Glycosylation influences voltage-dependent gating of cardiac and skeletal muscle sodium channels.

The role of glycosylation on voltage-dependent channel gating for the cloned human cardiac sodium channel (hH1a) and the adult rat skeletal muscle isoform (microl) was investigated in HEK293 cells transiently transfected with either hH1a or microl cDNA. The contribution of sugar residues to channel gating was examined in transfected cells pretreated with various glycosidase and enzyme inhibitors to deglycosylate channel proteins. Pretreating transfected cells with enzyme inhibitors castanospermine and swainsonine, or exo-glycosidase neuroaminidase caused 7 to 9 mV depolarizing shifts of V(1/2) for steady-state activation of hH1a, while deglycosylation with corresponding drugs elicited about the same amount of depolarizing shifts (8 to 9 mV) of V(1/2) for steady-state activation of microl. Elevated concentrations of extracellular Mg(2+) significantly masked the castanospermine-elicited depolarizing shifts of V(1/2) for steady-state activation in both transfected hH1a and microl. For steady-state activation, deglycosylation induced depolarizing shifts of V(1/2) for hH1a (10.6 to 12 mV), but hyperpolarizing shifts for microl (3.6 to 4.4 mV). Pretreatment with neuraminidase had no significant effects on single-channel conductance, the mean open time, and the open probability. These data suggest that glycosylation differentially regulates Na channel function in heart and skeletal muscle myocytes.

Environmental signals controlling sexual development of the corn Smut fungus Ustilago maydis through the transcriptional regulator Prf1.

Environmental signals induce and coordinate discrete morphological transitions during sexual development of Ustilago maydis. In this fungus, mating of two compatible haploid sporidia is a prerequisite for plant infection. Cell fusion is governed by the action of pheromones and receptors, whereas the subsequent pathogenicity program is controlled by the combinatorial interaction of homeodomain proteins. The U. maydis pheromone response factor (Prf1) is a central regulator of both processes. We have analyzed the regulation of the prf1 gene and demonstrate that pheromone and cAMP signaling regulate prf1 post-transcriptionally. Transcriptional activation of prf1 was observed in the presence of carbon sources, such as glucose and fructose, allowing us to define the cis-acting element in the prf1 promoter that mediates these effects. The same element provides for negative control of prf1 gene transcription at high cAMP levels. A protein that specifically binds to this element was purified and analyzed for its role in prf1 gene regulation. On the basis of these results, we present a model in which prf1 integrates different environmental signals to control development in U. maydis.

Effects of halothane and isoflurane on fast and slow inactivation of human heart hH1a sodium channels.

BACKGROUND: Cloning and heterologous expression of ion channels allow biophysical and molecular studies of the mechanisms of volatile anesthetic interactions with human heart sodium channels. Volatile anesthetics may influence the development of arrhythmias arising from cardiac sodium channel dysfunction. For that reason, understanding the mechanisms of interactions between these anesthetics and cardiac sodium channels is important. This study evaluated the mechanisms of volatile anesthetic actions on the cloned human cardiac sodium channel (hH1a) alpha subunit. METHODS: Inward sodium currents were recorded from human embryonic kidney (HEK293) cells stably expressing hH1a channels. The effects of halothane and isoflurane on current and channel properties were evaluated using the whole cell voltage-clamp technique. RESULTS: Halothane at 0.47 and 1.1 mM and isoflurane at 0.54 and 1.13 mM suppressed the sodium current in a dose- and voltage-dependent manner. Steady state activation was not affected, but current decay was accelerated. The voltage dependence of steady state fast and slow inactivations was shifted toward more hyperpolarized potentials. The slope factor of slow but not fast inactivation curves was reduced significantly. Halothane increased the time constant of recovery from fast inactivation. The recovery from slow inactivation was not affected significantly by either anesthetic. CONCLUSIONS: In a heterologous expression system, halothane and isoflurane interact with the hH1a channels and suppress the sodium current. The mechanisms involve acceleration of the transition from the open to the inactivated state, stabilization of the fast and slow inactivated states, and prolongation of the inactivated state by delayed recovery from the fast inactivated to the resting state.

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.

CuZnSOD and MnSOD immunoreactivity in brain stem motor neurons from amyotrophic lateral sclerosis patients.

Motor neurons from the brain stems of amyotrophic lateral sclerosis (ALS) and control patients were examined with immunoantibodies to CuZn-superoxide dismutase (CuZnSOD) and Mn-superoxide dismutase (MnSOD). We found that there was a marked staining for CuZnSOD in all the motor nuclei, the hypoglossus, ambiguus, facialis and trigeminus from the ALS patients, but not in the controls. The same neurons from the ALS patients also stained very intensely for MnSOD, whereas the neurons from the control patients stained weakly or not at all. Loss of neurons was also a very consistent finding and was noted in all the motor nuclei from the ALS patients. There was a proliferation of glial cells which stained strongly both for CuZnSOD and for MnSOD accompanying the loss of the neurons. These results indicated that there was an apparent increase of superoxide dismutase immunoreactivity in motor neurons of ALS patients. We conclude that CuZnSOD and MnSOD immunoreactivity is increased in motor neurons and glia in the brain stems of patients with ALS, specific for the terminal phase of this disease.

Anomalous effect of permeant ion concentration on peak open probability of cardiac Na+ channels.

Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. Decreasing extracellular permeant ion concentration decreases outward Na+ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na+, Li+, or hydrazinium) is substituted for a relatively impermeant cation (K+, Rb+, Cs+, N-methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na+ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to -140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.

Cysteine mapping in the ion selectivity and toxin binding region of the cardiac Na+ channel pore.

Aqueous exposure of critical residues in the selectivity region of voltage gated Na+ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3) and IV (D4) of the human heart Na+ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate (MTSEA+) and (2-sulfonatoethyl)methanethiosulfonate (MTSES-) in all six of the Cys-substituted channels, including Trp --> Cys substitutions at homologous positions in D3 and D4 that were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification, each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >10(2). Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C (the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even though both were exposed. Neither position responded to MTSES- suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES- markedly increased, and MTSEA+ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp1713 acts electrostatically in an unrestricted region. These results suggest that Lys1418 lies in a restricted region favorable to cations, whereas Asp1713 is at a more peripheral location in the Na+ channel pore.

Two conformational states involved in the use-dependent TTX blockade of human cardiac Na+ channel.

We used a fast inactivation-deficient mutant (QQQ) of the human heart Na+ channel alpha-subunit (hH1a) to assess the influence of the inactivation gate on tetrodotoxin (TTX) use-dependent block (UDB) and postrepolarization block (PRB). PRB had similar time courses in both channels, suggesting no direct interaction of the inactivation gate with the TTX binding site. The UDB saturated with high concentrations of TTX in hH1a but not in QQQ, revealing the modulatory action of fast inactivation on UDB. TTX did not stabilize the inactivated states of QQQ, and the extra block developing during long depolarizations suggests a higher-affinity site involved in the gating of the channel. These results cannot be solely explained by a slow recovery from the block in the inactivated states. They suggest a common use-dependent block mechanism for hH1a and QQQ involving a high-affinity site. We propose that an activated state is primarily responsible for UDB during short depolarization in the range of the action potential plateau and that fast inactivation modulates the accessibility of the toxin to this site.

Multiple mechanisms of Na+ channel--linked long-QT syndrome.

Inheritable long-QT syndrome (LQTS) is a disease in which delayed ventricular repolarization leads to cardiac arrhythmias and the possibility of sudden death. In the chromosome 3-linked disease, one mutation of the cardiac Na+ channel gene results in a deletion of residues 1505 to 1507 (Delta KPQ), and two mutation result in substitutions (N1325S and R1644H). We compared all three mutant-channel phenotypes by heterologous expression in Xenopus oocytes. Each produced a late phase of inactivation-resistant, mexiletine- and tetrodotoxin-sensitive whole-cell currents, but the underlying mechanisms were different at the single-channel level. N1325S and R1644H showed dispersed reopenings after the initial transient, whereas Delta KPQ showed both dispersed reopenings and long-lasting bursts. Thus, two distinct biophysical defects underlie the in vitro phenotype of persistent current in Na+ channel-linked LQTS, and the additive effects of both are responsible for making the Delta KPQ phenotype the most severe.

The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis.

In Ustilago maydis, the a and b mating type loci regulate cell fusion, filamentous growth and pathogenicity. The a locus encodes a pheromone-based cell recognition system, and the b locus specifies two homeodomain proteins. The expression of all genes in the a and b loci is induced by pheromone. We have identified a HMG protein (Prf1) that binds sequence specifically to pheromone response elements present in the a and b loci. prf1 mutants do not express the a and b genes and are sterile. The disruption of prf1 in pathogenic haploid strains results in a loss of pathogenicity. The constitutive expression of the b genes restores pathogenicity and induces filamentous growth in the absence of the pheromone signal. These results provide evidence that pheromone signalling, filamentous growth and pathogenic development are linked through Prf1.

Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels.

We have probed a cysteine residue that confers resistance to tetrodotoxin (TTX) block in heart Na channels, with membrane-impermeant, cysteine-specific, methanethiosulfonate (MTS) analogs. Covalent addition of a positively charged group to the cysteinyl sulfhydryl reduced pore conductance by 87%. The effect was selectively prevented by treatment with TTX, but not saxitoxin (STX). Addition of a negatively charged group selectively inhibited STX block without affecting TTX block. These results agree with models that place an exposed cysteinyl sulfhydryl in the TTX site adjacent to the mouth of the pore, but do not support the contention that STX and TTX are interchangeable. The surprising differences between the two toxins are consistent with the hypothesis that the toxin-receptor complex can assume different conformations when STX or TTX bound.

Effects of III-IV linker mutations on human heart Na+ channel inactivation gating.

Na+ channel inactivation, a critical determinant of refractoriness, differs in cardiomyocytes and neurons. In rat brain type IIa (rB2a) Na+ channels, a critical residue in the cytoplasmic linker between domains III and IV regulates fast inactivation such that a Phe-->Gln substitution (F1489Q) inhibits inactivation by at least 85%. Since this residue is conserved in voltage-gated Na+ channels, we tested whether F1485Q, the analogous mutation in human heart (hH1a) Na+ channels, has a similar functional effect. We found that fast inactivation in wild-type (WT) channels expressed in Xenopus oocytes was complete within 15 milliseconds at a test potential of 0 mV, and its time course was biexponential with time constants of 0.4 and 2 milliseconds. But in contrast to rB2a, the FQ mutation inhibited inactivation by < 50% and increased mean single-channel open time by only twofold. Residual fast inactivation was monoexponential, with a time constant similar to that of the slower phase of normal inactivation (2 milliseconds). In the mutant channels, unlike WT, null tracings were absent at holding potentials in the range of -140 to -120 mV, and the voltage range of steady-state inactivation coincided exactly with that of activation, suggesting that residual inactivation was tightly coupled to the open state. As in rB2a, simultaneous mutations of I1484Q and M1486Q, in addition to mutation F1485Q, completely inhibited fast inactivation. Our results show that in heart Na+ channels, the IFM cluster controls the stability of both open- and closed-channel inactivation in a manner qualitatively similar to that in the brain. Structural differences in the putative inactivation receptor may explain the distinct gating patterns in channel subtypes.

Functional interactions between K+ pore residues located in different subunits.

The aqueous pore (P-region) of homotetrameric voltage-gated K+ channels has been modeled as a radially symmetrical eight-stranded antiparallel beta-barrel to which each of the four subunits contributes equally. This model has hydrogen bonding between residues located on adjacent subunits and predicts that subunit interactions might have functional consequences. Previously we have used point mutations and an electrophysiological assay to detect functional interactions between a pair of residues at positions 369 and 374 in the P-region, but we could not distinguish between intra- and intersubunit interactions. In the present paper, we present evidence for interaction across subunit boundaries after co-injecting two cRNAs encoding subunits differing from each other at either position 369 or 374. Comparison of the phenotypes of homo- and heterotetrameric channels suggests that pore residues residing in adjacent subunits form a closely packed structure which determines both ion conductance and stability of the open state of the channel. Our results are consistent with a structure in which pore residues 369 and 374 are located in close proximity on adjacent antiparallel strands to allow both intra- and intersubunit interactions.

Regulation of K+/Rb+ selectivity and internal TEA blockade by mutations at a single site in K+ pores.

A conservative reversion at position 374 in a chimeric K+ pore, CHM, switched the preferred ionic conductance from K+ to Rb+. To understand how selectivity was switched, codons for 18 different amino acids were substituted at position 374 in each of two different K+ channels CHM and Kv2.1, the host channel for CHM. After injection of cRNA into Xenopus oocytes, less than half of the substituted mutants expressed functional channels. In both CHM and Kv2.1, channels with the substituted hydrophobic residues Val or Ile expressed Rb(+)-preferring pores while channels with the substituted polar residues Thr or Ser expressed K(+)-preferring pores. Val or Ile stabilized while Thr or Ser destabilized blockade by internal tetraethylammonium (TEA) confirming the importance of hydrophobic interactions for blockade. TEA blockade was dependent upon the charge carrier and was more effective in the presence of the ion having the larger conductance. The results are consistent with a model in which the side chains at position 374 form a filter for K+ and Rb+ ions and a site for blockade by internal TEA.

Inactivation determined by a single site in K+ pores.

An N-terminus peptide or a C-terminus mechanism involving a single residue in transmembrane segment 6 produces inactivation in voltage-dependent K+ channels. Here we show that a single position in the pore of K+ channels can produce inactivation having characteristics distinct from either N- or C-type inactivation. In a chimeric K+ channel (CHM), the point reversion CHM V369K produced fast inactivation and CHM V369S had the additional effect of halving K+ conductance consistent with a position in the pore. The result was not restricted to CHM; mutating position 369 in the naturally occurring channel Kv2.1 also produced fast inactivation. Like N- and C-types of inactivation, pore or P-type inactivation was characterized by short bursts terminated by rapid entry into the inactivated state. Unlike C-type inactivation, in which external tetraethylammonium (TEA) produced a simple blockade that slowed inactivation and reduced currents, in P-type inactivation external TEA increased currents. Unlike N-type inactivation, internal TEA produced a simple reduction in current and K+ occupancy of the pore had no effect. External TEA was not the only cation to increase current; external K+ enhanced channel availability and recovery from inactivation. Additional features of P-type inactivation were residue-specific effects on the extent of inactivation and removal of inactivation by a point reversion at position 374, which also regulates conductance. The demonstration of P-type inactivation indicates that pore residues in K+ channels may be part of the inactivation gating machinery.

Regional mRNA changes in brain stem motor neurons from patients with amyotrophic lateral sclerosis.

An antisense oligonucleotide (54 mer) from the mRNA to the midsize neurofilament protein (NFM) was labeled with 35S on the 3' end and purified by polyacrylamide gel electrophoresis (PAGE). In situ hybridization was performed on sections from medulla oblongata of patients with amyotrophic lateral sclerosis (ALS). The slides were dipped in photographic emulsion, developed, and stained. Neurons from both nucleus hypoglossus and nucleus ambiguous showed a marked reduction of silver grains when compared to normal. This indicates a reduction of mRNA, which may precede the reduction of ribosomal RNA and the changes in neurofilament proteins that have been described by several investigators in ALS. It does not settle the question of whether the reduction of mRNA is owing to reduced transcription or increased decay of mRNA.

Deficiency of copper can cause neuronal degeneration.

The aim of this article is to emphasize the important role that copper plays in the function of nerve cells. We are reporting preliminary data which suggest that the swelling of axons which we produce in rats by iminodipropionitrile, IDPN, is due to its chelating action on copper, and how conversely supplementation with copper abolishes both symptoms and lesions. The copper values we obtained by atomic absorption spectrophotometry of the spinal cord and brain from the animals fully support this contention. In comparing these results with the diseases that are known to be due to copper deficiency, namely Menkes disease in man, swayback in lambs and several neurological mutant mice, we find not only similar axonal swellings, but also amelioration of symptoms and lesions by early administration of copper. Considering the main forms in which copper is present, we discuss the cuproproteins, i.e. ceruloplasmin and metallothionein, and their role in transport and delivery of copper to various organs. Further, the many cuproenzymes i.e. superoxide dismutase, tryptophan-2,3-dioxygenase, lysine oxidase, cytochrome oxidase, monoamine oxidases, tyrosinase, dopamine-beta-hydroxylase and d-amino levulinate dehydratase are noted for their roles in the nervous system. Finally, we suggest that neuronal copper deficiency should be more fully investigated as a possible etiological factor in the more common neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis, ALS.

Differential expression of the alpha 2 and beta messenger RNAs of Na,K-ATPase in developing brine shrimp as measured by in situ hybridization.

We used in situ hybridization histochemistry with synthetic oligonucleotide probes to localize the mRNAs encoding the alpha 2- and beta-mRNAs of Na,K-ATPase during development of the brine shrimp Artemia. The mRNAs of the alpha 2- and beta-subunit were of low abundance in the cysts; in addition, less mRNA of the beta-subunit was localized. During emergence (12 hr), there was an increase in alpha 2-subunit mRNA in the gut mucosa, but there was a burst in beta-subunit mRNA throughout. As development progressed, the mRNAs of both the alpha 2- and beta-subunits showed a distinct pattern of expression in which the mRNA in the salt gland was of greatest abundance, followed by epidermal cells and gut mucosa. After 36 hr the alpha 2-subunit mRNA began to decrease in all positive cells but still remained highest in the salt gland and the brain region, while the mRNA of the beta-subunit kept increasing in the gut mucosa. Finally, the greatest abundance of the beta-subunit mRNA shifted from the salt gland to the antenna gland and the epidermal cells in the tail region, but the alpha 2-subunit mRNA did not. The more widespread distribution of the beta-mRNA than alpha 2-mRNA at certain stages (e.g., there was no alpha 2-mRNA in the antenna gland at the adult stage) is in all likelihood due to the marked drop in the alpha 2-subunit and a rise in alpha 1-subunit previously seen by Peterson et al. on polyacrylamide gel electrophoresis, as development progresses.