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

Abstract

The canonical mechanism of gramicidin (gA) channel formation is a transmembrane dimerization of non-conducting subunits that reside in opposite bilayer leaflets. The channels do not open and close per se; rather they appear and disappear. This basic monomer-dimer mechanism is supported by conductance-concentration studies, fluorescence measurements, conductance relaxation experiments using voltage, and pressure and light-inactivation experiments to perturb the monomer-dimer equilibrium. Recently this mechanism was challenged by Jones et al. (BJ 98:1486, 2010). Using lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled, Jones et al. proposed that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. A key assumption in these experiments is that gA monomers do not partition between vesicles; see Kemp and Wenner (ABB 176:547, 1976) and Bruggemann and Kayalar (PNAS 83:4276, 1986). To explore this issue further, we added gA-containing 1,2-dierucoyl-sn-glycero-3-phosphocholine (DC22:1PC) vesicles to one or both sides of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) planar bilayers. This protocol produced gA activity that was 10-fold higher when gA-containing vesicles were added to both sides of the bilayer, as compared to one side. We also found that when gA-free DC22:1PC vesicles were added to both sides of planar DOPC bilayers pre-incubated with gA, channel activity would reduce about 10-fold. We conclude that gA subunits can exchange between lipid vesicles and planar bilayers. We similarly could demonstrate the exchange of gA between lipid vesicles using a fluorescence assay for gA channel activity (Ingólfsson and Andersen, ADDT 4:427, 2010). We conclude that the parsimonious interpretation of the results of Jones et al. is that their channel activity is due to gA exchange between the vesicles and the planar bilayer, and that gA channels form by the canonical mechanism- transbilayer association of non-conducting subunits.