membrane at 0.1 ng and 8 ng injection levels. Third, mutants P91L and A179T were detectable on the plasma membrane (0.1 ng) and yielded currents of 98% and 80% of wild-type, respectively, at 25 pg injection. S294C yielded currents that were 45% of wild-type and were detected both on and just below the plasma membrane at 0.1 ng injection. This study has unraveled three distinct mechanisms by which mutations in hROMK2 could impair channel function in Bartter syndrome. Future experiments on kidney epithelial cell lines will have to confirm this classification, after which specific pharmacological treatments could be considered for each group of mutations.     

ROMK1 (Kir1.1) causes apoptosis and chronic silencing of hippocampal neurons

Lentiviral vectors were constructed to express the weakly rectifying kidney K(+) channel ROMK1 (Kir1.1), either fused to enhanced green fluorescent protein (EGFP) or as a bicistronic message (ROMK1-CITE-EGFP). The channel was stably expressed in cultured rat hippocampal neurons. Infected cells were maintained for 2-4 wk without decrease in expression level or evidence of viral toxicity, although 15.4 mM external KCl was required to prevent apoptosis of neurons expressing functional ROMK1. No other trophic agents tested could prevent cell death, which was probably caused by K(+) loss. This cell death did not occur in glia, which were able to support ROMK1 expression indefinitely. Functional ROMK1, quantified as the nonnative inward current at -144 mV in 5.4 mM external K(+) blockable by 500 microM Ba(2+), ranged from 1 to 40 pA/pF. Infected neurons exhibited a Ba(2+)-induced depolarization of 7 +/- 2 mV relative to matched EGFP-infected controls, as well as a 30% decrease in input resistance and a shift in action potential threshold of 2.6 +/- 0.5 mV. This led to a shift in the relation between injected current and firing frequency, without changes in spike shape, size, or timing. This shift, which quantifies silencing as a function of ROMK1 expression, was predicted from Hodgkin-Huxley models. No cellular compensatory mechanisms in response to expression of ROMK1 were identified, making ROMK1 potentially useful for transgenic studies of silencing and neurodegeneration, although its lethality in normal K(+) has implications for the use of K(+) channels in gene therapy.     

Pharmacotyping of hypokalaemic salt-losing tubular disorders

Long standing confusion exists in the terminology of hypokalaemic salt-losing tubulopathies (SLTs). SLTs are autosomal recessively transmitted and characterized by normotensive secondary hyperreninism/hyperaldosteronism with hypokalaemic metabolic alkalosis. Historically, four phenotypical variants have been described: (1) the (classic) Bartter syndrome (cBS), (2) the hypomagnesaemic hypocalciuric Gitelman syndrome (GS), (3) the hypercalciuric hyperprostaglandin-E-syndrome (HPS) or antenatal Bartter syndrome (aBS) and (4) the hyperprostaglandin-E-syndrome with sensorineural deafness (HPS + SND). The latter two syndromes are the most severe variants with antenatal manifestation with polyhydramnios and life-threatening course of salt- and water-loss. Defects in five renal membrane proteins involved in electrolyte reabsorption have been identified: In HPS-patients mutations in (1) either the furosemide-sensitive sodium-potassium-chloride cotransporter NKCC2, or (2) in the potassium channel ROMK have been identified, and (3) HPS + SND is caused by mutations in the beta-subunit of the chloride channels ClC-Kb and -Ka (named barttin), all mimicking the major pharmacological effects of furosemide with minor potassium-wasting in ROMK-patients as seen in patients treated with simultaneous furosemide and amiloride, and minor calcium-wasting in Barttin-patients resembling the combination of furosemide and thiazides. (4) cBS is caused by mutations in the chloride channel ClC-Kb with similar clinical characteristics as seen under combination of thiazides and furosemide, (5) GS is caused by mutations in the thiazide-sensitive sodium-chloride cotransporter NCCT resembling the effect of long-term thiazide administration. Conclusion: The combination of pharmacology and genetics suggests a new terminology for the above described SLTs: Furosemide-like-SLT for HPS caused by NKCC2-mutations, furosemide/amiloride-like-SLT for HPS caused by ROMK-mutations, furosemide/thiazide-like-SLT for HPS + SND, thiazide/furosemide-like-SLT for cBS, and thiazide-like-SLT for GS.     

Mg(2+)-dependent inward rectification of ROMK1 potassium channels expressed in Xenopus oocytes.

1. ROMK1 potassium channel currents were examined in Xenopus oocytes by two-microelectrode voltage-clamp and patch-clamp techniques following injection of oocytes with in vitro transcribed ROMK1 cRNA. Macroscopic currents recorded from intact cells rectified inwardly at positive potentials. 2. In inside-out membrane patches rectification is manifested as an apparent reduction of single channel current (at 500 Hz) in the presence of 0.1-10 mM Mg2+, without a decrease in the channel open probability. No inward rectification is observed when membrane patches are isolated into solutions containing potassium as the only internal inorganic cation. 3. Mg2+ block can be described by a simple one-site model for Mg2+ binding with K0 ([Mg2+] causing half-maximal block at 0 mV) of 16.7 mM and delta (the fraction of the membrane field sensed by the blocking Mg2+) of 0.35. 4. The voltage dependence of channel block by cytoplasmic Mg2+ was shifted approximately -50 mV by a reduction in extracellular [K+] from 140 to 0 mM, corresponding to a decrease of K0 to 4.4 mM. 5. At negative membrane potentials, ROMK1 channels exhibit a single subconducting state that is approximately 4/10 of the full conductance. The incidence of subconductance states is not appreciably enhanced in the presence of Mg2+.     

Episodic ataxia/myokymia mutations functionally expressed in the Shaker potassium channel.

Episodic ataxia type 1 is a rare, autosomal dominant neurological disorder caused by missense mutations of the Kv1.1 gene from the Shaker K+ channel subfamily. To study the functional effects of the disease-causing mutations in a robust K+ channel background, we introduced seven different episodic ataxia type 1 substitutions into the corresponding, conserved residues of the Shaker K+ channel. K+ channel currents expressed in Xenopus oocytes were studied by electrophysiology. All episodic ataxia type 1 mutations produced functional K+ channels. In a Shaker N-terminal deletion mutant with fast inactivation removed, current amplitudes were significantly reduced in channels harboring an episodic ataxia type 1 mutation. Six of the seven mutations also showed depolarizing shifts (+9 to +36 mV) in the conductance voltage dependence. One mutation (F307I) shifted the midpoint of the conductance-voltage relationship by 23 mV in the hyperpolarizing direction. Episodic ataxia type 1 mutations were also expressed in ShakerH4 with intact N-terminal inactivation. In this construct, current amplitudes for episodic ataxia type 1 mutants were not significantly different from wild-type channels. All mutations altered the voltage range of steady-state inactivation; most changes were coupled to the changes in activation gating. Some episodic ataxia type 1 mutants also caused significant changes in the kinetics of N-type (F307I, E395D) or C-type (F307I, E395D, V478A) inactivation. These results suggest that episodic ataxia type 1 mutations may change K+ channel function by two mechanisms: (i) reduced channel expression and (ii) altered channel gating.     

The E326K mutation and Gaucher disease: mutation or polymorphism?

Gaucher disease is caused by mutations in the gene for human glucocerebrosidase, a lysosomal enzyme involved in the intracellular hydrolysis of glucosylceramide. While over 150 different glucocerebrosidase mutations have been identified in patients with Gaucher disease, not all reported mutations have been fully characterized as being causative. One such mutation is the E326K mutation, which results from a G to A nucleotide substitution at genomic position 6195 and has been identified in patients with type 1, type 2 and type 3 Gaucher disease. However, in each instance, the E326K mutation was found on the same allele with another glucocerebrosidase mutation. Utilizing polymerase chain reaction (PCR) screening and restriction digestions of both patients with Gaucher disease and normal controls, we identified the E326K allele in both groups. Of the 310 alleles screened from patients with Gaucher disease, the E326K mutation was detected in four alleles (1.3%). In addition, screening for the E326K mutation among normal controls from a random population revealed that three alleles among 316 screened (0.9%) also carried the E326K mutation. In the normal controls with the E326K allele, the glucocerebrosidase gene was completely sequenced, but no additional mutations were found. Because the E326K mutation may be a polymorphism, we caution that a careful examination of any allele with this mutation should be performed to check for the presence of other glucocerebrosidase mutations.     

Attenuated renal excretion in response to thiazide diuretics in Gitelman's syndrome: a case report

Gitelman's syndrome is a variant of Bartter's syndrome characterized by hypocalciuria and hypomagnesemia. The administration of thiazide diuretics may induce a subnormal increase of urinary Na+ and Cl- excretion in patients with Gitelman's syndrome, consistent with the hypothesis that less Na+ and Cl- than normal is reabsorbed by the thiazide-inhibitable transporter in Gitelman's syndrome. Specific mutations of NaCl cotransporter, coupled with mutant NaCl cotransporter expression studies clearly demonstrated that many of the characteristics of individuals with Gitelman's syndrome are explained by lack of function of NaCl cotransporter. We recently diagnosed a patient with Gitelman's syndrome by performing the thiazide and furosemide tests, and it is suggested that the clearance studies by diuretic administration may be of diagnostic help in Gitelman's syndrome.     

Novel mutations in type 2 Gaucher disease in Chinese and their functional characterization by heterologous expression

We investigated 10 unrelated Chinese patients with type 2 Gaucher disease and performed ex vivo expression for the novel mutations to characterize their functional defects. These patients were diagnosed by enzymatic assays and clinicopathologic features over the past five years in a national centre in China. Genomic DNA was sequenced by a two-stage PCR approach for mutations in the functional GBA gene. Novel mutations were expressed with baculovirus-transfected Sf21 cells. Six novel mutations were found (in traditional nomenclature): P122L, Y363C, N382K, L383R, L385P, and M416V. Review of reported mutations indicated clustering of type 2 mutations in three regions of the GBA gene. Expression of novel mutations revealed that the enzyme defect could arise from one of two mechanisms: loss of catalytic activity (Y363C and M416V) or enzyme instability (P122L and N382K).     

Identification of three novel mutations (E196K, V203I, E211Q) in the prion protein gene (PRNP) in inherited prion diseases with Creutzfeldt-Jakob disease phenotype

Inherited prion diseases are characterized by mutations in the PRNP gene encoding the prion protein (PrP). As the other sporadic or infectious prion disease forms, they are almost all characterized by the accumulation in the brain of an abnormal misfolded form of the patient's PrP. Brain extracts can often transmit the disease once inoculated in a recipient animal. Inherited prion diseases with Creutzfeldt-Jakob disease (CJD) phenotype are autosomal forms, although sporadic cases have been reported. We report three novel mutations of the PRNP gene in unrelated patients with clinical and histopathologic features of CJD. The three mutations were missense: c635G>A (E196K), c656G>A (V203I) and c680G>C (E211Q). Familial history of neurologic disorders was evidenced for patients carrying the E196K and E211Q mutations. E196K would be predicted to have more severe effects on protein stability than V203I and E211Q. These mutations expand the spectrum of mutations in PRNP and reduce the proportion of CJD patients in whom genetic alterations have not been found.     

Excretion of the mono- and divalent ions in relation to flow rate in bartter's and pseudo bartter's syndromes in parotid saliva. comparative studies to the syndrome of conn

Summary In three patients, one with Bartter's syndrome and two with pseudo-Bartter's syndrome, the excretion pattern of mono- and divalent ions of the parotid saliva was studied as a function of flow rate. The results were compared with those observed in normal adults and in two patients with Conn's syndrome.In both Bartter's and pseudo-Bartter's syndromes, salivary K⁺ secretion was more than two times higher if compared with the corresponding flow rates. Other symptoms of the complex alterations of electrolyte transport in Bartter's and pseudo-Bartter's syndromes were the increased salivary excretion of calcium, magnesium, bicarbonate and inorganic phosphorus. Na⁺ reabsorption was slightly decreased in the patient with true Bartter's syndrome, and moderately increased in pseudo-Bartter's syndrome.The typical feature in the two patients with Conn's syndrome showed to be an excessive increase of salivary sodium reabsorption. The total amount of sodium actively reabsorbed by the duct system (J*_ac), exceeded the normal range by about a factor of 2–3. Simultaneously, and in contrast to Bartter's syndrome, the flow-dependent concentrations of bicarbonate were markedly and those of magnesium slightly lower. The concentrations of potassium and inorganic phosphorus were moderately elevated, whereas those of calcium were markedly increased.Quantitative as well as qualitative differences in the salivary electrolyte patterns in these diseases provide strong arguments against an exclusive role of aldosterone in the pathogenesis of the electrolyte disturbances in Bartter's and pseudo-Bartter's syndrome.Experiments on human parotid glands clearly demonstrate that alterations of transepithelial electrolyte transport in this disease are not only confined to the different segments of the renal tubulus, but seem to be a symptom of a generalized disturbance of electrolyte transport.Supported by the Deutschen Forschungsgemeinschaft.     

The molecular genetic approach to ”Bartter’s syndrome”

 The term ”Bartter’s syndrome” comprises a set of autosomal recessively inherited renal tubular disorders characterized by hypokalemia, metabolic alkalosis, hyperreninism, and hyperaldosteronism but normal blood pressure. Additional clinical and biochemical features led to a classification into phenotypically different tubulopathies: Gitelman’s syndrome, hyperprostaglandin E syndrome (antenatal Bartter’s syndrome), and classic Bartter’s syndrome. Gitelman’s syndrome results from mutations in the SLC12A3 gene encoding the human thiazide-sensitive sodium chloride cotransporter, leading to impaired reabsorption of sodium chloride in the distal convoluted tubule. Genetic heterogeneity of hyperprostaglandin E syndrome has been demonstrated by identification of mutations in the SLC12A1 gene as well as in the KCNJ1 gene. Mutations in SLC12A1 coding for the bumetanide-sensitive sodium potassium 2 chloride cotransporter (NKCC2) cause defective reabsorption of sodium chloride in the thick ascending limb of Henle’s loop. Mutations in KCNJ1 leading to loss of function of the potassium channel ROMK disrupt potassium recycling back to the tubule lumen and inhibit thereby the NKCC2 activity. A third gene for hyperprostaglandin E syndrome has been mapped to the short arm of chromosome 1, and it remains to be evaluated whether other genes are involved in the pathogenesis of this disease. Classic Bartter’s syndrome has been demonstrated to result from defective chloride transport across the basolateral membrane in the distal nephron due to mutations in the chloride channel gene CLCNKB. This article reviews the molecular genetic approach that has led to identification of genetic defects underlying the different hypokalemic tubulopathies. Received: 23 April 1997 / Accepted: 21 August 1997     

C112R, W323S, N317K mutations in the vasopressin V2 receptor gene in patients with nephrogenic diabetes insipidus. Mutations in brief no. 165. Online

Nephrogenic diabetes insipidus (NDI) is a rare, mostly X-linked recessive disorder characterized by renal tubular resistance to the antidiuretic effect of arginine vasopressin. The gene responsible for the X-linked NDI, the G-protein-coupled vasopressin V2 receptor, has been localized on the Xq28 region. In this study we present three NDI families from Hungary with three different missense mutations in the vasopressin V2 receptor gene. After the mutations in the affected probands in each family had been characterized, other family members were screened by restriction enzyme analysis. The N317K and W323S mutations have not been detected previously. The C112R is an already known mutation. The N317K was a de novo mutation in the patient. The C112R and the W323S were found in the mothers of the patients as carriers and in all other patients, but not in the unaffected members of the families. Segregation of the mutations was consistent with the clinically observed symptoms as well as their severity. As conclusion, these findings further evidence that X-linked NDI results from defects in the V2 receptor gene.     

Generation and analysis of the thiazide‐sensitive Na+‐Cl− cotransporter (Ncc/Slc12a3) Ser707X knockin mouse as a model of Gitelman syndrome

Gitelman syndrome (GS) is characterized by salt‐losing hypotension, hypomagnesemia, hypokalemic metabolic alkalosis, and hypocalciuria. To better model human GS caused by a specific mutation in the thiazide‐sensitive Na⁺‐Cl^? cotransporter (NCC) gene SLC12A3, we generated a nonsense Ncc Ser707X knockin mouse corresponding to human p.Ser710X (c.2135C>A), a recurrent mutation with severe phenotypes in Chinese GS patients. Compared with wild‐type or heterozygous littermates, homozygous (Hom) knockin mice fully recapitulated the phenotype of human GS. The markedly reduced Ncc mRNA and virtually absent Ncc protein expression in kidneys of Hom mice was primarily due to nonsense‐mediated mRNA decay (NMD) surveillance mechanisms. Expression of epithelial Na⁺ channel (Enac), Ca²⁺ channels (Trpv5 and Trpv6), and K⁺ channels (Romk1 and maxi‐K) were significantly increased. Late distal convoluted tubules (DCT) volume was increased and DCT cell ultrastructure appeared intact. High K⁺ intake could not correct hypokalemia but caused a further increase in maxi‐K but not Romk1 expression. Renal tissue from a patient with GS also showed the enhanced TRPV5 and ROMK1 expression in distal tubules. We suggest that the upregulation of TRPV5/6 and of ROMK1 and Maxi‐K may contribute to hypocalciuria and hypokalemia in Ncc Ser707X knockin mice and human GS, respectively. Hum Mutat 31:1–13, 2010. © 2010 Wiley‐Liss, Inc.     

Cation permeation and blockade of ROMK1, a cloned renal potassium channel

 ROMK1 is an inwardly rectifying K⁺ channel cloned from the outer medulla of rat kidney. We have determined the permeation and blocking characteristics for several monovalent cations in ROMK1 when expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique. The selectivity sequence for monovalent cations as determined by reversal potential changes under bi-ionic conditions was K⁺ > Rb⁺ > Cs⁺ = NH4⁺ >> Na⁺ = Li⁺. The conductivity for the two permeant ions K⁺ and Rb⁺ was a saturable function of the external concentration, with K _m values of 11.5 ± 1.3 mmol l^–1 (n = 19) and 47.3 ± 4.8 mmol l^–1 (n = 19), respectively. With mixtures of K⁺ and Rb⁺, the conductance went through a clear minimum as the concentration ratio [Rb]/[K+Rb] was varied between 0 and 1. ROMK1 was blocked by both Cs⁺ and Ba²⁺ in a concentration- and voltage-dependent manner. The electrical distance (δ) at which Ba²⁺ and Cs⁺ blocked the channel was 0.41 and 0.69, respectively, suggesting that these two ions block at different sites within the pore. Taken together with previous reports, the results indicate that ROMK1 has a multi-ion pore, and that the N-terminus contributes to the pore structure. Received: 30 September 1996 / Received after revision: 30 January 1997 / Accepted: 18 February 1997