A novel sodium channel mutation causing a hyperkalemic paralytic and paramyotonic syndrome with variable clinical expressivity

BACKGROUND: The CarboMedics valve is a relatively new, low-profile, bileaflet, mechanical prosthesis. The results of a prospective follow-up study after valve replacement with this prosthesis in a university hospital are presented. METHODS: We implanted 640 CarboMedics prostheses in 583 patients in the aortic (n = 359), mitral (n = 167), or aortic and mitral positions (double valve replacement; n = 57). Patient ages ranged from 11 to 81 years (mean age, 58 +/- 12.3 years). RESULTS: Overall hospital mortality was 9.0%; however, when high-risk urgent cases were removed from the calculation, the operative mortality fell to 4.5%. Follow-up was 98% complete, comprising 2,027 patient-years for a mean follow-up of 44 months (range, 6 to 72 months). Actuarial freedom from complications (linearized rates in parentheses) was as follows: late mortality, 85% +/- 2.0% (2.3%/patient-year); thromboembolism, 92% +/- 1.1% (1.6%/patient-year); anticoagulation-related hemorrhage, 87% +/- 1.2% (2.8%/patient-year); prosthetic valve endocarditis, 98% +/- 0.5% (0.1%/patient-year); and overall valve-related morbidity and mortality, 76% +/- 2.1% (4.3%/ patient-year). CONCLUSIONS: The CarboMedics valve shows a low rate of valve-related complications comparable with other new mechanical heart valve prostheses.     

Functional communication between cardiac ATP-sensitive K+ channel and Na/K ATPase

Insecticide resistance often is blamed for failures of insecticides to control cat fleas, Ctenocephalides felis (Bouché). Yet the genetics and adaptive advantage of resistance traits remain unexamined. Lethal doses of insecticides that kill 50% of the population fluctuate 7-fold within a cat flea strain. Many reports of flea resistance may be attributable to variable mortality from effects of solvents, substrates, humidities, temperatures, colonization, and ages of fleas. Resistance ratios (ratios of lethal doses of a resistant to a susceptible strain) are < 690-fold in fleas; lower than many other arthropods. This, plus strain variability, hinders resistance detection. Relationships between resistance levels, control failures, and health threats are unclear. Insensitive acetylcholinesterase, knockdown recovery, glutathione transferase conjugation, and mixed function oxidase/cytochrome P450 are demonstrated resistance mechanisms in cat fleas. Ecological genetics of resistance in cat fleas probably involves flea transfer among hosts, host movements, refugia, founder effects, and mortality from abiotic factors. Understanding cat flea resistance requires population monitoring before, during, and after insecticide treatments using conventional and rapid molecular bioassays. Sustained insecticide release devices such as flea collars and long-lived insecticide residues for premises possibly contribute to the development of resistance. New systemic and topical insecticides, especially when given prophylactically, may act similarly. Eliminating insecticides prevents insecticide resistance but necessitates application of biorational tactics incorporating mechanical, environmental, and cultural controls. Using high temperatures, low humidities, host grooming and such tactics as decreasing doses, increasing action thresholds, rotating insecticides, and leaving spatial and temporal refugia may suppress cat flea resistance.     

dGNaC1, a gonad-specific amiloride-sensitive Na+ channel

Amiloride-sensitive sodium channels have been implicated in reproductive and early developmental processes of several species. These include the fast block of polyspermy in Xenopus oocytes that follows the sperm binding to the egg or blastocoel expansion in mammalian embryo. We have now identified a gene called dGNaC1 that is specifically expressed in the gonads and early embryo in Drosophila melanogaster. The corresponding protein belongs to the superfamily of cationic channels blocked by amiloride that includes Caenorhabditis elegans degenerins, the Helix aspersa FMRF-amide ionotropic receptor (FaNaC), the mammalian epithelial Na+ channel (ENaC), and acid-sensing ionic channels (ASIC, DRASIC, and MDEG). Expression of dGNaC1 in Xenopus oocytes generates a constitutive current that does not discriminate between Na+ and Li+, but is selective for Na+ over K+. This current is blocked by amiloride (IC50 = 24 microM), benzamil (IC50 = 2 microM), and ethylisopropyl amiloride (IC50 = 49 microM). These properties are clearly different from those obtained after expression of the previously cloned members of this family, including ENaC and the human alphaENaC-like subunit, deltaNaC. Interestingly, the pharmacology of dGNaC1 is not very different from that found for the Na+ channel characterized in rabbit preimplantation embryos. We postulate that this channel may participate in gametogenesis and early embryonic development in Drosophila.     

Functional consequences of a posttransfection mutation in the H2-H3 cytoplasmic loop of the alpha subunit of Na,K-ATPase

During kinetic studies of mutant rat Na,K-ATPases, we identified a spontaneous mutation in the first cytoplasmic loop between transmembrane helices 2 and 3 (H2-H3 loop) which results in a functional enzyme with distinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 to A substitution, which resulted in the replacement of glutamate at 233 with a lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cells and their kinetic behavior was compared. Transport studies carried out under physiological conditions with intact cells indicate that the E233K mutant and alpha1 have similar apparent affinities for cytoplasmic Na+ and extracellular K+. In contrast, distinct kinetic properties are observed when ATPase activity is assayed under conditions (low ATP concentration) in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1 microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase of E233K. This distinctive behavior of E233K is due to its faster rate of formation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as well as 6-fold higher affinity for ATP at the low affinity ATP binding site. A lower ratio of Vmax to maximal level of phosphoenzyme indicates that E233K has a lower catalytic turnover than alpha1. These distinct kinetics of E233K suggest a shift in its E1/E2 conformational equilibrium toward E1. Furthermore, the importance of the H2-H3 loop in coupling conformational changes to ATP hydrolysis is underscored by a marked (2 orders of magnitude) reduction in vanadate sensitivity effected by this Glu233 --> Lys mutation.     

Andersen''s syndrome: a distinct periodic paralysis

PURPOSE: To develop a stable, transluminally created abdominal aortic aneurysm (AAA) within a live animal model. MATERIALS AND METHODS: Eight mongrel dogs were utilized to evaluate a new, catheter-based technique for the creation of an AAA. With use of a standard angioplasty balloon and a balloon-expandable intravascular metallic stent, the infrarenal abdominal aorta was overdilated to twice its measured diameter into a fusiform shape AAA in eight dogs. At 30 days, aortography was performed, the dogs were killed, and the aorta was resected and evaluated for histopathology. RESULTS: Seven of the eight dogs that underwent transluminal AAA creation survived the initial procedure. A stable, fusiform AAA was successfully created in these seven dogs. At 30 days, repeat angiography and histologic examination confirmed that the seven AAAs were still twice the diameter of the normal aorta (a four-fold increase in luminal area), that the branch arteries remained patent, and that the lumen was endothelialized. One of the eight dogs was killed 9 hours after the procedure because of inability to awaken from anesthesia. Gross and histopathologic results in this one dog also demonstrated an intact aorta containing an AAA. CONCLUSIONS: A stable, infrarenal AAA model can be successfully created in the canine species with use of standard catheter-based techniques and equipment. This model can be used to test emerging endovascular treatments of AAA.     

Novel LQT-3 mutation affects Na+ channel activity through interactions between alpha- and beta1-subunits

This study describes mesial and distal enamel thickness of the permanent posterior mandibular dentition. The sample comprised 98 Caucasian adults (59 males, 39 females) 20 to 35 years old. Bitewing radiographs of the right permanent mandibular premolars and first and second molars were illuminated and transferred to a computer at a fixed magnification via a video camera. Enamel and dentin thicknesses were identified and digitized on the plane representing the maximum mesiodistal diameter of each tooth. The results showed that there were no significant sex differences in either mesial or distal enamel thickness. Enamel on the second molars was significantly thicker (0.3 to 0.4 mm) than enamel on the premolars. Distal enamel was significantly thicker than mesial enamel. There was approximately 10 mm of total enamel on the four teeth combined. Assuming 50% enamel reduction, the premolars and molars should provide 9.8 mm of additional space for realignment of mandibular teeth.     

Mutations causing neurodegeneration in Caenorhabditis elegans drastically alter the pH sensitivity and inactivation of the mammalian H+-gated Na+ channel MDEG1

The mammalian degenerin MDEG1 belongs to the nematode degenerin/epithelial Na+ channel superfamily. It is constitutively activated by the same mutations that cause gain-of-function of the Caenorhabditis elegans degenerins and neurodegeneration. ASIC and DRASIC, which were recently cloned, are structural homologues of MDEG1 and behave as H+-gated cation channels. MDEG1 is also a H+-activated Na+ channel, but it differs from ASIC in its lower pH sensitivity and slower kinetics. In addition to the generation of a constitutive current, mutations in MDEG1 also alter the properties of the H+-gated current. Replacement of Gly-430 in MDEG1 by bulkier amino acids, such as Val, Phe, or Thr, drastically increases the H+ sensitivity of the channel (half-maximal pH (pHm) approximately 4.4 for MDEG1, pHm approximately 6.7 for the different mutants). Furthermore, these replacements completely suppress the inactivation observed with the wild-type channel and increase the sensitivity of the H+-gated channel to blockade by amiloride by a factor of 10 without modification of its conductance and ionic selectivity. These results as well as those obtained with other mutants clearly indicate that the region surrounding Gly-430, situated just before the second transmembrane segment, is essential for pH sensitivity and gating.     

Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.     

Subunit stoichiometry of a core conduction element in a cloned epithelial amiloride-sensitive Na+ channel

The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.     

A putative ATP-activated Na+ channel involved in sperm-induced fertilization

Extracellular application of adenosine triphosphate (ATP) to defolliculated Xenopus laevis oocytes activated a saturating inward current with a maximal amplitude E(max) of 2.4 +/- 0.2 microamperes and an apparent Michaelis constant of 197.6 micromolar. The current was carried predominantly by sodium ions and potently inhibited by amiloride, guanosine triphosphate (GTP), and its nonhydrolyzable analogs guanosine 5'-[beta,gamma-imido]triphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate). Likewise, in vitro fertilization using mature eggs and Xenopus sperm was inhibited by amiloride, GTP, and GppNHp. Hence, an ATP receptor on the egg membrane may be the recipient target for ATP originating in sperm, suggesting that an ATP-induced increase in sodium permeability mediates the initial sperm to egg signal in the fertilization process.     

Na+ channel modulation and force-frequency relationship in human myocardium

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line Cl8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of beta-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.     

Functional consequences of a decreased potassium affinity in a potassium channel pore. Ion interactions and C-type inactivation

We have shown previously that the inactivation of macrophages in nonobese diabetic (NOD) mice results in the prevention of diabetes; however, the mechanisms involved remain unknown. In this study, we found that T cells in a macrophage-depleted environment lost their ability to differentiate into beta cell-cytotoxic T cells, resulting in the prevention of autoimmune diabetes, but these T cells regained their beta cell-cytotoxic potential when returned to a macrophage-containing environment. To learn why T cells in a macrophage-depleted environment lose their ability to kill beta cells, we examined the islet antigen-specific immune response and T cell activation in macrophage-depleted NOD mice. There was a shift in the immune balance, a decrease in the T helper cell type 1 (Th1) immune response, and an increase in the Th2 immune response, due to the reduced expression of the macrophage-derived cytokine IL-12. As well, there was a deficit in T cell activation, evidenced by significant decreases in the expression of Fas ligand and perforin. The administration of IL-12 substantially reversed the prevention of diabetes in NOD mice conferred by macrophage depletion. We conclude that macrophages play an essential role in the development and activation of beta cell-cytotoxic T cells that cause beta cell destruction, resulting in autoimmune diabetes in NOD mice.     

Role of the actin cytoskeleton on epithelial Na+ channel regulation

The regulatory role of actin filament organization on epithelial Na+ channel activity is reviewed in this report. The actin cytoskeleton, consisting of actin filaments and associated actin-binding proteins, is essential to various cellular events including the maintenance of cell shape, the onset of cell motility, and the distribution and stability of integral membrane proteins. Functional interactions between the actin cytoskeleton and specific membrane transport proteins are, however, not as well understood. Recent studies from our laboratory have determined that dynamic changes in the actin cytoskeletal organization may represent a novel signaling mechanism in the regulation of ion transport in epithelia. This report summarizes work conducted in our laboratory leading to an understanding of the molecular steps associated with the regulatory role of the actin-based cytoskeleton on epithelial Na+ channel function. The basis of this interaction lies on the regulation by actin-binding proteins and adjacent structures, of actin filament organization which in turn, modulates ion channel activity. The scope of this interaction may extend to such relevant cellular events as the vasopressin response in the kidney.     

Modulation of Na+ channel inactivation by the beta 1 subunit: a deletion analysis

Na+ currents recorded from Xenopus oocytes expressing the Na+ channel alpha subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while co-expression with the beta 1 subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (approximately 30-fold vs approximately 5-fold). A re-assessment of steady-state inactivation, h infinity (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the beta 1 subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.     

A thyrotropin-secreting pituitary adenoma as a cause of thyrotoxic periodic paralysis

We describe a patient with thyrotoxic periodic paralysis (TPP) caused by a thyrotropin-secreting pituitary adenoma. The diagnosis TPP was based on the combination of episodes of reversible hypokalaemic paralysis, hyperthyroidism and electrophysiological findings. A thyrotropin-secreting pituitary adenoma was diagnosed on the basis of endocrinological function tests and MRI of the pituitary gland. Before transsphenoidal resection of the adenoma, treatment with octreotide restored euthyroidism both clinically and biochemically. Immunocytochemistry of the pituitary adenoma was positive for TSH exclusively. Incubation with octreotide or quinagolide induced decreased TSH and alpha-subunit production by the cultured adenoma cells, in agreement with the pre-operative in vivo data. This paper is the first to describe in vivo and in vitro characteristics of a thyrotropin-secreting pituitary adenoma in a patient presenting with periodic paralysis.     

Functional expression of a GLT-1 type Na+-dependent glutamate transporter in rat pinealocytes

We recently showed that treatment with actin antagonists perturbed stomatal behavior in Commelina communis L. leaf epidermis and therefore suggested that dynamic changes in actin are necessary for signal responses in guard cells (M. Kim, P.K. Hepler, S.O. Eun, K.-S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Here we show that actin filaments of guard cells, visualized by immunofluorescence microscopy, change their distribution in response to physiological stimuli. When stomata were open under white-light illumination, actin filaments were localized in the cortex of guard cells, arranged in a pattern that radiates from the stomatal pore. In marked contrast, for guard cells of stomata closed by darkness or by abscisic acid, the actin organization was characterized by short fragments randomly oriented and diffusely labeled along the pore site. Upon abscisic acid treatment, the radial pattern of actin arrays in the illuminated guard cells began to disintegrate within a few minutes and was completely disintegrated in the majority of labeled guard cells by 60 min. Unlike actin filaments, microtubules of guard cells retained an unaltered organization under all conditions tested. These results further support the involvement of actin filaments in signal transduction pathways of guard cells.     

Amiloride-insensitive Na+-H+ exchange: a candidate mediator of erythrocyte Na+-Li+ countertransport

Erythrocyte Na+-Li+ countertransport shows an increased activity in essential hypertension and diabetic nephropathy, but its nature remains unknown. This amiloride-insensitive membrane transport may not be a mode of operation of the amiloride-sensitive NHE1, the only Na+-H+ exchange isoform found in human erythrocytes. Whether an independent, although unknown, amiloride-insensitive isoform mediates Na+-Li+ countertransport is unclear. Na+-H+ exchange activity was measured in acid-loaded erythrocytes. Dimethylamiloride, a specific inhibitor of Na+-H+ exchange and phloretin, a known inhibitor of Na+-Li+ countertransport, gave a reduction in H+-driven Na+ influx (by 31 and 37%, respectively). This effect was additive, and a 66% reduction in H+-driven Na+ influx was found in the presence of both inhibitors. Internal acidification, a stimulus for Na+-H+ exchange, enhanced Na+-Li+ countertransport activity (from 287 +/- 55 to 1213 +/- 165 micromol x Lcell(-1) h(-1), mean +/- SEM, P = 0.003). This transport remained sensitive to phloretin under both conditions. Conversely, external acidification decreased Na+-Li+ countertransport activity (as expected for a Na+-H+ exchanger). Competition between internal H+ and Li+ or Na+ for a common binding site was present. Finally, similar kinetic parameters for external Na+ characterized Na+-Li+ countertransport and the phloretin-sensitive component of H+-driven Na+ influx. These findings suggest that both Na+-Li+ countertransport and the amiloride-insensitive, phloretin-sensitive component of H+-driven Na+ influx can be mediated by a previously unrecognized novel amiloride-insensitive Na+-H+ exchange isoform in human erythrocytes.