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We investigate the genetic etiology in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of a mild and transient form of pseudohypoaldosteronism type 1 (PHA1). Twelve patients with PHA1 from four different families with clinical and biochemical data were analyzed. The coding regions of NR3C2 and SCNN1A genes were sequenced. Human α-epithelial sodium channel (ENaC) wild-type (wt), αPhe226Cys and aPhe226Ser ENaC variants were expressed in Xenopus laevis oocytes to evaluate ENaC activity. The protein expression of α-ENaC wt and mutants was determined by Western blot. All patients were homozygotes for the p. Phe226Cys mutation of the α subunit of ENaC. In functional studies in X. laevis oocytes, p.Phe226Cys caused a significant reduction of ENaC activity (83% reduction), reduced the number of active ENaC mutant channels, and reduced the basal open probability compared with wt. Quantitative Western blot analysis revealed that the reduced activity of ENαC mutant channels was due to a reduced ENaC protein expression for the αPhe226Cys compared with wt. We present 12 patients from four different families with a mild and transient autosomal recessive PHA1 due to a novel homozygous missense mutation in the SCNN1A gene. Functional studies showed that the p.Phe226Cys substitution mutation in ENaC leads to a partial loss of function resulting mainly from both a decrease in the intrinsic ENaC activity and a reduction in channel expression at the protein level. The partial loss of ENaC function could explain the mild phenotype, variable expressivity, and the transient course of the disorder in these patients. [ABSTRACT FROM AUTHOR]

Harmonic nanoparticles (HNPs) have emerged as appealing exogenous probes for optical bioimaging due to their distinctive features such as long-term photostability and spectral flexibility, allowing multiphoton excitation in the classical (NIR-I) and extended near-infrared spectral windows (NIR-II and -III). However, like all other optical labels, HNPs are not suitable for whole-body imaging applications. In this work, we developed a bimodal nonlinear optical/magnetic resonance imaging (MRI) contrast agent through the covalent conjugation of Gd-(III) chelates to coated lithium niobate HNPs. We show that the resulting nanoconjugates exert strong contrast both in T1-weighted MRI of agarose gel-based phantoms and in cancer cells by harmonic generation upon excitation in the NIR region. Their capabilities for dual T1/T2 MRI were also emphasized by the quantitative mapping of the phantom in both modes. The functionalization protocol ensured high stability of the Gd-functionalized HNPs in a physiological environment and provided a high r1 relaxivity value per NP (5.20 × 105 mM–1 s–1) while preserving their efficient nonlinear optical response. [ABSTRACT FROM AUTHOR]

The epithelial [Na.sup.+] channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial [Na.sup.+] transport, mechano-transduction, and neurotransmission. The role of ENaC in [Na.sup.+] homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC [Beta] and [Gamma] subunits causing hypertension. The function of ENaC in [Na.sup.+] reabsorption depends critically on its ability to discriminate between [Na.sup.+] and other ions like [K.sup.+] or [Ca.sup.2+]. ENaC is virtually impermeant to [K.sup.+] ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC [Alpha] subunit ([Alpha]S589), which when mutated allows larger ions such as [K.sup.+], [Rb.sup.+], [Cs.sup.+], and divalent cations to pass through the channel. The relative ion permeability of each of the [Alpha]S589 mutants is related inversely to the ionic radius of the permeant ion, indicating that [Alpha]S589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate [Na.sup.+] and [Li.sup.+] ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.

Gly and Ser, the amino acid residues present in the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC), reduce the channel's affinity for amiloride block and prevent ion permeation, on mutation. The alpha subunit determines the pharmacological properties of the channel. The mutations in beta and gamma unit alter amiloride affinity and single channel conductances and activate binding sites for channel block by divalent cations. This indicates that these subunits participate in ion permeation through the channel.

Liddle syndrome is a mendelian form of hypertension characterized by constitutively elevated renal Na reabsorption that can result from activating mutations in the [beta] or [gamma] subunit of the epithelial Na channel. All reported mutations have deleted the last 45-76 normal amino acids from the cytoplasmic C terminus of one of these channel subunits. While these findings implicate these terminal segments in the normal negative regulation of channel activity, they do not identify the amino acid residues that are critical targets for these mutations. Potential targets include the short highly conserved Pro-rich segments present in the C terminus of [beta] and [gamma] subunits; these segments are similar to SH3-binding domains that mediate protein-protein interaction. We now report a kindred with Liddle syndrome in which affected patients have a mutation in codon 616 of the [beta] subunit resulting in substitution of a Leu for one of these highly conserved Pro residues. The functional significance of this mutation is demonstrated both by the finding that this is a de novo mutation appearing concordantly with the appearance of Liddle syndrome in the kindred and also by the marked activation of amiloride-sensitive Na channel activity seen in Xenopus oocytes expressing channels containing this mutant subunit (8.8-fold increase compared with control oocytes expressing normal channel subunits; P = 0.003). These findings demonstrate a de novo missense mutation causing Liddle syndrome and identify a critical channel residue important for the normal regulation of Na reabsorption in humans.

Alpha, beta and gamma subunits of the rat epithelial sodium channel and mec-4, mec-10 and deg 1 proteins belong to a new gene family which controls cellular volume. The epithelial Na+ channel is sensitive to amiloride and has ion-selective permeability, gating properties and pharmacological profile, decided by the coexpression of component subunits. The gene family may also be crucial for mechano-sensitive ion channels in various organisms and animal cells.

Abstract Background Rare mutations of the epithelial sodium channel (ENaC) result in the monogenic hypertension form of Liddle's syndrome. We decided to screen for common variants in the ENaC βand γ subunits in patients with essential hypertension and to relate their occurrence to the activity of circulating renin-angiotensin-aldosterone system. Methods Initially, DNA samples from 27 patients with low renin/low aldosterone hypertension were examined. The DNA variants were subsequently screened for in 347 patients with treatment-resistant hypertension, 175 male subjects with documented long-lasting normotension and 301 healthy Plasma renin and aldosterone levels were measured under baseline conditions and during postural and captopril challenge tests. Results Two commonly occurring βENaC variants (G589S and a novel intronic i12-17CT substitution) and one novel γENaC variant (V546I) were detected. One of these variants occurred in a heterozygous form in 32 patients, a prevalence (9.2%) significantly higher than that in normotensive males (2.9%, p = 0.007) and blood donors (3.0%, p = 0.001). βENaC i12-17CT was significantly more prevalent in the hypertension group than in the two control groups combined (4.6% vs. 1.1%, p = 0.001). When expressed in Xenopus oocytes, neither of the two ENaC amino acid-changing variants showed a significant difference in activity compared with ENaC wild-type. No direct evidence for a mRNA splicing defect could be obtained for the βENaC intronic variant. The ratio of daily urinary potassium excretion to upright and mean (of supine and upright values) plasma renin activity was higher in variant allele carriers than in non-carriers (p = 0.034 and p = 0.048). Conclusions At least 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γENaC, a three times higher prevalence than in the normotensive individuals or in random healthy controls. Patients with the variant alleles showed an increased urinary potassium excretion rate in relation to their renin levels.