BTX Binding Sites Research Articles

Epithelial Sodium Channel (ENaC) Tagged with Bungarotoxin Binding Site to Study Channel Plasma Membrane Expression and Trafficking

Transepithelial sodium reabsorption through ENaC plays a key role in electrolyte and extracellular volume homeostasis and regulation of blood pressure. ENaC is a heterotrimeric ion channel formed by α, β and γ subunits. Channel activity is tightly regulated by many local and circulating factors, which in turn control channel cell surface expression (N) and open probability (Po). ENaC upregulation resulting from decreased endocytosis or enhanced delivery to the membrane have been described. However, there are several technical challenges in the study of ENaC trafficking, including low levels of plasma membrane expression. The aim of this study was to develop a new tool to directly study the dynamics of ENaC trafficking to and from the plasma membrane. To that end we inserted an α‐bungarotoxin (BTX) binding site (BBS) in the extracellular domain of the a subunit and used fluorescently labeled BTX to study ENaC plasma membrane expression and trafficking in Xenopus laevis oocytes. Mutant αBBSβγ channels do not shown any functional abnormality in the absence or presence of the toxin. Confocal imaging of Alexa Fluor 488 (AF488)‐labeled BTX showed specific labeling in plasma membrane of oocytes expressing αBBSβγ channels. ENaC retrieval was detected by imaging AF488‐BTX labeled oocytes at different time points whereas ENaC insertion rate was detected by blocking αBBSβγ‐expressing oocytes with unlabeled BTX and then staining with AF488‐BTX at different times. Our results suggest that tagged αBBS subunits provide a useful tool to study plasma membrane ENaC abundance and trafficking to and from the membrane.

Illuminating Trafficking of KCNQ1/KCNE1 Channels in Heart

In human heart, pore-forming KCNQ1 subunits associate with auxiliary KCNE1 subunits to generate the slowly activating, delayed rectifier potassium current, IKs. Decreases in IKs, due to either congenital mutations in KCNQ1/KCNE1 subunits or the adverse neuro-hormonal milieu accompanying heart failure, delay cardiac repolarization, leading to long QT syndrome (LQTS). LQTS predisposes to lethal ventricular arrhythmias and sudden cardiac death. How KCNQ1/KCNE1 channel trafficking in the heart proceeds and is regulated is unknown. However, understanding this process is critical both for fundamental mechanistic insights into LQTS, and for rational development of potential new therapies for this condition. We sought to develop optical tools that permit direct visualization of KCNQ1/KCNE1 subunit assembly and trafficking in heart cells. KCNQ1 and KCNE1 were tagged intra-cellularly with YFP and CFP, respectively, and extra-cellularly with a 13-residue high affinity α-bungarotoxin (BTX) binding site (BBS). The tagged proteins were viable as determined by functional electrophysiological assays in Chinese hamster ovary (CHO) cells– BBS-KCNQ1-YFP expressed alone gave rise to rapidly-activating outward currents, and co-expression with KCNE1-CFP resulted in the slowly activating kinetic signature of IKs. Cell surface BBS-tagged channel subunits were selectively detected with quantum dot. Optical pulse chase assays in human embryonic kidney (HEK 293) cells revealed that surface KCNQ1 channels undergo rapid endocytosis and recycling, and this process is largely unaffected by KCNE1. Tagged KCNQ1/KCNE1 subunits expressed in guinea pig heart cells using adenovirus were targeted to the surface sarcolemma, t-tubules and intercalated disks, similar to endogenous channels. Surprisingly, IKs amplitude remained unchanged with over-expression of either KCNQ1 or KCNE1, suggesting a tightly regulated system for maintaining KCNQ1/KCNE1 surface density.

Synthesis and evaluation of hermitamides A and B as human voltage-gated sodium channel blockers

Hermitamides A and B are lipopeptides isolated from a Papau New Guinea collection of the marine cyanobacterium Lyngbya majuscula. We hypothesized that the hermitamides are ligands for the human voltage-gated sodium channel (hNaV) based on their structural similarity to the jamaicamides. Herein, we describe the nonracemic total synthesis of hermitamides A and B and their epimers. We report the ability of the hermitamides to displace [3H]-BTX at 10μM more potently than phenytoin, a clinically used sodium channel blocker. We also present a potential binding mode for (S)-hermitamide B in the BTX-binding site and electrophysiology showing that these compounds are potent blockers of the hNav1.2 voltage-gated sodium channel.

Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

GABA(B) receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABA(B) receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA(B) receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, alpha-bungarotoxin. By using the alpha-bungarotoxin binding site-tagged GABA(B) R1a subunit (R1a(BBS)), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, alpha-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA(B) receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors.

The Tumor Suppressor eIF3e Mediates Calcium-Dependent Internalization of the L-Type Calcium Channel CaV1.2

Voltage-gated calcium channels (VGCCs) convert electrical activity into calcium (Ca2+) signals that regulate cellular excitability, differentiation, and connectivity. The magnitude and kinetics of Ca2+ signals depend on the number of VGCCs at the plasma membrane, but little is known about the regulation of VGCC surface expression. We report that electrical activity causes internalization of the L-type Ca2+ channel (LTC) CaV1.2 and that this is mediated by binding to the tumor suppressor eIF3e/Int6 (eukaryotic initiation factor 3 subunit e). Using total internal reflection microscopy, we identify a population of CaV1.2 containing endosomes whose rapid trafficking is strongly regulated by Ca2+. We define a domain in the II-III loop of CaV1.2 that binds eIF3e and is essential for the activity dependence of both channel internalization and endosomal trafficking. These findings provide a mechanism for activity-dependent internalization and trafficking of CaV1.2 and provide a tantalizing link between Ca2+ homeostasis and a mammalian oncogene.

Discovery of diphenyl amine based sodium channel blockers, effective against hNa v1.2

The development of new therapies for chronic pain is an area of unmet medical need. Central to pathways of chronic pain is the upregulation of voltage-gated sodium channels. The use of tricyclic antidepressants, which also have sodium channel activity, in chronic pain therapy prompted us to develop novel compounds from this scaffold. Herein, we show that the tricyclic moiety is not needed for effective inhibition of the [ 3H]-BTX binding site and sodium currents of hNa v1.2. Our lead compound 6, containing a diphenyl amine motif, demonstrated a 53% inhibitory block of Na v1.2 currents at 10 μM, which is greater than 50% increase in current block in comparison to the amitriptyline standard. Altogether our study establishes that the tricyclic motif is unnecessary for hNa v1.2 activity and modification of the amine portion is detrimental to sodium channel block.

Fluorophore‐assisted light inactivation produces both targeted and collateral effects on N‐type calcium channel modulation in rat sympathetic neurons

Fluorophore-assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein-conjugated polypeptide, alpha-bungarotoxin (BTX) and a 13 amino acid BTX-binding site engineered into the N-terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G-protein-coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of calcium currents (I(Ca)) using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labelled neurons, mGluR8a-mediated I(Ca) inhibition was greatly attenuated whereas holding current and basal I(Ca), measures of non-specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, alpha(2)-adrenergic receptor-mediated I(Ca) modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium channel beta2 subunits or ionotropic 5-HT(3) receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR-independent I(Ca) modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore-tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.

Imaging of receptor trafficking by using α-bungarotoxin-binding-site-tagged receptors

α-Amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors mediate excitatory synaptic transmission and are dynamically regulated during synaptic plasticity in the CNS. The membrane trafficking of AMPA receptors to synapses is critical for the regulation of the efficacy of excitatory synaptic transmission. Direct imaging of AMPA receptors in various cell compartments is important to dissecting the regulation of distinct steps in receptor membrane trafficking. In this study, we have developed an approach for the imaging of receptor trafficking with subunits tagged with a 13-aa α-bungarotoxin (BTX)-binding site (BBS). The small polypeptide neurotoxin BTX has been used for decades to study the nicotinic acetylcholine receptor. Similar high-affinity ligands are rarely available for most receptors. Engineering the BBS tag into receptor subunits allowed the high-affinity binding of fluorescent, radioactive, and biotinylated BTX to the tagged receptor subunits. By using this approach, the total receptor expression, surface expression, internalization, and insertion of receptors into the plasma membrane could be visualized and quantified in fixed or live cells including cultured neurons. The BBS tag is a flexible approach for labeling membrane proteins and studying their dynamic trafficking.

Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine.

1. Chronic administration of nicotinic agonists in vivo increases the density of brain nicotinic binding sites. It has been proposed that this up-regulation results from agonist-induced functional blockade of nicotinic receptors. This hypothesis was tested by examining post mortem [3H]-nicotine and [125I]-alpha-bungarotoxin ([125I]-alpha BTX) binding following treatment in vivo with the quasi-irreversible and insurmountable CNS nicotinic blocker chlorisondamine, given either alone or in combination with chronic nicotine administration. 2. In rats that had not received chlorisondamine pretreatment, chronic nicotine administration (0.6 mg kg-1 s.c., twice daily for 12 days) increased [3H]-nicotine binding density (Bmax) in forebrain tissue sections by 19%, with no change in the apparent dissociation constant (KD). Chlorisondamine (10 mg kg-1, s.c.), given once prior to the chronic treatment phase, neither increased [3H]-nicotine binding by itself, nor altered the extent of nicotine-induced up-regulation. Nevertheless, chlorisondamine pretreatment resulted in a persistent blockade of CNS nicotinic receptors, as demonstrated by complete block of acute locomotor responses to nicotine. 3. In a second experiment, [3H]-nicotine and [125I]-alpha BTX binding was measured in tissue homogenates prepared from several brain regions. In the absence of chlorisondamine pretreatment, chronic nicotine administration (1 mg kg-1 s.c., twice daily for 12 days) increased the Bmax of [3H]-nicotine binding in the cerebral cortex (by 34%), striatum (by 28%), midbrain (by 16%) and hippocampus (by 36%); KD was unchanged. As before, this up-regulation was neither mimicked nor blocked by chlorisondamine pretreatment (10 mg kg-1, s.c., given twice), despite persistent blockade of acute locomotor responses to nicotine. Chronic nicotine treatment also increased the Bmax (but not KD) of [125I]-alpha BTX binding in cerebral cortex (by 35%), hippocampus (by 46%) and midbrain (by 35%). Chlorisondamine altered neither Bmax nor KD when given alone, but significantly attenuated the nicotine-induced up-regulation of toxin binding sites in midbrain, with a similar trend in the other two regions.4. The finding that chronic receptor blockade neither mimicked nor blocked the agonist-induced up-regulation of [3H]-nicotine binding sites suggests that up-regulation of these receptors is not determined by their functional status. In contrast, it appears that chronic nicotine-induced up-regulation of[125I]-alpha BTX binding sites may result from receptor activation.

Amino Acid Residues Within the Sequence Region α55–74 of Torpedo Nicotinic Acetylcholine Receptor Interacting with Antibodies to the Main Immunogenic Region and with Snake α-Neurotoxins

The sequence region 55-74 of the alpha-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment--residues alpha 67-76--forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence alpha 55-74 of Torpedo AChR binds alpha-bungarotoxin (alpha BTX), suggesting that amino acid residues within this sequence region may contribute to formation of an alpha BTX binding site. Using single-residue substituted synthetic analogues of the sequence alpha 55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with alpha-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg55, Arg57, Trp60, Arg64, Leu65, Arg66, Trp67, or Asn68 strongly inhibited alpha-toxin binding, whereas substitutions of Ile61, Val63, Pro69, Ala70, Asp71, or Tyr72 had marginal effects. Substitutions within the region alpha 68-72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp60 substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp62 slightly but consistently affected binding of mAbs 6 and 22.

Effect of cold stress on cholinergic receptors in the rat adrenal gland

The effects of cold exposure on cholinergic binding sites in the rat adrenal gland were assessed by examining the binding of [ 125I]α-bungarotoxin (BTX), a nicotonic receptor antagonist and [ 3H]quinuclidinyl benzilate (QNB), a muscarinic receptor antagonist, to adrenal tissue homogenates. Cold exposure resulted in significant alterations in both nicotinic and muscarinic binding. Exposure to cold for 4 and 7 days resulted in a significant decrease in QNB binding. Scatchard analysis indicates that this alteration is due to a decrease in binding sites ( B max) rather than a change in ligand affinity ( K d). In contrast, chronic cold exposure produced a significant increase in BTX binding sites. These results indicate that adrenal cholinergic receptors are altered in reciprocal fashion by chronic cold exposure, and that this change may represent a key event in the sympathoadrenal system's adaptive response to chronic cold stress.

Acetylcholine receptors and sodium channels in denervated and botulinum‐toxin‐treated adult rat muscle.

1. The number of acetylcholine (ACh) receptors and Na channels was measured in adult rat hind-limb muscles after denervation or injection of botulinum toxin type A (BoTX), using specific binding of radiolabelled neurotoxins. 2. Denervation by sciatic nerve section increased the number of [125I]iodo-alpha-bungarotoxin ([125I]BTX) binding sites from low, unmeasurable levels to 39 +/- 3 fmol of toxin bound per milligram muscle protein at 21 days. 3. Subcutaneous injection of BoTX produced complete neuromuscular blockade for 11-14 days over which time the number of [125I]BTX binding sites increased with the same time course and to the same extent as following denervation. 4. Neither denervation nor BoTX treatment significantly altered the number of tritiated saxitoxin ([3H]STX) binding sites from normal values of 7.8 fmol/mg muscle weight or 57 +/- 3 fmol/mg homogenate protein. This may, however, correspond to a lower density of [3H]STX sites in the muscle membrane. 5. It was concluded that neuromuscular blockade with BoTX is equivalent to denervation in its effects on synthesis of ACh receptors. Numbers of Na channels are more stable than ACh receptors but may also be modulated by neuromuscular activity.

Affinity directed reactions of 3-trimethylaminomethyl catechol with the acetylcholine receptor from Torpedo californica

Summary 3-Trimethylaminomethyl catechol (TMC) was synthesized and shown maximally to inactivate 50% of the α-bungarotoxin (B tx) binding to the acetylcholine receptor (AcChR) from Torpedo californica electroplax. Incubation of the receptor with unsubstituted catechol produced no toxin binding inactivation of the receptor. Further, co-incubation of the receptor with 1 mM hexamethonium and 30 mM TMC effectively blocked the affinity-directed inactivation of Btx binding produced by incubation with 30 mM TMC alone. This degree of protection was consistent with the relative binding constants for hexamethonium (4.8 × 10−6 M) and TMC (2.8 × 10−5 M) with the AcChR. The affinity directed inactivation is suggested to be due to the covalent reaction of one or more of the quinone intermediates formed during the spontaneous oxidation of TMC with the Btx binding sites of the AcChR.