Frequent SLC12A3 mutations in Chinese Gitelman syndrome patients: structure and function disorder

Purposes: This study was conducted to identify the frequent mutations from reported Chinese Gitelman syndrome (GS) patients, to predict three-dimensional structure change of human Na-Cl co-transporter (hNCC), and to test the activity of these mutations and some novel mutations in vitro and in vivo. Methods: SLC12A3 gene mutations in Chinese GS patients previously reported in the PubMed, CNKI and Wanfang database were summarized. Predicted configurations of wild type (WT) and mutant proteins were achieved using the I-TASSER workplace. Six missense mutations (T60M, L215F, D486N, N534K, Q617R and R928C) were generated by site-directed mutagenesis. 22Na+ uptake experiment was carried out in the Xenopus laevis oocyte expression system. 35 GS patients and 20 healthy volunteers underwent the thiazide test. Results: T60M, T163M，D486N, R913Q, R928C and R959 frameshift were frequent SLC12A3 gene mutations (mutated frequency >3%) in 310 Chinese GS families. The protein’s three-dimensional structure was predicted to be altered in all mutations. Compared with WT hNCC, the thiazide-sensitive 22Na+ uptake was significantly diminished for all 6 mutations: T60M 22±9.2%, R928C 29±12%, L215F 38±14%, N534K 41±15.5%, Q617R 63±22.1% and D486N 77±20.4%. In thiazide test, the net increase in chloride fractional excretion in 20 healthy controls was significantly higher than GS patients with or without T60M or D486N mutations. Conclusions: Frequent mutations (T60M, D486N, R928C) and novel mutations (L215F, N534K and Q617R) lead to protein structure alternation and protein dysfunction verified by 22Na+ uptake experiment in vitro and thiazide test on patients.

Structure and transport mechanism of the sodium/proton antiporter MjNhaP1

Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 in two complementary states. The inward-open state was obtained by x-ray crystallography in the presence of sodium at pH 8, where the transporter is highly active. The outward-open state was obtained by electron crystallography without sodium at pH 4, where MjNhaP1 is inactive. Comparison of both structures reveals a 7° tilt of the 6 helix bundle. 22Na+ uptake measurements indicate non-cooperative transport with an activity maximum at pH 7.5. We conclude that binding of a Na+ ion from the outside induces helix movements that close the extracellular cavity, open the cytoplasmic funnel, and result in a ∼5 Å vertical relocation of the ion binding site to release the substrate ion into the cytoplasm.

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K+ ingestion or rest after exercise. Force can be restored by muscle work or treatment with β2-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na+ channel (Nav1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K+]o. In resting mutant soleus, tetrodotoxin (TTX)-suppressible 22Na uptake and [Na+]i were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na+,K+ pump–mediated 86Rb uptake was 83% larger than in WT. Salbutamol stimulated 86Rb uptake and reduced [Na+]i both in mutant and WT soleus. Stimulating Na+,K+ pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na+]i with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na+,K+ pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na+]i on the synthesis of Na+,K+ pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na+ influx and show that contractility can be restored by acute stimulation of the Na+,K+ pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in 86Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP.

Peripheral blood mononuclear cells express mutated NCCT mRNA in Gitelman's syndrome: evidence for abnormal thiazide-sensitive NaCl cotransport.

Genetic analysis has demonstrated complete linkage between the human thiazide-sensitive sodium chloride cotransporter gene (NCCT or TSC) and Gitelman's syndrome (GS). Several genomic NCCT mutations have been reported. This study was performed to determine whether peripheral blood mononuclear cells (PBMC) express NCCT mRNA and whether defective PBMC NaCl cotransport could be demonstrated in GS. PBMC were isolated from two brothers with GS, their parents, and healthy control subjects. Northern analysis revealed that NCCT mRNA is expressed in PBMC. The sequence of full-length NCCT cDNA amplified from normal PBMC was identical to human renal NCCT cDNA. Two different mutations were detected in the patients' NCCT cDNA (compound heterozygote). In cDNA derived from the patient's maternal allele, exon 24 was deleted, resulting in a premature stop codon (after amino acid 920). cDNA derived from the patient's paternal allele had an additional 119-bp insertion between exons 3 and 4, generating a premature stop codon (after amino acid 187). The patient's genomic DNA had a previously described 5' splice site mutation in intron 24, GGT --> GTT (maternal allele), and a new 3' splice site mutation in intron 3, CAG --> CAA (paternal allele), which resulted in the activation of a nearby cryptic splice site in intron 3. The latter mutation was not present in 300 normal chromosomes. To determine the functional significance of these findings, chlorothiazide-inhibitable 22Na uptake was measured in PBMC from control subjects, the parents, and the patients with GS in the presence of bumetanide. In control PBMC, chlorothiazide inhibited 22Na uptake by approximately 9%. PBMC from the two patients with GS failed to respond to chlorothiazide. These results demonstrate that PBMC can be used for mutational analysis of NCCT mRNA in patients with GS. Furthermore, functional evidence is provided that the underlying cause of GS is defective NCCT NaCl cotransport.

Inhibition of amiloride-sensitive Na+ channel by isothiouronium derivatives

The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.