Basal Open Probability Research Articles

A mild and transient form of autosomal recessive pseudohypoaldosteronism type 1 caused by a novel mutation in the SCNN1A gene.

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 αPhe226Ser 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 ENαC 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.NEW & NOTEWORTHY This paper demonstrates that mild autosomal recessive pseudohypoaldosteronism type 1 (PHA1) due to p.Phe226Cys missense mutation in the extracellular domain of ENαC α subunit can be transient, with phenotypic variability even with the normal sweat test, and incomplete penetrance. Functional studies explain the phenotype and denote the importance of the location on the extracellular domain of the SCNN1A p.Phe226Cys mutation for the intrinsic ENaC activity and the channel expression at the protein level.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during β-adrenergic stimulation, and in heart failure. We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and β-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVβ to α1C I-II loop and eliminated β-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to β-adrenergic agonists when reconstituted heterologously with β2B and Rad or transgenically expressed in cardiomyocytes. Ca2+ channel activity is dynamically modulated under basal conditions, during β-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVβ binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the β-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by β-adrenergic agonists/PKA also requires this rigid linker and β-binding to α1C.

Genotype‒Phenotype Correlation of TRPV3-Related Olmsted Syndrome

We have previously shown that gain-of-function variations in transient receptor potential vanilloid-3 (TRPV3) underlay Olmsted syndrome, a rare hyperkeratotic skin channelopathy. In this study, we attempt to establish a genotype‒phenotype correlation in Olmsted syndrome, which has been unclear owing to the rarity and heterogeneity of the condition. We identified five previously unreported TRPV3 variations (R416Q, R416W, L655P, W692S, and L694P) and three recurrent variations (G568D, G568V, and L673F) in nine unrelated patients. Seven variants were expressed in human embryonic kidney 293 cells, and channel behavior was characterized electrophysiologically, with results compared with the clinical severity. These variant TRPV3 channels, in either homomeric or heteromeric form, exhibited differentially elevated basal open probability, increased voltage sensitivity, and cytotoxicity. Functional changes were particularly pronounced in variants corresponding to severer Olmsted syndrome (e.g., L673F and W692S) but not in mild Olmsted syndrome variants (e.g., R416Q). Interestingly, the extent of functional rescue by wild-type TRPV3 invitro was also consistent with the clinical severity of the variants. These findings, in combination with all reported cases, indicate a preliminary genotype‒phenotype correlation, that is, variations in the S4‒S5 linker and transient receptor potential domain of TRPV3 significantly enhance channel function, causing severe phenotype, whereas other variations appear to exert milder effects on channel function and disease phenotype.

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels.

We recently showed that lovastatin attenuates cyclosporin A (CsA)-induced damage of cortical collecting duct (CCD) principal cells by reducing intracellular cholesterol. Previous studies showed that, in cell expression models or artificial membranes, exogenous cholesterol directly inhibits inward rectifier potassium channels, including Kir1.1 (Kcnj1; the gene locus for renal outer medullary K(+) [ROMK1] channels). Therefore, we hypothesized that lovastatin might stimulate ROMK1 by reducing cholesterol in CCD cells. Western blots showed that mpkCCDc14 cells express ROMK1 channels with molecular masses that approximate the molecular masses of ROMK1 in renal tubules detected before and after treatment with DTT. Confocal microscopy showed that ROMK1 channels were not in the microvilli, where cholesterol-rich lipid rafts are located, but rather, the planar regions of the apical membrane of mpkCCDc14 cells. Furthermore, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], an activator of ROMK channels, was detected mainly in the microvilli under resting conditions along with the kinase responsible for PI(4,5)P2 synthesis, phosphatidylinositol-4-phosphate 5-kinase, type I γ [PI(4)P5K I γ], which may explain the low basal open probability and increased sensitivity to tetraethylammonium observed here for this channel. Notably, lovastatin induced PI(4)P5K I γ diffusion into planar regions and elevated PI(4,5)P2 and ROMK1 open probability in these regions through a cholesterol-associated mechanism. However, exogenous cholesterol alone did not induce these effects. These results suggest that lovastatin stimulates ROMK1 channels, at least in part, by inducing PI(4,5)P2 synthesis in planar regions of the renal CCD cell apical membrane, suggesting that lovastatin could reduce cyclosporin-induced nephropathy and associated hyperkalemia.

Basal calcium entry in vascular smooth muscle

Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring 45Ca 2+ uptake into cultured rat aortic smooth muscle cells. Wash solution (0 °C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular 45Ca 2+ retention more effectively than EGTA (0.2 mM). Basal Ca 2+ entry was 1.45×10 9 Ca 2+·cell −1·min −1. This translated to ∼250 μmol −1·min −1 given cell volumes of 4–15 pl as determined by 3-D image reconstruction. Gadolinium (100 μM) blocked 80% of the leak and exhibited a biphasic concentration–response relation (IC 50s=1 μM and 2 mM). Organic ion channel blockers also inhibited ∼80% of the leak; 45% by nifedipine (10 μM), 7% was exclusively blocked by SKF 96365 (1-[ b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole) (50 μM) and 23% was exclusively sensitive to 2-aminoethoxy-diphenylborate (2-APB, 75 μM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca 2+ entry is mainly due to a basal open probability of excitable Ca 2+-channels.

Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters.

Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex, causes cardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Using cultured myotubes prepared from skeletal muscle of normal and Bio14.6 hamsters (J2N-k strain), we investigated the possibility that the delta-SG deficiency may lead to alterations in ionic conductances, which may ultimately lead to myocyte damage. In cell-attached patches (with Ba(2+) as the charge carrier), an approximately 20-pS channel was observed in both control and Bio14.6 myotubes. This channel is also permeable to K(+) and Na(+) but not to Cl(-). Channel activity was increased by pressure-induced stretch and was reduced by GdCl(3) (>5 microM). The basal open probability of this channel was fourfold higher in Bio14.6 myotubes, with longer open and shorter closed times. This was mimicked by depolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes, the unidirectional basal Ca(2+) influx was enhanced compared with control. This Ca(2+) influx was sensitive to GdCl(3), signifying that stretch-activated cation channels may have been responsible for Ca(2+) influx in Bio14.6 hamster myotubes. These results suggest a possible mechanism by which cell damage might occur in this animal model of muscular dystrophy.

Regulation of Large Calcium-activated Potassium Channels by Protein Phosphatase 2A

Vasodilating agents induce relaxation of mesangial cells, in part through cGMP-mediated activation of large calcium-activated potassium channels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cGMP (Bt2cGMP) is characterized by a biphasic increase and then decrease ("rundown") in open probability. Using the patch-clamp method in conjunction with phosphatase inhibitors, we investigated whether the run-down phase was the result of dephosphorylation by an endogenous protein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentrations, caused a significantly greater and sustained response of BKCa to Bt2cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt2cGMP was greater than the response to these agents added separately. Incubation of mesangial cells with okadaic acid for 20 min at a concentration (5 nM) specific for PP2A increased the basal open probability of BKCa and completely inhibited rundown after activation by Bt2cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BKCa activity. In inside-out patches, Bt2cGMP plus MgATP caused a sustained activation of BKCa that was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common substrate for endogenous cGMP-activated protein kinase, which activates BKCa, and PP2A, which inactivates BKCa, in human mesangial cells.

Permselectivity of a porous phospholipid-cholesterol artificial membrane. Calcium and lanthanum effects

Basal calcium leak into smooth muscle was identified 30 years ago yet remains poorly understood. We characterized this leak measuring 45Ca2+ uptake into cultured rat aortic smooth muscle cells. Wash solution (0 °C) containing lanthanum (3 mM) removed extracellular tracer and increased cellular 45Ca2+ retention more effectively than EGTA (0.2 mM). Basal Ca2+ entry was 1.45×109 Ca2+·cell−1·min−1. This translated to ∼250 μmol−1·min−1 given cell volumes of 4–15 pl as determined by 3-D image reconstruction. Gadolinium (100 μM) blocked 80% of the leak and exhibited a biphasic concentration–response relation (IC50s=1 μM and 2 mM). Organic ion channel blockers also inhibited ∼80% of the leak; 45% by nifedipine (10 μM), 7% was exclusively blocked by SKF 96365 (1-[b-[3-(4-Methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole) (50 μM) and 23% was exclusively sensitive to 2-aminoethoxy-diphenylborate (2-APB, 75 μM). Reverse transcriptase polymerase chain reaction revealed TrpC1, 4 and 6 mRNA, and we propose that 2-APB may selectively block TrpC4-containing channels. We conclude that basal Ca2+ entry is mainly due to a basal open probability of excitable Ca2+-channels.