Hydrophobic coupling of lipid bilayer energetics to channel function

Abstract

The hydrophobic coupling between membrane-spanning proteins and the lipid bilayer core causes the bilayer thickness to vary locally as proteins and other "defects" are embedded in the bilayer. These bilayer deformations incur an energetic cost that, in principle, could couple membrane proteins to each other, causing them to associate in the plane of the membrane and thereby coupling them functionally. We demonstrate the existence of such bilayer-mediated coupling at the single-molecule level using single-barreled as well as double-barreled gramicidin channels in which two gramicidin subunits are covalently linked by a water-soluble, flexible linker. When a covalently attached pair of gramicidin subunits associates with a second attached pair to form a double-barreled channel, the lifetime of both channels in the assembly increases from hundreds of milliseconds to a hundred seconds--and the conductance of each channel in the side-by-side pair is almost 10% higher than the conductance of the corresponding single-barreled channels. The double-barreled channels are stabilized some 100,000-fold relative to their single-barreled counterparts. This stabilization arises from: first, the local increase in monomer concentration around a single-barreled channel formed by two covalently linked gramicidins, which increases the rate of double-barreled channel formation; and second, from the increased lifetime of the double-barreled channels. The latter result suggests that the two barrels of the construct associate laterally. The underlying cause for this lateral association most likely is the bilayer deformation energy associated with channel formation. More generally, the results suggest that the mechanical properties of the host bilayer may cause the kinetics of membrane protein conformational transitions to depend on the conformational states of the neighboring proteins.

Figure 1.

Schematic model of side-by-side, double-barreled gramicidin channels that assemble from two pairs of linked subunits in a lipid bilayer. Each individual subunit spans one leaflet of the DPhPC bilayer and is joined at its COOH-terminal to an adjacent subunit, in the same leaflet, by means of a hydrophilic peptide linker, which extends into the aqueous solution. The two peptide strands in the linker are joined by a lysine, which is extended by three more carboxy-terminal residues to further increase the linker's water solubility (and also were necessary for efficient synthesis). The flexible linkers are shown to scale in an arbitrary extended conformation. Channel formation requires that two subunits in opposite leaflets associate to form a hydrogen bond–stabilized cation-conducting channel. In a double-barreled channel, the two covalently linked subunits in one leaflet form bilayer-spanning channels with two covalently linked subunits in the opposite leaflet. The lipids distant from the channel barrels are represented as DPhPC molecules with fully extended acyl chains, which will tend to exaggerate the hydrophobic mismatch between the channel and the bilayer. To illustrate how the bilayer adapts to the bilayer-spanning channels, the lipids adjacent to or near a channel barrel are depicted with acyl chains that have been shortened by two carbons (second “shell”) or four carbons (first “shell”), which represents the bilayer thinning. Carbon and hydrogen atom colors reflect the particular molecular components: gramicidin subunits, green/orange; lipids, gray; linkers, light blue. The colors for other atoms are: O, red; N, dark blue; P, magenta.

Figure 2.

Characterization of the tandem gramicidin analogues. (A) RP-HPLC elution profile obtained with the 15-residue–linked tandem gramicidin gA. (B) MALDI spectra for the same compounds. The primary peak is an analogue-H⁺ peak with a molecular weight of 5,210, equivalent to the predicted molecular weight of 5,209. Despite the rather broad elution profile the compound is of high purity as evident in the mass spectrum.

Figure 3.

Circular dichroism spectra for the gramicidin analogues used in this study: gA, gA with the 7-residue eda tail, tandem gA with the 15-residue linker, and tandem gA with the 23-residue linker. DMPC vesicles.

Figure 4.

Single-channel current traces obtained with the gramicidin analogues used in this study (the channel type in D is shown at higher current and time resolution in Fig. 6). (A) gA. (B) gA with the 7-residue tail. (C) Tandem gA with the 15-residue linker. (D and E) Tandem gA with the 23-residue linker. 1.0 M CsCl, 200 mV, 200 Hz.

Figure 5.

Current transition amplitude histograms (left) and lifetime distributions (right) for channels formed by: A, gA; B, gA with the 7-residue tail; C, 15-residue–linked tandem gA; and D, 23-residue–linked tandem gA. The current transition histograms were determined at 200 mV. The lifetime distributions for gA, gA with the 7-residue tail, and the 15-residue-linked tandem were also determined at 200 mV; the lifetime distributions for the 23-residue-linked tandem gA is based on results obtained at 100 and 200 mV. 200 Hz.

Figure 6.

Bursting channel activity and formation and disappearance of channels formed by the 23-residue–linked tandem gA. The top two rows of traces show two different examples of double-barreled channel appearance/disappearance patterns in which the transition to the fully conducting level proceeds through an intermediate conductance level. In each row, the traces to the right and left of the complete channel show the appearance/disappearance transitions at higher time resolution. To better visualize the various conductance states, the interrupted lines in each trace are drawn through the baseline, when no channel is conducting. (Top row) Channel formation is preceded by bursting channel activity with no bursting activity when the channel disappears. (Middle row) There is no bursting channel activity preceding channel formation, but there is bursting activity after the channel disappearance—as well as an isolated bursting event that follows closely after the disappearance of the double-barreled channel. (Bottom three panels) Isolated bursting channel activity. The histograms to the right show that the burst reflects transition within a bilayer-spanning dimer; the two current level histograms at the right were recorded during the bursting channel activity (top histogram) and just before (bottom histogram). 200 mV, 200 Hz.

Figure 7.

Current transition amplitude and lifetime histograms for the appearance and disappearance of the double-barreled channels formed by the 23-residue–linked tandem gA. (Top) Current transition amplitude histograms. (Left) Histogram for the initial transition from the baseline to the intermediary current level. (Right) Histogram for the final transition from the double-barreled to intermediary current level. (Bottom) Lifetime distributions. (Left) The interval distribution for the durations of bursting transition when the double-barreled channel appears. (Right) The interval distribution for the durations of bursting transition when the double-barreled channel disappears. The results, as well as those for the bursting and flickery channels are summarized in Table III. 1.0 M CsCl, 200 mV, 200 Hz.

Figure 8.

Heterodimer experiment with the 23-residue–linked tandem gA, and gA. The two gramicidins were added to opposite sides of a bilayer, and the gA-containing solution is the electrical reference. Left, current traces; right, current transition amplitude histograms. Top, results at +200 mV; bottom, results at −200 mV. The smaller peak, marked by an asterisk, denotes homodimeric gA channels due to an avoidable, if slow, “leak” of gA across the bilayer. The results are summarized in Table IV. 200 Hz.

SCHEME I

SCHEME II

Figure 9.

Schematic representation of the formation of double-barreled gramicidin channels in a bilayer with a thickness that is larger than the channel length. (Left) top view. (Right) side view. (A) Two independent tandem gramicidins, one on each side of a bilayer, in which the subunits are separated by a flexible linker. (B) The initial event is the formation of a single bilayer-spanning pore with the monomeric subunits at opposite sides of the conducting dimer. The dimerization causes a local thinning of the bilayer around the bilayer-spanning dimer. (C) For the second barrel to form, the subunits diffuse in each monolayer (and the linker bends), causing the nonconducting monomers to approach each other and dimerize, which causes a further bilayer deformation around the second bilayer-spanning dimer. (D) To minimize the overall bilayer deformation energy, the two bilayer-spanning dimers (conducting pores) will tend to associate closely. The figure is drawn with the two barrels separated by a distance corresponding to one phospholipid molecule, but we have no direct information about the distance separating the two barrels.