Single Channel and Ensemble Measurements of CLIC1 in Lipid Bilayers

Abstract

AbstractChloride intracellular channel 1 (CLIC1) is an ion channel protein which has been hypothesized to play a fundamental role in neurodegenerative diseases like Alzheimer's disease (AD) [1]. To date, single-channel characteristics of this protein have been obtained by reconstituting it in lipid bilayers and whole cell patch clamp measurements of overexpressing cells. CLIC1 protein can exist in both soluble and integral membrane form[2] and localizes on the plasma membrane and intracellular organelles. Obtaining ensemble measurements of CLIC1 by whole cell patch clamp is very difficult, due to its subcellular localization and its solubility in cytoplasm [3]. We report single channel and ensemble measurements of reconstituted CLIC1 in artificial lipid bilayers formed using sessile droplets, a measurement platform amenable to parallel and automated ion channel studies.[4] We also measured dose dependent inhibition of CLIC1 multi-channel currents by a known blocker, IAA94. This work may be applicable to measurement and screening of other intracellular ion channels as well.[1] Stephania Averaimo, Rosemary H. Milton, Michael R. Duchen, Michele Mazzanti. ‘Chloride intracellular channel 1 (CLIC1): sensor and effector during oxidative stress’. FEBS Letters 584 (2010) 2076-2084.[2] Singh, H. ‘Two decades of dimorphic chloride intracellular channels’. FEBS Letters 584 (2010) 2112-2121.[3] Michele Mazzanti et al. ‘Involvement of the Intracellular Ion Channel CLIC1 in Microglia-Mediated -Amyloid-Induced Neurotoxicity’. The Journal of Neuroscience (2004) 5322-5330.[4] J L Poulos, S A Portonovo, H Bang and J J Schmidt. ‘Automatable Lipid Bilayer Formation and ion Channel Measurement Using Sessile Droplets’. Journal of Physics: Condensed Matter (2010) 22 (45), 454105.