ION CHANNEL FORMATION BY THE MARINE SPONGE GEODIA CORTICOSTYLIFERA EXTRACT IN BILAYER MEMBRANES

Rangel, M.(1), Procopio, J.(2), Konno, K.(3), Freitas, J.C.(1)

(1)Departamento de Fisiologia, Instituto de Biociências e CEBIMar, USP, São Paulo, (2)Departamento de Fisiologia, Instituto de Ciências Biomédicas, USP, São Paulo, (3)Centro de Estudo de Insetos Sociais, Instituto de Biociências, UNESP, Rio Claro.

Objectives: In the present work we studied ion channel formation into bilayer membranes by fractions of the extract of G. corticostylifera.

Methods: The methanol-water extract prepared from G. corticostylifera specimens collected in São Paulo State coast, Brazil, was fractionated in an acetonitrile-water gradient in a Sep Pak Vac C₁₈ cartridge, resulting in 9 fractions, each three were solubilized  in 20, 50 and 90% CH₃CN, respectively. The haemolytic fraction (the second one with 50% CH₃CN solubility) was tested, in concentrations of 1-4mg/ml, in azolecitin membranes formed in a circular hole of a polypropylene wall coupled to an acrylic chamber. Electrical measurements were made with a patch-clamp amplifier, set into voltage-clamp. Data were converted to digital form through an A/D interface and acquired by a computer program.

Results: Macroscopic and unitary current-voltage relations of the channels were non-linear, showing pronounced rectification, favouring current passage from the CIS (were the fraction was added) to the TRANS side. The channels were shown to be cation-selective when Na⁺ was tested against Cl^-, and more selective to Na⁺ against K⁺. Channel conductance was 18pS (-100mV pulse, 145mM NaCl + 10mM CaCl₂ solution). Channel openings apparently were modulated by Ca⁺⁺.

Conclusions: The channels formed by the haemolytic fraction may present an assymetry related to the side of incorporation into the membrane, as suggested by the current-voltage relations. Further experiments with pure ionophore isolated from the sponge will be performed and a model for the channel will be proposed.

Granted by FAPESP

CORRESPONDENCE TO:

José Carlos de Freitas, Rua do Matão, trav. 14 no. 101 Depto. de Fisiologia – IB, Cidade Universitária, São Paulo, SP, CEP: 05508-900, Brasil, Email: jfreitas@usp.br