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. 2004 Apr;86(4):2129-36.
doi: 10.1016/S0006-3495(04)74272-1.

Purification and functional reconstitution of N- and C-halves of the MscL channel

Kyu-Ho Park  1 , Catherine Berrier, Boris Martinac, Alexandre Ghazi
Affiliations

Purification and functional reconstitution of N- and C-halves of the MscL channel

Kyu-Ho Park et al. Biophys J. 2004 Apr.

Abstract

MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100 mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity.

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Figures

FIGURE 1
FIGURE 1
Production of the N-half and C-half part of MscL. (A) Membrane topology of the E. coli MscL subunit, based on PhoA fusion experiments (Blount et al., 1996a) and on the structure of M. tuberculosis MscL (Chang et al., 1998) (left), and of the two halves of MscL produced in E. coli (right). Residual amino acids after GST affinity tag removal are shown in gray: GSLEHRENNM for the N-half and GS for the C-half. (B) Tricine-SDS-PAGE (16.5% acrylamide/bis-acrylamide) patterns showing the fusion proteins produced in E. coli and the resulting cleaved proteins. Both halves of the protein MscL have been produced as N-terminal fusion proteins with glutathion-S-transferase. After fixation to affinity beads, fusion proteins could be displaced by reduced glutathion (lanes 1 and 3). Cleavage by thrombin of the fusion proteins fixed on the column led to elution of the N-half and C-half proteins (lanes 2 and 4). A time dependant band is observed near 21 kDa, corresponding to additional cleavage of GST on its carboxyl end, as verified by N-terminal sequencing. For the C-half part, thrombin cleavage was less efficient (C-half, lane 4). Both fusion proteins have a similar size as expected, but peptides corresponding to each moiety migrate quite differently, and they are not stained efficiently by Coomassie dye. Molecular weight markers are indicated as small bars and related migration values in kilodaltons on the left.
FIGURE 2
FIGURE 2
Spontaneous channel activity recorded in patches made on giant proteoliposomes reconstituted with the N-half part of MscL. Traces show the most commonly observed activity. Pipette voltage was −20 mV. No pressure was applied to the patch. (A, C, and D) The protein was purified from vesicles and reconstituted at a lipid/protein ratio of 500 (w/w). (B) The peptide was purified from inclusion bodies and reconstituted at a lipid/protein ratio of 1000. Time and conductance scales are indicated by small bars. Arrows indicate the closed levels for all channels in the patches. Bath medium: 100 mM KCl, 10 mM HEPES-KOH, pH 7.4. Pipette medium: similar to bath medium with, in addition, 2 mM CaCl2 and 5 mM MgCl2.
FIGURE 3
FIGURE 3
N- and C-halves coreconstituted together form mechanosensitive channels. (A) The N-half and C-half moieties of the protein were incubated together in detergent in equivalent amount and reconstituted in liposomes for patch-clamp experiments. The lipid/protein ratio was 1000 (w/w). Upper trace shows current and lower trace shows pressure applied to the membrane patch. Application of negative pressure activated ion channels that closed upon release of suction. (B) Electrophysiological activity of the native MscL purified and reconstituted in liposomes. (C) Mixed activities of N-half protein and recovered MscL in the same patch under −34 mm Hg suction. MscL activity appears with application of pressure, whereas N-half protein activity is observed before suction (not shown). All recordings were carried out at pipette voltage of −10 mV. Other conditions as in Fig. 2.
FIGURE 4
FIGURE 4
Pressure dependence of the reassembled MscL channel. (A) Channel activity of N- and C-halves coreconstituted together at various levels of suction. Levels of pressure are indicated at the bottom of each trace. The lipid/protein ratio was 1000 (w/w). Pipette voltage was −10 mV. Other conditions as in Fig. 2. (B) Open probability P0 versus the applied pressure, at fixed membrane potential (−10 mV pipette), of the channel whose activity is shown in A. The data, obtained from 10-s segments of recording at each pressure, were fitted to a Boltzmann distribution of the form P0 = (1 + exp α (p1/2 − p))−1, where P0 is the open probability, p is the pressure, p1/2 is the pressure at which the open probability is 0.5, and α is the sensitivity. For these data, 1/α = 0.37 mm Hg, p1/2 = 22.9 mm Hg. By way of comparison, the Boltzmann distribution describing the pressure dependence of native MscL channels is shown as a dotted line. The parameters of this distribution (1/α = 4.85 mm Hg, p1/2 = 44 mm Hg) are the average of parameters obtained from three different patches.
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