RESUMO
Snake alpha-neutotoxins of Elapidae venoms are grouped into two structural classes, short-chain and long-chain alpha-neutotoxins. While these two classes share many chemical and biological characteristics, there are also distinct dissimilarities between them, including their binding site on the nicotinic acetylcholine receptor (nAChR), specificity among species of Chordata, and the associated pharmacological effects. In the present study we test the hypothesis that structural motifs that evolved to confer natural resistance against conspecific long-chain alpha-neurotoxins in Elapidae snakes also interfere with the biological action of short-chain alpha-neurotoxins. We expressed functional nAChRs that contains segments or single residues of the Elapidae nAChR ligand binding domain and tested the effect of short-chain alpha-neurotoxin erabutoxin-a (ETX-a) from the Erabu sea snake Laticauda semifasciata on the acetylcholine-induced currents as measured by two-microelectrode voltage clamp. Our results show that the Elapidae nAChR alpha subunit segment T(154)-L(208) ligand binding domain has an inhibitory effect on the pharmacological action of ETX-a. This effect is primarily attributed to the presence of glycosylation at position N(189). If the glycosylation is removed from the T(154)-L(208) segment, the nAChR will be inhibited, however, to a lesser extent than seen in the mouse. This effect correlates with the variations in alpha-neurotoxin sensitivity of different species and, importantly, reflects the evolutionary conservation of the binding site on the nAChR polypeptide backbone per se. Phylogenetic analysis of alpha-neurotoxin resistance suggests that alpha-neurotoxin-resistant nAChR evolved first, which permitted the evolution of snake venom alpha-neurotoxins. A model describing alpha-neurotoxin resistance in Elapidae snakes is presented.
Assuntos
Elapidae/genética , Erabutoxinas/antagonistas & inibidores , Evolução Molecular , Receptores Nicotínicos/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Sequência Conservada/genética , Análise Mutacional de DNA , Eletrofisiologia/métodos , Erabutoxinas/farmacologia , Glicosilação , Camundongos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Mutação Puntual/genética , Estrutura Terciária de Proteína/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/fisiologia , XenopusRESUMO
The effects of histamine on the acetylcholine (ACh) receptor-channel complex were examined by means of voltage-clamp at the frog endplate. ACh was ionophoretically applied to the endplate. Histamine was added to the perfusate. Histamine (100 nM - 1 mM) reversibly depressed the peak amplitude of the ACh-induced inward current in a dose-dependent manner. The double reciprocal plot of the dose-response relationship between the peak ACh current and the amount of ACh applied suggested that histamine (100 microM) depressed the ACh-induced current in a competitive manner. Histamine prevented the specific ACh binding site within the receptor-channel complex from binding erabutoxin, a sea-snake venom, which binds irreversibly to the specific ACh binding site. Histamine had no detectable effects on the equilibrium potential of the endplate current but shortened the half-decay time of the endplate current in a voltage-dependent manner. It was therefore concluded that histamine blocks not only the specific ACh binding site but also interacts with the ACh-channel site. The present experiments strongly suggest that histamine can act as an antagonist to modulate nicotinic cholinergic transmission.
