ICln is a cytoplasmic water-soluble protein forming a PH domain, which can be intro-duced into the cellular membrane, and form ion channels. We set out to test, whether or not disulphide bridging and consecutive dimerization is important for channel formation. Wt-ICln ion channels when expressed in cellular systems, mediate a chloride current resembling that activated after cell swelling (IClswell ), which allows the cells to perform regulatory volume decrease (RVD). Knocking-down of ICln impairs IClswell, therefore supporting the hypothesis of ICln being an important entity for IClswell. In order to test the possible involvement of disulfide bonds in ICln dimerization, we examined the effect of the reducing agent dithiothreitol (DTT) on ICln dimerization using Western-blots we show that the ICln dimer is absent in samples treated with DTT. ICln has only two cystines (C128 and C130) located at the c-terminal end of the α-helix. To determine the specificity of DTT on ICln dimerization a double and single cystine mutant (C128A/C130A, C128A and C130A) were constructed and their effect on ICln dimeriza-tion under renaturing or non-denaturing conditions was analysed. Western-blots show that the C128A/C130A mutant annihilates the dimer formation. In addition to Western-blots, we performed a biophysical characterization of the mutants after their reconstitu-tion in black lipid bilayers built of a sphingomyeline mixture. By comparing ICln-wt and the different cystine mutants we found a dramatic decrease of channel formation, if disulphide bridging is impeded. Our experiments indicate that the format of disulphide bridging is essential for ICln function as a channel.
Forming of ICln-dimers by disulphide bridging / S. Dopinto, M. Boccazzi, C. Zanoni, G. Bottà, G. Meyer, M. Paulmichl. ((Intervento presentato al convegno Paracelsus Science Get Together-Life Science tenutosi a Salisburgo nel 2008.
|Titolo:||Forming of ICln-dimers by disulphide bridging|
BOCCAZZI, MARTA (Secondo)
|Data di pubblicazione:||13-giu-2008|
|Citazione:||Forming of ICln-dimers by disulphide bridging / S. Dopinto, M. Boccazzi, C. Zanoni, G. Bottà, G. Meyer, M. Paulmichl. ((Intervento presentato al convegno Paracelsus Science Get Together-Life Science tenutosi a Salisburgo nel 2008.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|