Forming of ICln-dimers by disulphide bridging

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

Membrane thickness changes ion-selectivity of channel-proteins

The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become

The ICln interactome

The many different functional phenotypes described in mammalian cells can only be explained by an intense interaction of the underlying proteins, substantiated by the fact that the number of independently expressed proteins in living cells seems not to exceed 25 K, a number way too small to explain the >250 K different phenotypes on a one-protein-one-function base. Therefore, the study of the interactome of the different proteins is of utmost importance. Here, we describe the present knowledge of the ICln interactome. ICln is a protein, we cloned and whose function was reported to be as divers as (i) ion permeation, (ii) cytoskeletal organization, and (iii) RNA processing. The role of ICln in these different functional modules can be described best as being a 'connector hub' with 'date hub' functio