Affinity labeling of the digitalis-binding site.
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Affinity Labels↗
Biomedical subjects
M Lazdunski
Publications and source records attributed to M Lazdunski.
Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger.
Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure--activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmacological profiles. 5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+/H+ exchange system. Conversely, amiloride derivatives that are substituted on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.
Amiloride↗
Purification and subunit structure of a putative K+-channel protein identified by its binding properties for dendrotoxin I.
The binding protein for the K+-channel toxin dendrotoxin I was purified from a detergent extract of rat brain membranes. The purification procedure utilized chromatography on DEAE-Trisacryl, affinity chromatography on a dendrotoxin-I-Aca 22 column, and wheat germ agglutinin-Affigel 10 with a final 3800- to 4600-fold enrichment and a recovery of 8-16%. The high affinity (Kd, 40-100 pM) and specificity of the binding site are retained throughout the purification procedure. Analysis of the purified material on silver-stained NaDodSO4/polyacrylamide gel revealed three bands of Mr 76,000-80,000, 38,000, and 35,000. Interestingly, the binding site for 125I-labeled mast cell degranulating peptide, another putative K+-channel ligand from bee venom, which induces long-term potentiation in hippocampus, seems to reside on the same protein complex, as both binding sites copurify through the entire purification protocol.
Animals↗
Vasopressin modulates the spontaneous electrical activity in aortic cells (line A7r5) by acting on three different types of ionic channels.
A7r5 smooth muscle (aorta) cells have a spontaneous electrical activity. Application of vasopressin produces a hyperpolarization accompanied by an interruption of the spontaneous activity, which is followed by a depolarization associated with a recovery of the spiking activity. Vasopressin action is produced by an action of the peptide on three different types of ionic channels. Vasopressin activates a Ca2+-sensitive K+ conductance, presumably by producing inositol 1,4,5-trisphosphate intracellularly and liberating Ca2+ from internal stores. This activation is transient (0.5-4 min) and is related to the vasopressin-induced hyperpolarization. Intracellular perfusion of inositol trisphosphate triggers by itself a transient K+ current and prevents subsequent activation by vasopressin. Vasopressin inhibits an L-type Ca2+ channel through both protein kinase C activation and a [Ca2+]i-dependent inactivation mechanism triggered by inositol trisphosphate production. The addition of the activation of a Ca2+-sensitive K+ channel and of the inhibition of a voltage-sensitive Ca2+ channel is responsible for the transient blockade of the spontaneous activity. Vasopressin also provokes the activation of an inward current (2-20 min) due to a nonselective channel able to transfer Ca2+, Na+, K+, and Cs+ across the membrane. This effect of the peptide is associated with the depolarization following the hyperpolarization phase.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Characterization, purification, and affinity labeling of the brain [3H]glibenclamide-binding protein, a putative neuronal ATP-regulated K+ channel.
Sulfonylurea and particularly glibenclamide are potent blockers of ATP-regulated K+ channels in insulin-secreting cells. A very good correlation exists between binding of sulfonylurea to brain and insulinoma cell membranes. The [3H]glibenclamide-binding component from pig brain microsomes was solubilized with digitonin with a complete retention of its properties of interaction with glibenclamide and other sulfonylureas. A four-step purification was achieved that used (i) hydroxylapatite chromatography, (ii and iii) affinity chromatographies on ADP-agarose and wheat germ agglutinin-agarose columns, and (iv) a final chromatographic step on a mixture of AMP-agarose/GMP-agarose/hydroxylapatite. This procedure led to a 2500-fold purification. NaDodSO4/polyacrylamide gel electrophoresis of the purified material in reducing and nonreducing conditions showed that the sulfonylurea-binding component is made of a single major polypeptide chain of Mr 150,000 +/- 10,000. Direct photoaffinity labeling of the receptor with [3H]glibenclamide at different steps of the purification also showed that radioactivity was specifically incorporated into a polypeptide of Mr 150,000 +/- 5000, thus confirming the subunit structure indicated by the purification.
Adenosine Triphosphate↗
ATP-sensitive K+ channels that are blocked by hypoglycemia-inducing sulfonylureas in insulin-secreting cells are activated by galanin, a hyperglycemia-inducing hormone.
The action of the hyperglycemia-inducing hormone galanin, a 29-amino acid peptide named from its N-terminal glycine and C-terminal amidated alanine, was studied in rat insulinoma (RINm5F) cells using electrophysiological and 86Rb+ flux techniques. Galanin hyperpolarizes and reduces spontaneous electrical activity by activating a population of ATP-sensitive K+ channels with a single-channel conductance of 30 pS (at -60 mV). Galanin-induced hyperpolarization and reduction of spike activity are reversed by the hypoglycemia-inducing sulfonylurea glibenclamide. Glibenclamide blocks the galanin-activated ATP-sensitive K+ channel. 86Rb+ efflux from insulinoma cells is stimulated by galanin in a dose-dependent manner. The half-maximum value of activation is found at 1.6 nM. Galanin-induced 86Rb+ efflux is abolished by glibenclamide. The half-maximum value of inhibition is found at 0.3 nM, which is close to the half-maximum value of inhibition of the ATP-dependent K+ channel reported earlier. 86Rb+ efflux studies confirm the electrophysiological demonstration that galanin activates an ATP-dependent K+ channel.
Action Potentials↗
Molecular properties of structure and regulation of the calcium channel.
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Animals↗
The structure of the amiloride sensitive apical Na+ channel from pig kidney.
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Amiloride↗
A novel high affinity class of Ca2+ channel blockers.
Benzolactams (HOE 166 and analogs) form a new class of molecules acting on the 1,4-dihydropyridine-sensitive L-type Ca2+ channels. The main binding properties of HOE 166 and analogs to rabbit skeletal muscle membranes are as follows. (i) The compounds have a specific binding site to which they associate with a high affinity (0.25 nM for HOE 166). (ii) Unlabeled HOE 166 and analogs completely inhibit 1,4-dihydropyridine binding [(+)-[3H]PN 200-110] in a competitive way. (iii) Affinity values measured for HOE 166 inhibition of (+)-[3H]PN 200-110 (K0.5 = 0.25 nM and K1 = 0.55 nM) and of [3H]HOE 166 binding (K0.5 = 0.5 nM) are in good agreement. They also fit with results from direct binding experiments with tritiated HOE 166 (Kd = 0.27 nM) and from kinetic experiments (Kd = 0.39 nM). (iv) HOE 166 completely inhibits the specific binding of other classes of Ca2+ channel antagonists such as phenylalkylamines [(-)[3H] desmethoxyverapamil], benzothiazepines (d-cis-[3H]diltiazem), diphenylbutylpiperidines ([3H]fluspirilene), and [3H]bepridil. In all these cases the binding inhibition is of a noncompetitive nature. (v) The maximum binding capacity for [3H]HOE 166 binding to transverse tubule membranes, 65 pmol/mg of protein, is the same as that found for other classes of Ca2+ channel antagonists. 45Ca2+ uptake experiments performed with the rat aortic cell line A7r5 and the insulin-secreting cell line RINm5F demonstrate that HOE 166 and analogs fully inhibit the 1,4-dihydropyridine-sensitive 45Ca2+ influx elicited by depolarization. There is a good correlation between inhibitory potencies of compounds in the HOE 166 series measured on (+)-[3H]PN 200-110 binding to A7r5 membranes and on the activity of Ca2+ channels followed by 45Ca2+ fluxes with the same cells. Structure-function relationships of HOE 166 and analogs for Ca2+ channel blockade in A7r5 and RINm5F cells were also in good correlation. Finally, voltage-clamp experiments confirmed that voltage-dependent L-type Ca2+ channels are completely blocked by 100 nM HOE 166 even at a membrane potential held at -80 mV.
Animals↗
The receptor for antidiabetic sulfonylureas controls the activity of the ATP-modulated K+ channel in insulin-secreting cells.
Sulfonylureas are powerful hypoglycemic drugs that have been used for decades to treat diabetic patients. This paper describes a 86Rb+ flux technique that permits one to study easily the properties of ATP-modulated K+ channels in RINm5F insulinoma cells. Sulfonylureas inhibit this type of K+ channel under conditions of intracellular ATP depletion. The most potent sulfonylureas (glibenclamide, glipizide, and gliquidone) are acting in the nanomolar range of concentration. Inhibition of the single ATP-modulated K+ channels by low concentrations of sulfonylureas was also observed using the patch-clamp technique. The sulfonylurea receptor has been biochemically identified with [3H]glibenclamide. For 10 different sulfonylureas (or sulfonylurea analogs) there was an excellent correlation between efficacy of blockade of ATP-modulated K+ channels and efficacy of binding to the sulfonylurea receptors using the 3H-ligand.
Adenoma, Islet Cell↗
Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system.
The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.
Affinity Labels↗
Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels.
Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.
Affinity Labels↗
Receptors for diphenylbutylpiperidine neuroleptics in brain, cardiac, and smooth muscle membranes. Relationship with receptors for 1,4-dihydropyridines and phenylalkylamines and with Ca2+ channel blockade.
Neuroleptic molecules of the diphenylbutylpiperidine series (DPBP), such as fluspirilene, penfluridol, pimozide and clopimozide, antagonize binding of (-)[3H]desmethoxyverapamil ((-)[3H]D888) and (+)[3H]PN 200-110 to rabbit brain, heart and smooth muscle membranes. The diphenylbutylpiperidine binding site in all these tissues is distinct but is allosterically related to the 1,4-dihydropyridine binding site and to the phenylalkylamine binding site. High and low affinity binding sites for (-)D888 were identified. (-)[3H]D888 binding at both types of sites was inhibited following the saturation of a single type of diphenylbutylpiperidine binding site. Half-maximal inhibition (K0.5) of brain, heart and smooth muscle membranes binding by different diphenylbutylpiperidines was in the range of 10-100 nM. These K0.5 values were one to two orders of magnitude higher than those found for the high affinity diphenylbutylpiperidine receptor in skeletal muscle membranes. The K0.5 values found in binding experiments in smooth muscle were similar to the (IC50) values for half-maximal inhibition by diphenylbutylpiperidine of voltage-dependent 45Ca2+ influx through the slow Ca2+ channel.
Animals↗
The brain response to the bee venom peptide MCD. Activation and desensitization of a hippocampal target.
The mast cell-degranulating peptide (MCD) isolated from bee venom has been found previously to have receptor sites in rat brain. Behavioral and electrocorticographic responses following intracerebroventricular injections of various doses of MCD have been analyzed. MCD produced a quasi-permanent hippocampal theta rhythm in the motionless animal alternating with epileptiform spike waves and paroxystic seizures. At a dose of 70 pmol seizures occurred for half of the treated rats. At a dose of 100 pmol generalized paroxystic crises were observed for all the rats. These effects were not antagonized by naloxone, morphine, diazepam and progabide. Rats recovered 24 h after a 100 pmol injection of MCD. A second ipsilateral injection to these rats showed the occurrence of a desensitization phenomenon. Desensitization was not observed when the second injection was contralateral. These physiological responses were studied in relation with a biochemical approach on membrane sites of action of MCD using [125I]MCD and their behavior in the desensitization process. The target of [125I]MCD is the ipsilateral hippocampus. Recovery from MCD effects was not due to MCD degradation. Desensitization was not due to down-regulation of the MCD receptor level.
Animals↗
Identification in mammalian brain of an endogenous substance with Na+ channel blocking activities similar to those of tetrodotoxin.
A substance with Na+ channel blocking activities has been isolated from pig brain after extraction and purification on sulfopropyl-Sephadex C-25, reversed-phase and carboxymethyl Synchropak high pressure liquid chromatography columns. The peptidic material i) displaces [3H]ethylenediamine tetrodotoxin ([3H]en-TTX) from its binding sites on rat brain membranes, (ii) it blocks 22Na+ influx induced by veratridine and sea anemone toxin on neuroblastoma and embryonic chick heart cells in culture, (iii) it specifically decreases the height of the action potential generated in frog sciatic nerve, and (iv) it blocks the fast Na+ current in voltage-clamped neuroblastoma cells. These properties are similar to those of tetrodotoxin while the endogenous factor is a peptide that is destroyed by proteases. These results suggest the presence in pig brain of a potent Na+ channel modulation activity.
Action Potentials↗
Autoradiographic analysis in rat brain of the postnatal ontogeny of voltage-dependent Na+ channels, Ca2+-dependent K+ channels and slow Ca2+ channels identified as receptors for tetrodotoxin, apamin and (-)-desmethoxyverapamil.
The postnatal development of the distribution of 3 different ionic channel proteins in rat brain was studied using light microscopic autoradiography. [3H]Ethylenediaminetetrodotoxin, [125I]apamin and (-)-[3H]desmethoxyverapamil were used to label one class of voltage-dependent Na+ channel proteins, one class of Ca2+-dependent K+ channel proteins, and the slow Ca2+ channel protein, respectively. Ca2+-dependent K+ channel proteins are detected very early in the germinative zone. They are associated to neuronal somas during their migration and their maturation. In hippocampus and cerebral cortex, apamin binding sites are already present at birth and their density increases to day 20 postnatal when the adult localization is established. Slow Ca2+ channel protein development occurs later in CNS ontogenesis. The development of slow Ca2+ channels seems to follow the development of dendrites. Density of these channel proteins increases regularly until adult age. At the resolution level of this analysis, Na+ channel proteins are absent in diencephalon at birth. Their appearance and their increase in density are strictly correlated to the synaptogenesis in particular in cerebral and cerebellar cortex and hippocampus. Although cerebellum, neocortex and hippocampus have been particularly analyzed, other brain structures have also been examined.
Animals↗
Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel.
Binding studies indicate that ciguatoxin and brevetoxin allosterically enhance in a very similar way the binding of [3H]batrachotoxinin A 20-alpha-benzoate to the neuronal Na+ channel protein. Moreover ciguatoxin competitively inhibits the binding of [3H]brevetoxin-3 to rat brain membranes. The affinity of ciguatoxin for the Na+ channel is at least 20-50-times higher than that of brevetoxin. These results indicate that ciguatoxin and brevetoxins act at the same binding site on the sodium channel.
Animals↗
The antidiabetic sulfonylurea glibenclamide is a potent blocker of the ATP-modulated K+ channel in insulin secreting cells.
The ATP-sensitive K+ channel of RINm5F insulinoma cells is activated after an intracellular ATP depletion. This activation can be followed by 86Rb+ efflux. Once activated by ATP depletion, the K+ channel can be blocked by the hypoglycemic drug, glibenclamide. The blockade is of a high-affinity type (K0.5 = 0.06 nM). Recording of the activity of ATP-sensitive K+ channels with the patch-clamp technique confirmed that they could be completely blocked with 20 nM glibenclamide.
Adenosine Triphosphate↗