Exchange of Gramicidin between Lipid Bilayers: Implications for the Mechanism of Channel Formation

Abstract

The canonical mechanism of gramicidin (gA) channel formation is transmembrane dimerization of nonconducting subunits that reside in opposite bilayer leaflets. The channels do not open and close; they appear and disappear due to subunit association and dissociation. Many different types of experiments support this monomer ↔ dimer mechanism. Recently, however, this mechanism was challenged, based on experiments with lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled. In these experiments, sustained channel activity was observed long after fusion had been terminated, which led to the proposal that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. This proposal is at odds with 40 years of experiments, but involves the key assumption that gA monomers do not exchange between bilayers. We tested the possibility of peptide exchange between bilayers using three different types of experiments. First, we demonstrated the exchange of gA between 1,2-dierucoyl-sn-glycero-3-phosphocholine (DCPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DCPC) lipid vesicles using a fluorescence assay for gA channel activity. Second, we added gA-free DCPC vesicles to both sides of planar DCPC bilayers preincubated with gA, which reduced channel activity up to 10-fold. Third, we added gA-containing DCPC vesicles to one or both sides of DCPC planar bilayers, which produced much higher channel activity when the gA-containing vesicles were added to both sides of the bilayer, as compared to one side only. All three types of experiments show that gA subunits can exchange between lipid bilayers. The exchange of subunits between bilayers thus is firmly established, which becomes a crucial consideration with respect to the mechanism of channel formation.

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

“…1 and 2), but these are likely to depend on the types of lipids in the vesicles, and their negligible magnitude cannot be taken for granted. Indeed, the energy cost of gA dimer formation increases with increasing bilayer thickness, meaning that the monomer-to-dimer equilibrium of gA is shifted towards the monomeric state [40,53], and in such cases the gA monomers have been shown to more easily exchange between vesicles [54]. This could result in a loss of the protein during the CDmediated lipid exchange between donor and acceptor vesicles.…”

Molecular Mechanism for Gramicidin Dimerization and Dissociation in Bilayers of Different Thickness

“…The bilayer-spanning gramicidin channel forms by transmembrane dimerization of two nonconducting monomers (1,2), and the antiparallel dimer structure is stabilized by six hydrogen bonds at the formyl-N-termini (3)(4)(5). Each hydrogen bond contributes $4 k B T to the channel stability (6)(7)(8)(9), indicating that gramicidin channel dissociation occurs on a timescale far beyond microseconds.…”

Model Biological Membranes and Possibilities of Application of Electrochemical Impedance Spectroscopy for their Characterization

“…Different types of EECs were proposed for the experimental features of the electrochemical impedance spectra of the model membrane, but the values of the EEC parameters are difficult to be connected with the physical properties of the system . The fundamental parameter for a successful application of t‐BLMs in a concept of biosensing is the incorporation of functional membrane proteins and the creation of pores (the ion‐selective gramicidin channels , α‐hemolysin , etc.). The pore can be used to detect polymers and proteins and can be modified as a receptor for divalent metal ions, organic compounds, etc.…”