Nanoparticles used to Study Allosteric Control of Ion Channels

Abstract

Nanoscale apolipoprotein bound bilayers (NABBs) are stable, homogeneous discoidal lipid bilayers, approximately 10 nm in diameter, formed by a stoichiometric mixture of zebrafish apo A-I protein (zap1) and lipids [1]. We showed previously using KcsA incorporated into NABBs that the NABBs can serve as carriers to efficiently deliver ion channels to a distinct bilayer [2]. We now report the use of NABB technology to study binding events in more complex ion channels. We optimized reconstitution of several new channels into NABBs. Here we present data obtained from a prokaryotic cyclic nucleotide modulated potassium channel, MloK1, using NABBs. We performed characterization studies of MloK1-NABB complexes using gel-filtration chromatography, electron microscopy imaging and isothermal titration calorimetry. We compare the binding affinity of cyclic nucleotides to MloK1 in NABBs with that of isolated cyclic nucleotide binding domains (CNBDs). We discuss the use of these complexes as an ideal platform for discovering allosteric modulators of these channels. The functions of NABBs highlighted here extend the versatility of discoidal lipoproteins for studying membrane proteins outside the cellular environment. [1] S. Banerjee, T. Huber, T.P. Sakmar. 2008. Rapid Incorporation of Functional Rhodopsin into Nanoscale Apolipoprotein Bound Bilayer (NABB) Particles. J. Mol. Biol. 377, 1067-81. [2] S. Banerjee, C.M. Nimigean 2010. Ion Channels in Nanoscale Apolipoprotein Bound Bilayers. Biophys. J. 98, 600a.