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

<div><p>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 (Na_v) NaChBac in a well-defined membrane environment. Na_vs 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 Na_vs 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 <i>in vivo</i> ensemble studies.</p></div

Ionic selectivity of NaChBac at single-molecule BLM recording.

<p>(A) Schematic representation of the bi-ionic setup. (B) Schematic representation of the voltage ramp protocol. (C) Current traces under the Na⁺/K⁺ condition. A total of 100 current traces are overlaid and shown in black. The average trace is highlighted in red. Arrowhead indicates the E_rev. An exemplary trace showing no K⁺ conductance is highlighted in blue. (D) Current traces under the Na⁺/Ca²⁺ condition. Note there are two proteins in this recording. Highlighted traces and arrowhead have the same meaning as (C).</p

Single-molecule recording of NaChBac for Na⁺ conductance determination.

<p>(A) The voltage protocol used for the recording. The channel was first hold at -80 mV for 0.2 sec, followed by a step to an incremented voltage (from -80 mV to 80 mV) for 10 sec and then back to -80 mV. In one experiment the voltage protocol was repeated 5 times and the data pooled for analysis. (B) A representative trace of the recording at +35 mV. Three conducting levels are observed. (C-E). Zoom-in image of (B) as indicated by the blue bars in (B) to show each of the three conducting levels.</p

Single-channel conductance and open probability analysis of NaChBac.

<p>(A) Representative I-V plot of NaChBac obtained from single-channel recording. Data points are summarized from single-channel event analysis. If no data point displayed, it means that not enough events were found in that particular voltage step. While L1 (red square) opened in both positive and negative step voltage, L3 (green triangle) and L2 (blue diamond) open less frequently at negative step voltage. (B) Distribution of the open probability of the three sub-states at different voltage based on the same experiment of (A). Data were analyzed by all-point current histogram. (C) The total open probability of NaChBac depends on the holding voltage.</p

Macroscopic current recording of NaChBac under varied buffer conditions.

<p>(A) The voltage step protocol used for the recording. (B) An exemplary current trace when a voltage step of +60 mV was applied under the buffer condition of 150/150 mM NaCl. Note that majority of the channels stay open at -120 mV for the first ~3.4 sec. (C) A magnified view of the dotted blue box in (B) showing opening events of individual channels. (D) The I-V plots of single opening events obtained from macroscopic current recording. (E) Relationship between the unitary conductance and the [NaCl] in the buffer.</p

Schematic representation of the BLM setup and the structure of prokaryotic Na_v.

<p>(A) The BLM sample chamber and the amplifier. The reference electrode is connected to the <i>trans</i> chamber through a salt bridge and the command electrode is connected to the <i>cis</i> chamber. (B) Top-view of the structure of a full-length prokaryotic Na_v. The four monomers, highlighted in different colors, assemble along each other to form the ionic conducting pore in the center of the tetrameric structure. Picture is generated based on the X-ray crystal structure of Na_vRh (PDB ID 4DXW) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188861#pone.0188861.ref007" target="_blank">7</a>]. (C) Side-view of the same structure in (B). (D) Schematic illustration comparing the structure of eukaryotic and prokaryotic Na_vs. The four arginine residues on S4 that are responsible for the voltage sensing are denoted by “+” signs. N and C denote the N- and C- termini, respectively.</p