Single-molecule study of full-length NaChBac by planar lipid bilayer recording.

Andrew Jo,Carlo D Montemagno,Hiofan Hoi,Manisha Gupta,Hang Zhou,Bruce R Donald

doi:10.1371/journal.pone.0188861

Andrew Jo, Carlo D Montemagno + Show 4 more

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https://doi.org/10.1371/journal.pone.0188861

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Abstract

Planar lipid bilayer device, alternatively known as BLM, is a powerful tool to study functional properties of conducting membrane proteins such as ion channels and porins. In this work, we used BLM to study the prokaryotic voltage-gated sodium channel (Nav) NaChBac in a well-defined membrane environment. Navs are an essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Navs in recent years has greatly promoted the understanding of the molecular mechanism that underlies these proteins and their eukaryotic counterparts. In this work, we investigated the single-molecule conductance and ionic selectivity behavior of NaChBac. Purified NaChBac protein was first reconstituted into lipid vesicles, which is subsequently incorporated into planar lipid bilayer by fusion. At single-molecule level, we were able to observe three distinct long-lived conductance sub-states of NaChBac. Change in the membrane potential switches on the channel mainly by increasing its opening probability. In addition, we found that individual NaChBac has similar permeability for Na+, K+, and Ca2+. The single-molecule behavior of the full-length protein is essentially highly stochastic. Our results show that planar lipid bilayer device can be used to study purified ion channels at single-molecule level in an artificial environment, and such studies can reveal new protein properties that are otherwise not observable in in vivo ensemble studies.

Highlights

Planar lipid bilayer device, alternatively known as black lipid membrane (BLM), is an attractive technique for characterization of channel-forming proteins such as ion channels and porins [1,2,3]
The cDNA coding for NaChBac was cloned from a NaChBac expressing E. coli strain[19] into a pET28a vector (Novagen) between the Nde I and EcoR I restriction sites
Reconstitution of NaChBac into planar lipid bilayer Direct insertion of ion-channels has been challenging, and its incorporation into the planar lipid bilayer is usually achieved by fusion of pre-formed protein vesicles with the planar bilayer [13,14,15, 20] (S1 Fig)

Summary

Introduction

Alternatively known as black lipid membrane (BLM), is an attractive technique for characterization of channel-forming proteins such as ion channels and porins [1,2,3]. Isolated membrane protein was inserted into the bilayer [2, 3]. Planar lipid bilayer study of NaChBac a transmembrane voltage is applied to induce an ionic current through the protein of interest (Fig 1A). The technique was initially limited to physiochemical studies of lipid membrane due to the lack of understanding on membrane proteins and their isolation at the time. With the recent advancement in membrane protein biotechnology, there is an increasing interest in revisiting this old method for channel protein characterization. BLM is useful for studies of conductance, sub-conductance states, transition rates between states, ion selectivity, ionic strength dependence and inhibitor binding behavior for ion channels

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