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Determination of membrane potential with lipophilic cations: correction of probe binding
Authors:Makoto Demura   Naoki Kamo  Yonosuke Kobatake
Affiliation:2. Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark;3. Sydney Medical School, Charles Perkins Centre, University of Sydney, Sydney, Australia;1. Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de maio, 100, CEP 04044-020, Vila Clementino, São Paulo, SP, Brazil;2. Escola de Educação Física e Esporte, Universidade de São Paulo, Avenida Professor Mello Moraes, 65, CEP 05508-030, Cidade Universitária, São Paulo, SP, Brazil;3. Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Professor Lineu Prestes, 1374, CEP 05508-900, Cidade Universitária, São Paulo, SP, Brazil;4. Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Pedro de Toledo, Rua Pedro de Toledo, 669, CEP 04039-032, Vila Clementino, São Paulo, SP, Brazil;5. Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Avenida Professor Lineu Prestes, 748, CEP 05508-000, Cidade Universitária, São Paulo, SP, Brazil;6. CNRS, France;7. Instituto Serrapilheira-Rua Dias Ferreira, 78 s202, CEP 22431-050, Leblon, Rio de Janeiro, RJ, Brazil
Abstract:The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0–4) and tetraphenylphosphonium (TPP+). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of for various probes are plotted against the binding coefficient. Here, Ciapp is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential.
Keywords:Ion-selective electrode   Phosphonium cation   Membrane potential   Lipophilic cation binding   Envelope vesicles   (H. halobium)
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