Characterization of the α‐Bungarotoxin Receptor in Chick‐Embryo Retina

Abstract

Primary cultures and membranes fractions from chick embryo retina bind iodinated a-bungarotoxin, a highly selective ligand for nicotinic acetylcholine receptors fromskeletal muscle and fishelectric organ. The binding issaturable with anequilibrium dissociationconstant (Kd) of0.75 i 0.09nM. The pseudo-first-order rate constant(k + , )for binding at 37°C is 1.76 x lo5 M-' s-', the dissociation rate constant (k,) at 37,'C is 1.15 x s-'. Nicotinic cholinergic ligands and local anaesthetics inhibit a-bungarotoxin binding. In the case of carbamoylcholine, the inhibition of binding depends on the time of exposure to this cholinergic agonist. a-Bungarotoxin has no effect on the carbamoylcholine-induced stimulation in sodium permeability of cultured retinal neurons. On sucrose density gradients containing Triton X-I 00, the toxin binding site sediments with an apparent sedimentation coefficient of 10.5 -11 S. Detergent-solubilized a-bungarotoxin receptor crossreacts with antisera raised against nicotinic acetylcholine receptors from Torpedo murmoruta. These results are interpreted as indicating that a-bungarotoxin binds to retinal nicotinic acetylcholine receptors without affecting cholinergic receptor function.During the past decade, the nicotinic acetylcholine receptor from fish electric organ and skeletal muscle has been extensively purified and characterized in several laboratories (for reviews see [I, 21). This progress has become possible mainly through the use of a-toxins from the venoms of various elapid snake species which are highly selective ligands for this protein.One of these toxins, a-bungarotoxin from Bungurus multirinctus, has been most commonly used and shown to block the function of the muscle and fish acetylcholine receptor by binding to the cholinergic ligand recognition site of the receptor protein [I -31.Binding sites for a-bungarotoxin of nicotinic specificity are also widely distributed in the peripheral and central nervous systems of both vertebrates and invertebrates (for review see [4]). The nature of these toxin binding sites, though otten referred to as neuronal nicotinic acetylcholine receptors, remains, however, unsolved since their identification as cholinergic receptors relies almost exclusively on pharmacological binding experiments. Both electrophysiological and sodiumflux studies on spinal cord and sympathetic neurons have shown that a-bungarotoxin is unable to antagonize the activation of neuronal nicotinic acetylcholine receptors [5 -101. As an important argument against the identity of the neuronal a-bungarotoxin binding protein with a nicotinic acetylcholine receptor, Patrick and Stallcup [7] reported that antibodies to the cholinergic receptor from Electrophorus electricus failed to recognize the detergent-solubilized a-bungarotoxin binding site of PC12, a rat phaeochromocytoma cell line. The same antibodies, however, were effective in blocking the function of the nicotinic acetylcholine receptor present on these PC12 cells. Controversial observations suggesting a crossreactivity...

"Receptores nicotínicos de acetilcolina no desenvolvimento da retina de pinto em cultura: modulação por melatonina endógena"

“…A possibilidade de haver aumento de número de sítios de α-BTX, do quarto para o quinto dia de cultivo foi descartada por resultados obtidos nos experimentos de saturação. Esses resultados estão de acordo com outros trabalhos que mostram diminuição do número de sítios de α-BTX em culturas de retina de pinto a partir do quarto dia de cultivo (Betz, 1981;De Pomerai e cols., 1983), oposto do observado no desenvolvimento do embrião, como já descrito. Os valores de Kd encontrados nas duas idades, 17,6 nM (C4) e 5,9 nM (C5), são semelhantes aos estimados para o receptor α8 em retinas de pinto pós eclosão (5,5 nM) (Gotti e cols., 1997) (Wonnacot, 1986;Lippiello, 1987 (Lippiello, 1987 Luzindole inibiu a eficácia da acetilcolina (1mM) na mesma faixa de concentração em que inibiu a mobilização de fosfoinositídeo de membrana em fatias de cérebro de pinto, efeito esse atribuído ao receptor ML2 (Popova e Dubocovich, 1995), atual MT3/QR2 o qual é bloqueado por luzindole em doses acima de 1µM (Nosjean, 2001 Uma possibilidade interessante para explicar a diminuição da eficácia da acetilcolina, quando as culturas foram tratadas cronicamente com luzindole, baseia-se no fato de que o bloqueio crônico de receptores de melatonina com luzidole pode estar provocando um estado de claro-constante na retina, para todas as ações da melatonina via receptor de membrana, que passam a estar inibidas.…”