Solution structure of the sodium channel inactivation gate

The sodium channel initiates action potentials by opening in response to membrane depolarization. Fast channel inactivation, which is required for proper physiological function, is mediated by a cytoplasmic loop proposed to occlude the ion pore via a hinged lid mechanism with the triad IFM serving as a hydrophobic "latch". The NMR solution structure of the isolated inactivation gate reveals a stably folded core comprised of an alpha-helix capped by an N-terminal turn, supporting a model in which the tightly folded core containing the latch motif pivots on a more flexible hinge region to occlude the pore during inactivation. The structure, in combination with substituted cysteine mutagenesis experiments, indicates that the IFM triad and adjacent Thr are essential components of the latch and suggests differing roles for the residues of the IFMT motif in fast inactivation.     

Developmental changes in glucocorticoid receptor and 11beta-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells

Glucocorticoids directly regulate testosterone production in Leydig cells through a glucocorticoid receptor (GR)-mediated repression of the genes that encode testosterone biosynthetic enzymes. The extent of this action is determined by the numbers of GR within the Leydig cell, the intracellular concentration of glucocorticoid, and 11beta-hydroxysteroid dehydrogenase (11betaHSD) activities that interconvert corticosterone (in the rat) and its biologically inert derivative, 11-dehydrocorticosterone. As glucocorticoid levels remain stable during pubertal development, GR numbers and 11betaHSD activities are the primary determinants of glucocorticoid action. Therefore, in the present study, levels of GR and 11betaHSD messenger RNA (mRNA) and protein were measured in rat Leydig cells at three stages of pubertal differentiation: mesenchymal-like progenitors (PLC) on day 21, immature Leydig cells (ILC) that secrete 5alpha-reduced androgens on day 35, and adult Leydig cells (ALC) that are fully capable of testosterone biosynthesis on day 90. Numbers of GR, measured by [3H]dexamethasone binding, in purified cells were 6.34 +/- 0.27 (x 10(3) sites/cell; mean +/- SE) for PLC, 30.45 +/- 0.74 for ILC, and 32.54 +/- 0.84 for ALC. Although GR binding was lower in PLC, steady state levels for GR mRNA were equivalent at all three stages (P > 0.05). Oxidative and reductive activities of 11betaHSD were measured by assaying the conversion of radiolabeled substrates in incubations of intact Leydig cells. Both oxidative and reductive activities were barely detectable in PLC, intermediate in ILC, and highest in ALC. The ratio of the two activities favored reduction in PLC and ILC and oxidation in ALC (oxidation/reduction, 0.33 +/- 0.33 for PLC, 0.43 +/- 0.05 for ILC, and 2.12 +/- 0.9 for ALC, with a ratio of 1 indicating equivalent rates for both activities). The mRNA and protein levels of type I 11betaHSD in Leydig cells changed in parallel with 11betaHSD reductive activity, which increased gradually during the transition from PLC to ALC, compared with the sharp rise that was seen in oxidative activity. We conclude that Leydig cells at all developmental stages have GR and that their ability to respond to glucocorticoid diminishes as net 11betaHSD activity switches from reduction to oxidation. This provides a mechanism for the Leydig cell to regulate its intracellular concentration of corticosterone, thereby varying its response to this steroid during pubertal development.     

Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction

Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the alpha1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca2+ concentrations. The relative transmitter release of injected neurons was reduced by approximately 25% at physiological Ca2+ concentration, whereas injection of the corresponding region of the L-type Ca2+ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. Our data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca2+ dependence to higher values.     

Immunochemical identification and differential phosphorylation of alternatively spliced forms of the alpha 1A subunit of brain calcium channels

Biochemical properties of the alpha 1 subunits of class A brain calcium channels (alpha 1A) were examined in adult rat brain membrane fractions using a site-directed anti-peptide antibody (anti-CNA3) specific for alpha 1A. Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive alpha 1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa alpha 1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of alpha 1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of alpha 1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct alpha 1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.     

Identification of benz(othi)azepine-binding regions within L-type calcium channel alpha1 subunits

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.     

Adenocarcinoma of the stomach: radiological and pathological correlation of effects of treatment with fast neutrons

During the past six years 40 patients have been treated with fast neutrons for inoperable adenocarcinoma of the stomach. A number of these patients have been studied in detail both radiologically and pathologically with regard to the effects of fast neutrons on both the tumour and normal stomach. After treatment there was a constant reduction in size of the stomach capacity when compared with a control group and the motility of the stomach was lost. Mucosal changes, which were noted radiologically, appeared to be dose related. Histologically most of the tumour was destroyed. Small clumps of cells were, however, found in all except one of the patients but only in areas of the stomach which had not received the standard dose. Marked fibrosis occurred and it is our impression that the amount of fibrosis is in excess of that seen with other forms of radiotherapy.     

Surface carbohydrates involved in the adhesive interactions of metastatic cells

Extracellular proteases can trigger intracellular events via at least two members of the super-family of G protein coupled receptors: the thrombin receptor and PAR-2. The two receptors have a similar structure, share a common mechanism of activation and may have arisen by gene duplication, but there are differences as well as similarities in their tissue distribution, the proteases by which they are activated and potentially in the consequences of their activation. Human platelets express the thrombin receptor, but not PAR-2, while at least some types of human endothelial cells express both. This review briefly summarizes current information about the two receptors and considers their roles in platelet and endothelial cell biology, their interactions with proteases, and the known mechanisms for receptor redistribution and replacement.     

Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na+ channel alpha subunit

alpha-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na+ channel and inhibit fast inactivation. Basic amino acids of the toxins and domains I and IV of the Na+ channel alpha subunit have been previously implicated in toxin binding. To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na+ channel alpha subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125I-alpha-scorpion toxin binding. Conversion of Glu1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable alpha-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity. Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of alpha-scorpion toxin or sea anemone toxin. Moreover, chimeric Na+ channels in which amino acid residues at the extracellular end of segment IVS3 of the alpha subunit of cardiac Na+ channels were substituted into the type IIa channel sequence had reduced affinity for alpha-scorpion toxin characteristic of cardiac Na+ channels. Electrophysiological analysis showed that E1613R has 62- and 82-fold lower affinities for alpha-scorpion and sea anemone toxins, respectively. Dissociation of alpha-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. alpha-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation. These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in alpha-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in alpha-scorpion toxin binding affinity between cardiac and neuronal Na+ channels. The results also support the hypothesis that this region of the Na+ channel is important for coupling channel activation to fast inactivation.     

Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.     

Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses

We studied how Ca2+ influx through different subtypes of Ca2+ channels couples to release at a calyx-type terminal in the rat medial nucleus of the trapezoid body by simultaneously measuring the presynaptic Ca2+ influx evoked by a single action potential and the EPSC. Application of subtype-specific toxins showed that Ca2+ channels of the P/Q-, N-, and R-type controlled glutamate release at a single terminal. The Ca2+ influx through the P/Q-type channels triggered release more effectively than Ca2+ influx through N- or R-type channels. We investigated mechanisms that contributed to these differences in effectiveness. Electrophysiological experiments suggested that individual release sites were controlled by all three subtypes of Ca2+ channels. Immunocytochemical staining indicated, however, that a substantial fraction of N- and R-type channels was located distant from release sites. Although these distant channels contributed to the Ca2+ influx into the terminal, they may not contribute to release. Taken together, the results suggest that the Ca2+ influx into the calyx via N- and R-type channels triggers release less effectively than that via P/Q-type because a substantial fraction of the N- and R-type channels in the calyx is localized distant from release sites.