PROTON CONDUCTION IN GRAMICIDIN-A AND IN ITS DIOXOLANE-LINKED DIMER IN DIFFERENT LIPID BILAYERS

Gramicidin A (gA) molecules were covalently linked with a dioxolane ri ng. Dioxolane-linked gA dimers formed ion channels, selective for mono valent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers an d HCl concentrations (10-8000 mM). Two ion channels with different gat ing and conductance properties were identified in bilayers from the pr oduct of dimerization reaction. The most commonly observed and most st able gramicidin A dimer is the main object of this study. This gramici din dimer remained in the open state most of the time, with brief clos ing flickers (tau(closed)approximate to 30 mu s). The frequency of clo sing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximate to 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine -phosphatidylcholine) bilayers, single-channel current-voltage relatio nships had an ohmic behavior at low voltages, and a marked sublinearit y at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bi layers, the linear conductance of single-channel proton currents at di fferent proton concentrations was essentially the same for both natura l and gA dimers. g(max) and K-D, obtained from fitting experimental po ints to a Langmuir adsorption isotherm, were similar to 1500 pS and 30 0 mM, respectively, for both the natural gA and its dimer. In GMO bila yers, however, proton affinities of gA and the dioxolane-dimer were si gnificantly lower (K-D of similar to 1 and 1.5 M, respectively), and t he g(max) higher (similar to 1750 and 2150 pS, respectively) than in P EPC bilayers, Furthermore, the relationship between single-channel con ductance and proton concentration was linear at low bulk concentration s of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. I t is concluded that 1) The mobility of protons in gramicidin A channel s in different lipid bilayers is remarkably similar to proton mobiliti es in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by o nly 25%. 2) Differences between proton conductances in gramicidin A ch annels in GMO and PEPC cannot be explained by surface charge effects o n PEPC membranes. It is proposed that protonated phospholipids adjacen t to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some ex perimental results cannot be reconciled with simple alterations in acc ess resistance to proton flow in gA channels. Said differences could b e explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by mo difications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channe l. However, other factors can also influence closing flickers.