Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Localization of epitopes for antibodies that differentially label sodium channels in skeletal muscle surface and T-tubular membranes

Summary We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1–30 (I^1–30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to II^1–30. L/D3 binding was quantitatively inhibited by oligopeptides 1–30, 7–30, 13–30, and 19–30 but not 25–30, while binding of A/B2 was blocked only by the intact I^1–30 peptide. This data implies that the epitope for L/D3 lies within residues 19–25 while the epitope for A/B2 is contained within residues 1–6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I^1–30 and immunoreactivity of a peptide corresponding to residues 1–12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.We wish to thank Dr. John Lambris for helpful discussions. We also thank Ms. Candace Mello and Mr. James Hills for their expert technical assistance. This work was supported in part by NIH Grant NS 18013 (RLB) and by a grant from the W.W. Smith Charitable Trust (SAC). SAC is a Scholar of the Pfizer Scholar's Program for New Faculty.     

Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.     

Localization of calmodulin binding sites on the ryanodine receptor from skeletal muscle by electron microscopy.

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by electron microscopy. Wheat germ CaM was labeled specifically at Cys-27 with a maleimide derivative of a 1.4-nm-diameter gold cluster, and the gold-cluster-labeled CaM was bound to the purified ryanodine receptor. The complexes were imaged in the frozen-hydrated state by cryoelectron microscopy with no stains or fixatives present. In the micrographs, gold clusters were frequently observed near the corners of the square-shaped images of the ryanodine receptors. In some images, all four corners of the receptor were occupied by gold clusters. Image averaging allowed the site of CaM binding to be determined in two dimensions with an estimated precision of 4 nm. No changes were apparent in the quaternary structure of the ryanodine receptor upon binding CaM to the resolution attained, about 3 nm. Side views of the ryanodine receptor, in which the receptor is oriented approximately perpendicular to the much more frequent fourfold symmetric views, were occasionally observed, and showed that the CaM binding site is most likely on the surface of the receptor that faces the cytoplasm. We conclude that the CaM binding site is at least 10 nm from the transmembrane channel of the receptor and, consequently, that long-range conformational changes are involved in the modulation of the calcium channel activity of the receptor by CaM.     

Localization of sodium pump sites in frog urinary bladder

[³H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and authoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na⁺ transport was completely reversible in toad bladder whereas, in frog bladder, [³H] ouabain was tightly bound and Na⁺ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [³H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of cecrtain (Na⁺?K⁺)-ATPase ligands (K⁺, ATP) to levels known to reduce ouabain binding. High K⁺ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [³H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na⁺ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significatly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.     

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-gated sodium channels are targets for toxins from the venom of the spider Heriaeus melloteei

Three novel peptides were isolated from the venom of the spider Heriaeus melloteei (Thomisidae) and characterized. The peptides named Hm-1, 2 and 3 blocked voltage-gated Na⁺ channels at concentrations in the order of 100 nM. Activity of the purified peptides was investigated in Na⁺ channel isoforms of mammals and insects. Hm-1 and 2 appeared to act as pore blockers, whereas Hm-3 modulated the channel activation process. The toxins described exhibit minor similarity with other known peptides and may therefore constitute new groups of Na⁺ channel ligands.     

Binding of scorpion toxin to receptor sites associated with sodium channels in frog muscle. Correlation of voltage-dependent binding with activation.

Purified scorpion toxin (Leiurus quinquestriatus) slows inactivation of sodium channels in frog muscle at concentrations in the range of 17-170 nM. Mono[125I]iodo scorpion toxin binds to a single class of sites in frog sartorius muscle with a dissociation constant of 14 nM and a binding capacity of 13 fmol/mg wet weight. Specific binding is inhibited more than 90% by 3 microM sea anemone toxin II and by depolarization with 165 mM K+. Half-maximal inhibition of binding is observed on depolarization to -41 mV. The voltage dependence of scorpion toxin binding is correlated with the voltage dependence of activation of sodium channels. Removal of calcium from the bathing medium shifts both activation and inhibition of scorpion toxin binding to more negative membrane potentials. The results are considered in terms of the hypothesis that activation of sodium channels causes a conformational change in the scorpion toxin receptor site resulting in reduced affinity for scorpion toxin.     

Grasping for calcium binding sites in sodium channels with an EF hand

Amino acid sequences near the carboxy terminal end of the Electrophorus electricus electric organ and rat brain sodium channel polypeptides were discovered to be putative EF hand calcium binding sites. This conclusion was made using the following criteria: the Tufty-Kretsinger and Szebenyi-Moffat EF hand tests, a computer generated analysis, the revised guidelines of Chou & Fasman, and sequence comparisons with other published EF hand calcium binding regions. These results suggest that the sodium channel may be a calcium binding protein.     

Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms.     

Reserpine: interactions with batrachotoxin and brevetoxin sites on voltage-dependent sodium channels

Reserpine inhibited batrachotoxin-elicited sodium influx in guinea pig brain synaptoneurosomes with an IC₅₀ of about 1 M. In the presence of brevetoxin the IC₅₀ increased to about 80 M. Reserpine inhibited binding of batrachotoxinin-A [³H]benzoate ([³H]BTX-B) binding in a complex manner causing a partial inhibition from 0.001 to 0.08 M, then a rebound stimulation from 0.1 to 0.8 M, followed by complete inhibition by 80 M. The stimulation was prevented by the presence of brevetoxin; reserpine then smoothly inhibited binding with an IC₅₀ of about 1 M. Reserpine at 1 M slightly reduced the off-rate of [³H]BTX-B binding measured in the presence of veratridine, while at a concentration of 50 M it enhanced the off-rate, presumably by an allosteric mechanism. Reserpine at 0.3–10 M elicited a partial inhibition of the binding of [³H]brevetoxin-3. The local anesthetic dibucaine had effects similar to reserpine: It partially inhibited binding of [³H]brevetoxin. The presence of brevetoxin reduced the potency of dibucaine as an inhibitor of batrachotoxin-elicited sodium influx from an IC₅₀ of about 2 M to an IC₅₀ of about 50 M. The results suggest that reserpine binds at both a local anesthetic site to cause allosteric inhibition of batrachotoxin-binding and action, but that it also binds to another site causing, like brevetoxin, an enhancement of batrachotoxin-binding and action. Local anesthetics also may bind to the brevetoxin site.     

Simultaneous modifications of sodium channel gating by two scorpion toxins.

The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan (1975, J. Physiol. [Lond.]. 244:511-534) for the crude scorpion venom. Both of these effects were observed in fibers treated with both of these toxins, and the kinetics of the induced current were modified in a way that showed that the same sodium channels were modified simultaneously by both toxins. Although the toxins can act on different sites, the time course of the action of C. sculpturatus toxin was accelerated in the presence of the L. quinquestriatus toxin, indicating some form of interaction between the two toxin binding sites.     

Proton block of rat brain sodium channels. Evidence for two proton binding sites and multiple occupancy

The acid titration function of bilayer-incorporated batrachotoxin (BTX)- modified sodium channels was examined in experiments in which the pH was decreased symmetrically, on both sides of the membrane, or asymmetrically, on only one side. In an attempt to minimize interpretational ambiguities, the experiments were done in 1.0 M NaCl (buffered to the appropriate pH) with channels incorporated into net neutral bilayers. When the pH was decreased symmetrically (from 7.4 to 4.5), the small-signal conductance (g) decreased in accordance with the predictions of a simple (single-site) titration function with a pK of approximately 4.9. As the pH was decreased below 6.5, the single- channel current-voltage (i-V) relation became increasingly rectifying, with the inward current being decreased more than the outward current. When the pH was decreased asymmetrically (with the pH of the other solution being held constant at 7.4), the titration behavior was different for extra- and intracellular acidification. With extracellular acidification, the reduction in g could still be approximated by a simple titration function with a pK of approximately 4.6, and there was a pronounced rectification at pHs < or = 6 (cf. Woodhull, A. M. 1973. Journal of General Physiology. 61:687-708). The voltage dependence of the block could be described by assuming that protons enter the pore and bind to a site with a pK of approximately 4.6 at an apparent electrical distance of approximately 0.1 from the extracellular entrance. With intracellular acidification there was only a slight reduction in g, and the g-pH relation could not be approximated by a simple titration curve, suggesting that protons can bind to several sites. The i-V relations were still rectifying, and the voltage-dependent block could be approximated by assuming that protons enter the pore and bind to a site with a pK of approximately 4.1 at an apparent electrical distance of approximately 0.2 from the intracellular entrance. Based on the difference between the three g-pH relations, we conclude that there are at least two proton binding sites in the pore and that they can be occupied simultaneously.     

Localization of the functional sites on the alpha chain of acetylcholine receptor

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to systematically screen the entire extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (AChR) for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered, AChR; the regions recognized by AChR-primed T cells; the regions that bind alpha-bungarotoxin and cobratoxin; and an acetylcholine-binding region. Eight continuous antigenic sites were localized in this part of the alpha chain by all of the antisera tested. The sites were independent of the host species from which the antisera were obtained and were also similar to antisera against the isolated pentameric AChR or against the membrane-sequestered AChR. Six regions were found to stimulate AChR-primed T cells (T sites). Three of the T sites coincided with regions recognized by antibodies. At least two T sites had no detectable antibody responses directed to them. Five toxin-binding regions were localized, and may constitute distinct sites or, alternatively, different faces in one (or more) sites. Some of these regions coincided with regions recognized by anti-AChR antibodies. One of the toxin-binding regions bound acetylcholine, and immunization with this peptide induced experimental autoimmune myasthenia gravis.     

Immunocytochemical localization of sodium channels in an insect central nervous system using a site-directed antibody

Antibodies to channel proteins and specific peptide sequences have been previously used to localize voltage-activated sodium channels in the rat brain. Here we describe the first localization of sodium channels in an insect nervous system using a site-directed antibody. The mesothoracic ganglion of the cockroach was stained with an antibody to the highly conserved SP19 sequence. Antibody labelling was visualized by light microscopy using the avidin/biotin method on was sections, and transmission electron microscopy of immunogold-labelled thin sections. Central ganglia of insects contain clearly separated regions of cell bodies, synaptic neuropil, axon tracts, and nerves. Antibody staining by light microscopy was limited to neurons, and was intense in axons throughout the ganglion and nerves. Staining was also strong in the cytoplasm, but not the nuclei, of many neuronal cell bodies. Neuropil regions were relatively lightly labelled. These findings can be correlated with the known electrophysiology of the ganglion. Electron microscopy detected sodium channels in areas surrounding axons, probably including axon membranes and enveloping glial cell membranes. Axonal mitochondria were also heavily labelled, suggesting a sodium channel transport function for these organelles. © 1993 John Wiley & Sons, Inc.