Apamin Receptor Research Articles

Increased K+ release during exercise in myotonic dystrophy type 1 (DM1) but not type 2 (DM2)

Background: Previously excessive K+ increase upon exercise has been described in myotonic dystrophy, but not in non-dystrophic myotonias or other muscular dystrophies. However, at that time the differentiation into myotonic dystrophy type 1 (DM1) or type 2 (DM2) was not yet possible. Objective: Resting venous and interstitial (intramuscular) lactate and K+ levels were measured in patients with genetically defined DM1 (n=6) and DM2 (n=6). Control groups included mitochondrial disorders (n=9) and healthy volunteers (n=9). In all individuals venous and interstitial lactate and K+ were analysed upon standardized forearm exercise using both an ischemic and nonischemic protocol. Results: There was no difference in resting venous and interstitial lactate and K+ levels between DM1, DM2, and both control groups. Maximal contraction force (MCF), workload, venous and interstitial lactate increase were significantly lower in DM1 than in the other groups. Patients with DM2 and mitochondrial disorders exhibited intermediate values. Venous and interstitial K+ increases were not different between DM1, DM2, and both control groups. Venous K+ increase related to workload (specific K+ production) was significantly higher in both protocols in patients with DM1, but not in those with DM2 compared to both control groups. In contrast venous lactate increase related to workload (specific lactate production) was not different in all four groups. Due to the used temporal resolution the interstitial increases were less pronounced than in venous blood. Conclusion: An abnormally high increase of venous K+ upon exercise seems to be specific for DM1 but could not be observed in DM2. Even if a higher K+ production upon exercise might be related to myotonia in DM1, other mechanisms seems to be responsible in DM2. For example, altered splicing has been shown for the small conductance Ca 2+-activiated K+-channel (apamin receptor) mRNA and might contribute to increased K+ release and subsequently to myotonic phenomenon in DM1.

Characterization and regulation of apamin-binding K+ channels in skeletal muscle.

The Pattern of development and regulation of the apamin receptor (afterhyperpolarization channel) was studied in cultures of skeletal muscle prepared from 1-2-day-old rat pups. Expression was measured by the specific binding of (125)I-apamin. Apamin binding was virtually undetectable until the time of fusion (3-4 days in culture) of single myoblasts into myotubes. Mature myotubes (5-7 days in vitro) displayed a Bmax of 7.4 fmol/mg protein and a Kd of 376 pmol/L. When studied in mature muscle cells apamin binding was found to increase twofold in response to tetrodotoxin (TTX) and elevated Ko, which resulted in decreased Na(i). In contrast, treatments causing an increase in Na(i), such as monensin and veratridine, caused a decrease in apamin binding. The increase in apamin binding following TTX treatment was due mainly to synthesis of new channels, as the effect was blocked by cycloheximide. Alterations in cytosolic Ca2+ by calcium ionophore or Ca-channel blockers were without effect on apamin-sensitive channel expression. We conclude that afterhyperpolarization channel expression is regulated by the level of intracellular Na+ ions.

Pharmacology of the high-affinity apamin receptor in rabbit heart

Apamin is a potent blocker of calcium-activated small conductance potassium (SK) channels in neurons, liver, skeletal muscle and ileum smooth muscle, but not in cardiac muscle. Cardiac muscle is devoid of SK channels; however, in isolated, single ventricular myocytes apamin is an extremely potent blocker of the L-type calcium current, and the anti-arrhythmic drug quinidine reverses apamin block. To characterize the receptor binding properties and pharmacology of the apamin receptor in heart. The binding properties of the apamin receptor were determined by rapid filtration of purified rabbit heart membranes. Monoiodinated apamin binds to a labile, membrane-bound protein in heart membranes at a single, high-affinity site (KD = 8.07 +/- 2.14 pM and Bmax = 686 +/- 167 fmoles/mg protein, significant run test at P = 0.05 for a one site fit). 125I-apamin binding is dose-dependently inhibited by apamin, scyllatoxin, quinidine, amiloride, as well as a variety of di- and trivalent cations that are classical blockers of L-type calcium channels (e.g. Co2+, Cd2+, Mn2+, La3+, Gd3+). The cardiac apamin receptor is also critically dependent upon pH, temperature and KCl, and co-purifies in the same membrane fraction as L-type cardiac Ca2+ channels. The apamin receptor in rabbit heart P2 membranes has pharmacological and biochemical properties in common with both an SK channel and an L-type Ca2+ channel.

Leiurotoxin I, a scorpion toxin specific for Ca(2+)-activated K+ channels. Structure-activity analysis using synthetic analogs.

Recently, we reported a structure-activity relationship study on P05, a novel leiurotoxin I-like scorpion toxin which is selective for the apamin-sensitive Ca(2+)-activated K+ channel [Sabatier et al. (1993) Biochemistry 32, 2763-2770]. Arg6, Arg7 and C-terminal His31 appeared to be key residues for P05 biological activity. Owing to the high sequence identity between P05 and leiurotoxin I (87%), several analogs of leiurotoxin I (Lei-NH2) with point mutations at these positions were designed and chemically synthesized using an optimized solid-phase technique. The synthesized peptides were [L6]Lei-NH2, [R7]Lei-NH2, Lei-OH and [R7]Lei-OH, as well as fragment [R7,Abu8]N4-S11-NH2. A chimeric analog ([M22,K24,R27]Lei-NH2), which possesses part of the iberiotoxin C-terminus, was also constructed. Circular dichroism analyses of these analogs, in agreement with their structural models obtained by molecular dynamics, showed that the point mutations did not significantly affect the overall secondary structures, as compared to natural Lei-NH2. All the peptides and natural toxins were compared in vitro for their capacity to inhibit binding of [125I]-apamin to rat brain synaptosomes, and in vivo for their specific neurotoxicity in mice. The Arg6 residue was essential for high biological activity of leiurotoxin I. Further, substitution of Met7 in the natural toxin by Arg7, or C-terminal amidation of His31, greatly increased affinity for the apamin receptor but did not significantly affect toxin neurotoxicity. Remarkably, the chimeric analog [M22,K24,R27]Lei-NH2 was found to retain leiurotoxin I-like activity, thus indicating that the negatively charged residues Asp24 and Glu27 (and Ile22) are not directly involved in the high toxin bioactivity. However, the chimeric molecule had no iberiotoxin-like effect on rat muscular maxi-K+ channels incorporated in lipid bilayers.

Solution structure of P05-NH2, a scorpion toxin analog with high affinity for the apamin-sensitive potassium channel.

The venom of the scorpion Androctonus mauretanicus mauretanicus contains a toxin--P05--which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. P05, a 31-residue polypeptide cross-linked by three disulfide bridges, also possesses binding and physiological properties similar to those of the bee venom toxin apamin (18 residues, two disulfides). However, the amino acid sequences of these two polypeptides are dissimilar, except for a common Arg-Arg-Cys-Gln motif which is located on an alpha-helix. P05-NH2, a synthetic analog of P05, unlike native P05, was found to bind irreversibly to the apamin receptor. The solution structure of P05-NH2 has been solved by conventional two-dimensional NMR techniques followed by distance geometry and energy minimization. The obtained conformation is composed of two and an half turns of alpha-helix (residues 5-14) connected by a tight turn to a two-stranded antiparallel beta-sheet (sequences 17-22 and 25-29). This beta-sheet has a right-handed twist as usual for such secondary structures. The beta-turn connecting the two strands belongs to type II'. This structure is homologous to all scorpion toxin structures known so far as well as to insect defensins. The three arginines known to be involved in the pharmacological activity, i.e., Arg6, Arg7, and Arg13, are all located on the solvent-exposed side of the helix and form a positively charged surface which includes Gln9. The calculated electrostatic potential is highly asymmetric with the greatest positive potential centered on the Arg-rich alpha-helix side.(ABSTRACT TRUNCATED AT 250 WORDS)

Colchicine alters apamin receptors, electrical activity, and skeletal muscle relaxation.

A low conductance calcium-activated K+ channel is thought to regulate the rate of firing of several excitable cells. In skeletal muscle the expression of this channel is under nerve control. Previously, we reported that axonal flow blockade of rat nerves, induced by colchicine, caused a transient increase in muscle apamin receptors, determined by 125I-apamin binding to membrane fractions. The increase in apamin receptors was correlated with repetitive discharges resembling myotonic potentials in the electromyogram, that were blockable by apamin. Here we show that the increase in muscle apamin receptors and the alteration of the electromyogram are followed closely by a slowing of the twitch relaxation, that in turn, is decreased by apamin. Furthermore, the presence of myotonic-like alterations in the electromyogram and a slowing of muscle relaxation when muscle apamin receptors are increased suggests that these channels may participate, among other factors, in the generation of some kinds of myotonia.

Increase of apamin receptors in skeletal muscle induced by colchicine: possible role in myotonia.

We have shown an increase of apamin receptors in rat skeletal muscle membranes following the application of colchicine to the sciatic nerve. 125I-apamin binding to partially purified membrane fractions was observed since day 4, reached a maximum around days 6-15, and was negligible at day 35 after the application of colchicine. Control muscles (nerves treated with buffer solution) showed low binding values (11 fmol/mg protein). Maximal 125I-apamin binding values to partially purified muscle membranes of colchicine-treated rats (42 fmol/mg protein) were lower than those obtained in denervated muscle (95 fmol/mg protein). The affinity binding constant values were 37 (colchicine) and 95 pM (denervation). No signs of muscle denervation were observed on histological examination of the nerve submitted to colchicine treatment nor in the muscles innervated by it. Muscle tension developed by indirect stimulation was the same as in controls. We here show also that partially purified membranes of normal untreated muscles have measurable amounts of 125I-apamin binding (13 fmol/mg protein), similar to those obtained in control muscles. Electromyographic recordings of the muscles after colchicine treatment of the nerve showed abnormal repetitive electrical discharges, similar to myotonic discharges, that were present with a similar temporal course as the increase in apamin receptors. The myotonic-like discharges were suppressed by the topical application of apamin to the muscle, whereas the toxin had no effect on anthracene-9-carbolytic acid-induced myotonia. Our results suggest that a neurotrophic factor that travels by axonal flow is involved in the regulation of the expression of apamin receptors in skeletal muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of apamin binding sites in rat intestinal mucosa

Apamin, a specific blocker of one class of Ca 2+-activated K + channels, was used to detect the apamin receptors associated with K + channels in the mucosa of the rat jejunum and colon. Two receptor sites for 125I-apamin have been identified. These sites differed in their affibity for apamin (jejunum: K D1 = 1.1 nM and K D2 = 170 nM; colon: K D1 = 0.5 nM and K D2 = 140 nM) and the maximum number of sites (jejunum: B max1 = 111 and B max2 = 4030; colon: B max1 = 187 and B max2 = 7550fmol/mg of protein). 125I-apamin binding was stimulated by K + ions with K 0.5 = 1.0 mM and inhibited by the neuromuscular blocker tubocurarine (K I = 50 μM ). We interpret these data to demonstrate that the high-affinity, low-capacity binding sites reflect the existence of apamin-sensitive K + channels in the intestinal mucosa.

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel

Photoaflinity labeling of rat brain membraines with [ 125l]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to b %glycoxylated. Partial protocolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. Thes results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but results from cleavage of the 86 kDa polypeptide

Polypeptide constitution of receptors for apamin, a neurotoxin which blocks a class of Ca2+-activated K+ channels

Affinity labelling experiments with different azido-125I-apamin derivatives were carried out to identify polypeptide components of the apamin-sensitive Ca2+-activated K+ channel in brain and pheochromocytoma cell membranes. Different polypeptides were labelled with different apamin derivatives. The major component has a molecular mass of about 30 kDa but other components at 45, 58 and 86 kDa were also identified. Results obtained with brain membranes on one hand and pheochromocytoma cells on the other were not exactly identical and suggest that there are sub-types of apamin receptors.

Different innervation mechanisms between the lesser and greater curvature of guinea pig antrum.

Transmural stimulation (TS) produced a frequency-dependent contraction of the longitudinal muscles from the lesser curvature of the guinea pig antrum, which was abolished by atropine. On the other hand, a response to TS of the strips from the greater curvature was biphasic: a rapid contraction followed by a relaxation, which was abolished by tetrodotoxin. By pretreatment with atropine, rapid contraction of the biphasic response evoked by TS in the greater curvature was abolished and relaxation was augmented. Relaxation to TS of the greater curvature was not affected by prazocine, yohimbine, phentolamine, propranolol, theophylline, apamin, alpha-chymotrypsin, and vasoactive intestinal polypeptide receptor antagonist. Different innervation mechanisms were suggested to be present in the longitudinal muscles between the lesser curvature (innervated with excitatory cholinergic neurons) and the greater curvature (innervated with excitatory cholinergic neurons and nonadrenergic inhibitory neurons) of the guinea pig antrum.

Detection and photoaffinity labeling of the Ca 2+-activated K + channel-associated apamin receptor in cultured astrocytes from rat brain

Apamin, an 18-amino acid bee venom peptide, is a specific blocker of a class of Ca 2+ activated K + channels. Mono 125I-iodoapamin was used to detect the K + channel-associated receptor site in cultured astrocytes from rat brain. Specific high-affinity binding to intact glial cells with a K d of about 90 pM at 1 °C and pH 7.5 was demonstrated by equilibrium and kinetic methods. The average receptor capacity was 3 fmol/mg cell protein which is 2 to 3-fold lower than in primary cultured neurons. Bindings was stimulated by K + ions, but to a lesser extent than with neuronal receptors. Photoaffinity labeling of receptor/ion channel components using an arylazide derivative of 125I-monoiodoapamin revealed the presence of the 86- and 33-kDa polypeptides, previously detected in neurones. However a 59-kDa peptide which is present in synaptic membrane preparations from adult rat brain, but not in cultured neurons, was also clearly labeled in intact astrocytes. This indicates that the 59-kDa polypeptide is not a proteolytic fragment of the 86-kDa chain but an associated subunit which is only accessible to photolabeling in certain apamin receptor preparations. Apamin-sensitive Ca 2+-activated K + channels in astrocytes may be one of the pathways by which glial cells redistribute K + in the central nervous system (CNS).

Solubilization of the apamin receptor associated with a calcium- activated potassium channel from rat brain

The apamin binding protein was solubilized from rat brain synaptic membranes using sodium cholate. Receptor yield and stability depended closely on the detergent/protein ratio. In optimum conditions the receptor retained high affinity for mono 125I-iodoapamin with Kd = 40 pM at pH 7.5 and 1 degree C and a binding capacity of 17 fmol/mg protein. 125I-apamin binding was stimulated by K+ ions with a K0.5 = 0.6 mM, demonstrating that the regulatory K+ site is also part of the soluble complex. Other ions could be substituted for K+ with an affinity sequence Tl+ = K+ = Rb+ greater than Cs+ greater than NH4+ greater than Li+ or Na+. Binding was inhibited by the neuromuscular blockers gallamine and tubocurarine and by the K+ channel blockers quinidine and tetraethylammonium chloride but not by 4-aminopyridine, in agreement with known pharmacological profile for inhibition of apamin-sensitive K+ permeability. Increasing the K+ concentration did not reverse inhibition by tetraethylammonium ions demonstrating that it does not bind competitively to the regulatory cationic site. Analysis of the covalently labeled apamin binding protein/sodium cholate complex by density gradient centrifugation indicated a high molecular weight with S20,w = 20 S.

Quantitative autoradiographic mapping in rat brain of the receptor of apamin, a polypeptide toxin specific for one class of Ca 2+-dependent K + channels

The localization of the receptor for apamin, a specific toxin for one class of sensitive Ca 2+-dependent K + channel, was studied in rat brain using an in vitro autoradiographic technique. Radiolabeled monoiodoapamin binds specifically to rat brain sections with a high affinity ( K d = 25 pM) to a single class of sites. Autoradiograms demonstrated a very heterogeneous distribution of the apamin receptor throughout the brain. Very high grain densities were localized on the habenula, lateral septum, supraoptic and supraschiasmatic nuclei. Areas containing high levels of apamin binding sites included anterior olfactory nucleus, stratum oriens of hippocampus, pontine nuclei and granular layer of the cerebellar cortex and inferior olive. The thalamus, some nuclei of hypothalamus, hippocampus, tegmental area, red and oculomotor nuclei, vestibular nuclei and superior olive, among others, presented intermediate grain densities. In the other main areas, in particular basal ganglia, raphe, low to very low levels of apamin binding sites have been observed.

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative potassium channel subunits and target size analysis

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12 pheochromocytoma cells which hyper-produce the apamin receptor.

Undifferentiated PC12 cell produce high levels of apamin receptors (measured with 125I-apamin) after 7 days in culture. These levels are at least 50 times higher than those found in other cellular types which are also known to have apamin receptors and apamin-sensitive Ca2+-activated K+ channels in their membranes. Treatment of undifferentiated PC12 cells with nerve growth factor maintains these cells in a state having a low level (10 times less after 7 days of culture) of apamin receptors. Ca2+ injection into PC12 cells with the calcium ionophore A23187 has been used to monitor the activity of the Ca2+-activated K+ channel following 86Rb+ efflux. A large component of this Ca2+-activated 86Rb+ efflux is inhibited by apamin. Half-maximum inhibition by apamin of both 86Rb+ efflux and 125I-apamin binding was observed at 240 pM apamin. Another component of 86Rb+ efflux is due to another type of Ca2+-activated K+ channel which is resistant to apamin and sensitive to tetraethylammonium. The Ca2+ channel activator Bay K8644 also triggers an apamin-sensitive Ca2+-dependent 86Rb+ efflux. Bay K8644 has been used to analyze the internal Ca2+ concentration dependence of the apamin-sensitive channel activity. Under normal conditions, the internal Ca2+ concentration is 109 +/- 17 nM, and the apamin-sensitive channel is not activated. The channel is fully activated at an internal Ca2+ concentration of 320 +/- 20 nM.

Expression of apamin receptor in muscles of patients with myotonic muscular dystrophy

Myotonic muscular dystrophy, or Steinert disease, is a dominantly inherited disease of muscle which occurs with a frequency of between 1 in 18,000 and 1 in 7,500 people (refs 1, 2). One of the prominent clinical manifestations is muscle stiffness and difficulty in relaxation of muscles after voluntary contractions. Electrophysiological signs of myotonia include increased excitability with a tendency to fire trains of repetitive action potentials in response to direct electrical and mechanical stimulation. Most experimental and clinical data suggest that myotonic muscular dystrophy arises from genetically induced alterations of the muscle membrane. We show here for the first time that muscle membranes of patients with myotonic muscular dystrophy contain the receptor for apamin, a bee venom toxin known to be a specific and high-affinity blocker of one class of Ca2+-activated K+ channels in mammalian muscle. The apamin receptor is completely absent in normal human muscle as well as in muscles of patients with spinal anterior horn disorders.

Existence of a Ca 2+-dependent K + channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding

Apamin, a 18-amino acid neurotoxin isolated from bee venom, is a specific blocker of one class of the Ca 2+-dependent K + channels. The monoiodo derivative of the toxin with high specific radioactivity (1600 Ci/mmol) has been used to study its binding to synaptic. membrane (SM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brains. The B max (30.2 fmol/mg protein) for CTX-PSD is about twice that for CTX-SM (17.3 fmol/mg protein), suggesting a concentration of the apamin receptor protein in CTX-PSD over CTX-SM fractions. The lower value of B max for CL-PSD (12.3 fmol/mg protein), and the higher K d value (51 pM) than for CTX-SM (33 pM), CTX-PSD (24 pM), and and CL-SM (39 pM), may reflect the disruptive effect of Triton X-100 on these thin structures. The values of B max and K d for CTX-SM are similar to those (22.0 fmol/mg protein and 33 pM) for rat CTX-SM 33. Both Ca 2+ and NA + inhibit apamin binding to CTX-PSD with K 0.5 values of 14 and 31 mM, respectively, while the optimum concentration of KCl for activation is 5 mM. All these values are similar to those found for rat synaptosomes 30. Covalent labeling of the apamin binding protein, using the non-cleavable cross-linker, disuccinimidyl suberate, reveals an apamin binding polypeptide of 27 kdaltons under reducing and denaturing conditions in both the CTX-SM and CTX-PSD preparations, similar to that (28 kdaltons) reported for rat CTX-SM fractions 33. Prior phosphorylation of isolated CTX-PSD had no effect on apamin binding, nor did apamin binding influence subsequent phosphorylation of CTX-PSD. Calmodulin, an intrinsic PSD protein, may not play a role in apamin binding to PSD, since addition of calmodulin, or removal of the calmodulin by EGTA treatment, resulted in no change in the binding capacity of the PSD. The apamin binding protein seems to be bound quite firmly in the CTX-PSD fraction since treatments with 0.5% deoxycholate, 1% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, could only remove the apamin-receptor complexes from CTX-PSD by 40, 55, 52, 12 and 15%, respectively. These results contrast with the findings that the two detergents mentioned solubilize 80–93% of the receptor from the synaptosomal or synaptic membrane fractions, indicating that a good deal of the receptor in these fractions is membrane-bound and not connected to the PSD. The results suggest that the apamin binding protein, which spans the postsynaptic membrane, is an intrinsic PSD component. This finding is the first instance of a channel protein being found in a PSD fraction. The present results, together with previous ones showing the presence of GABA, flunitrazepam, and glutamate binding sites in the isolated PSD fraction, indicate that the PSDs are involved in not only the anchoring of membrane receptor proteins, but also in the anchoring of membrane channel proteins, and suggest a mediating role for the PSD in synaptic events.

The all-or-none role of innervation in expression of apamin receptor and of apamin-sensitive Ca2+-activated K+ channel in mammalian skeletal muscle.

The long-lasting after-hyperpolarization(s) (AHP) that follows the action potential in rat myotubes differentiated in culture is due to Ca2+-activated K+ channels. These channels have the property to be specifically blocked by the bee venom toxin apamin at low concentrations. Apamin has been used in this work to analyze, by electrophysiological and biochemical techniques, the role of innervation in expression of these important channels. The main results are as follows: (i) Long-lasting AHP that follows the action potential in rat myotubes in culture disappears when myotubes are cocultured with nerve cells from the spinal cord under the conditions of in vitro innervation. (ii) Extensor digitorum longus muscles from adult rats have action potentials that are not followed by AHP but AHP are systematically recorded after muscle denervation and they are blocked by apamin. (iii) Specific 125I-labeled apamin binding is undetectable in innervated muscle fibers but it becomes detectable 2-4 days after muscle denervation to be maximal 10 days after denervation. (iv) Apamin receptors detected with 125I-labeled apamin are present at fetal stages with biochemical characteristics identical to those found in myotubes in culture. The receptor number decreases as maturation proceeds and 125I-labeled apamin receptors completely disappear after the first week of postnatal life, in parallel with the disappearance of multi-innervation. All these results taken together strongly suggest an all-or-none effect of innervation on the expression of apamin-sensitive Ca2+-activated K+ channels.

Molecular properties of the apamin-binding component of the Ca2+-dependent K+ channel. Radiation-inactivation, affinity labelling and solubilization.

Radiation-inactivation was used to assess the functional size of the apamin-binding component of the Ca2+-dependent K+ channel. The amount of specific binding of 125I-apamin to receptors in synaptic membranes of rat cortex decayed exponentially with increasing doses of ionizing radiation and target size analysis was consistent with a relative molecular mass of 250 000 +/- 20 000 for the 125I-apamin receptor. Analysis on sodium dodecyl sulfate gels following covalent cross-linking of 125I-apamin to its receptor in a synaptosomal membrane preparation from rat cortex revealed a single labelled polypeptide chain of Mr = 33 000 +/- 2000 in the presence of protease inhibitors. Our results suggest that the Ca2+-dependent K+ channel from rat cortex is an oligomeric structure of Mr = 250 000 +/- 20 000 containing an apamin-binding subunit of Mr = 33 000 +/- 2000. The apamin-binding component of the Ca2+-dependent K+ channel from rat synaptosomes was solubilized using detergents such as sodium cholate or 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate. Phospholipids did not increase the stability of the apamin-binding component during the solubilization. Binding of apamin to its solubilized receptor is reversible and saturable. The dissociation constant of the apamin-receptor complex is 40-150 pM, the rates constants of association and dissociation being 3.2 X 10(6) M-1s-1 and 1.4 X 10(-4)s-1 respectively. These binding characteristics are similar to those found for the membrane-bound apamin receptor.