In situ, Reversible Gating of a Mechanosensitive Ion Channel through Protein-Lipid Interactions

Abstract

Understanding the functioning of ion channels, as well as utilizing their properties for biochemical applications requires control over channel activity. Herein we report a reversible control over the functioning of a mechanosensitive ion channel by interfering with its interaction with the lipid bilayer. The mechanosensitive channel of large conductance from Escherichia coli is reconstituted into liposomes and activated to its different sub-open states by titrating lysophosphatidylcholine (LPC) into the lipid bilayer. Activated channels are closed back by the removal of LPC out of the membrane by bovine serum albumin (BSA). Electron paramagnetic resonance spectra showed the LPC-dose-dependent gradual opening of the channel pore in the form of incrementally increasing spin label mobility and decreasing spin-spin interaction. A method to reversibly open and close mechanosensitive channels to distinct sub-open conformations during their journey from the closed to the fully open state enables detailed structural studies to follow the conformational changes during channel functioning. The ability of BSA to revert the action of LPC opens new perspectives for the functional studies of other membrane proteins that are known to be activated by LPC.

Keywords: MscL; bovine serum albumin; electron spin resonance spectroscopy; lysophosphatidylcholines; mechanosensation; reversible gating.

Figure 1

Schematic representation of reversible activation of MscL. (A) Side view of homopentameric MscL from Mycobacterium tuberculosis (PDB access code: 2AOR). The approximate position of the lipid bilayer is marked with orange lines. A single subunit is highlighted in white. (TM: transmembrane domain). (B) MscL is reconstituted into the liposome. While the assymmetric insertion of L-α-lysophosphatidylcholine into the lipid bilayer triggers MscL opening, its selective removal by bovine serum albumin (BSA) closes the MscL channel. The open-close cycle can be repeated multiple times. For clarity, only four out of five identical subunits are shown.

Figure 2

Reversible activation of MscL by LPC and BSA. Black trace: Calcein loaded control MscL proteoliposomes treated with 40 mol% LPC reaches maximum calcein release within 5 min. When the duplicate of the control sample was treated with 25 mol% BSA (relative to LPC concentration) at t = 2 min, the calcein release stopped immediately (blue trace). The red trace shows repeated opening and closing cycles of MscL. Consecutive LPC and BSA additions indicated by black and red arrows, respectively. Calcein-loaded liposomes with no MscL channel were used as a negative control to test the effect of 40 mol% LPC (relative to the lipid concentration) and 25 mol% BSA (relative to LPC) on the lipid bilayer itself (green trace), showing that the indicated amounts of LPC and BSA did not cause any leakage through the lipid bilayer. The asterisk marks the time point of detergent addition to the samples for lysing the liposomes and releasing all calcein.

Figure 3

Reversible activation of MTSSL-labeled G22C MscL monitored by continuous wave EPR. (A) Schematic representation of MscL based on the crystal structure of the homologous MscL from Mycobacterium tuberculosis (PDB access code: 2AOR). The yellow area represents the lipid bilayer. (B) MscL channel activation by 40 mol% LPC (relative to the lipid concentration). Top and Middle: the spectra show MTSSL-labeled G22C MscL (MscL-SL, minimally labeled) in the absence of LPC (black); activated with LPC (red). Bottom: the spectra of LPC-activated channels after adding BSA to the proteoliposomes (blue). Spectra are normalized to the same number of spins. (C) EPR spectra of maximally labeled MscL-SL samples with increasing mol% of LPC, percentages are given relative to the lipid concentration. The magnetic field scale shown at the bottom of (B,C) refers to all spectra of the respective set.

Figure 4

Room temperature cw EPR spectra of minimally labeled and maximally labeled MscL-SL. (A) Black: maximally labeled, red: minimally labeled MscL. LPC concentrations in the liposomes are indicated. Spectra are normalized to the amplitude of the central line. The arrows indicate features of the spectra that derive from spin labels that are slowly rotating (see text). The features indicated by arrows diminish and disappear upon increasing the concentration of LPC. (B) Parameters describing spin label mobility as a function of LPC concentrations: Inverse central line width (ΔH⁻¹, red squares) and second moment (<ΔB²>, black dots) of the minimally labeled sample. Increased mobility is reflected in a narrower line, which results in a larger inverse central line width (ΔH⁻¹), and a smaller second moment (<ΔB²>) parameter.