David Busath

Tryptophan photolysis is responsible for gramicidin-channel inactivation by ultraviolet light.

The decay of gramicidin fluorescence resulting from ultraviolet exposure was compared to the decay of conductance from gramicidin-containing planar bilayer membranes under the same conditions of illumination. The decay rate was the same for both processes. The fluorescence decay was identical whether gramicidin was dissolved in methanol or incorporated into lipid vesicles, indicating that the peptide conformation does not affect the sensitivity of gramicidin to photolysis. The correlation of fluorescence decay and conductance decay imply that conductance loss from gramicidin-doped membranes illuminated with ultraviolet light is due to photochemical modifications of the channel tryptophans rather than simply to disturbance of the conformation of gramicidin channels.

Is a beta-barrel model of the K+ channel energetically feasible?

A mutation of Shaker D431 or T449 strongly affects external tetraethylammonium (TEA) binding (1) whereas a mutation of Shaker T441 (2) or NGK2/DRK1 chimera L374 (equivalent to Shaker V443) and chimera V369 (3) affect internal TEA blocking in the potassium channel. Changes in Shaker T449 (1), F433, T441, T442 (4), and chimera L374 and V369 (3) affect ion conductance in the K+ channel. These facts suggest that SS1 ( 430-440 in Shaker) and SS2 (440-450 in Shaker) line the channel, passing through the cell membrane and back. Mutations of several residues just outside of the SS1SS2 region affect external charybdotoxin block (5). Mutations of chimera residues M379 and V368 (3), and Shaker D431 (1) have little or no effect on channel conductance. The K+ channel is comprised of four equivalent monomers (6). Using molecular mechanics, we addressed the following questions: (a) Could four pairs of SS1 and SS2 hydrogen bond together to form a ,-barrel which could span the bilayer and form the pore-lining region of the channel? (b) Is there a model consistent with the assumption that site directed mutagenesis of residues with side chains projecting into the barrel would be most likely to affect permeability properties? (c) Would there be room in the p-barrel for the inward projecting side chains? (d) Would water and K+ fit in the channel?

Small iminium ions block gramicidin channels in lipid bilayers

Guanidinium and acetamidinium, when added to the bathing solution in concentrations of approximately 0.1M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.

Gramicidin channel selectivity. Molecular mechanics calculations for formamidinium, guanidinium, and acetamidinium

Empirical energy function calculations were used to evaluate the effects of minimization on the structure of a gramicidin A channel and to analyze the energies of interaction between three cations (guanidinium, acetamidinium, formamidinium) and the channel as a function of position along the channel axis. The energy minimized model of the gramicidin channel, which was based on the results of Venkatachalam and Urry (1983), has a constriction at the channel entrance. If the channel is not allowed to relax in the presence of the ions (rigid model), there is a large potential energy barrier for all three cations. The barrier varies with cation size and is due to high van der Waals and ion deformation energies. If the channel is minimized in the presence of the ions, the potential energy barrier to formamidinium entry is almost eliminated, but a residual barrier remains for guanidinium and acetamidinium. The residual barrier is primarily due, not to the expansion of the helix, but, to the disruption of hydrogen bonds between the terminal ethanoloamine and the next turn of the helix which occurs when the carbonyls of the outer turn of the helix librate inward toward the ion as it enters the channel. The residual potential energy barriers could be a possible explanation for the measured selectivity of gramicidin for formamidinium over guanidinium. The results of this full-atomic model address the applicability of the size-exclusion concept for the selectivity of the gramicidin channel.

Synthesis and channel properties of [Tau16]gramicidin A

Des(ethanolamine)-taurine16-gramicidin A ([Tau 16]gramicidin A) was synthesized by the solid phase method and its channel-forming behavior in planar lipid bilayers was examined. The purified monovalent anionic peptide formed channels when applied to the aqueous compartments on both sides of the bilayer, but not when applied to one side only. The single-channel conductance was measured for KCl concentrations between 0.1 and 1.0 M and was found to be higher than that of gramicidin A in each case. Single-channel lifetimes were similar to those of gramicidin A suggesting that the channels have the beta 6.3 helix structure.

Inhibition of gramicidin channel activity by local anesthetics

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LAs) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LAs were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LAs present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.

GUANIDINIUM AS A PROBE OF THE GRAMICIDIN CHANNEL INTERIOR

Guanidinium is a planar trigonal cation which is similar in size to the gramicidin channel pore. We measured the effect of guanidinium on the conductance properties of the gramicidin channel and theoretically evaluated its interactions with the β-6.3 channel interior using an energy minimization and conformational search approach. Guanidinium current (measured in the absence of other permeable ions) could not be detected directly (g(Guan)/g(K) < 0.004). However, guanidinium induces blocks in gramicidin channel potassium currents. The average block duration gets shorter with increased membrane potential suggesting that guanidinium can penetrate the ion channel. Energy minimization calculations indicate that, by reorienting along the pathway, the guanidinium should be able to penetrate the gramicidin channel. This finding is illustrated by a computer graphics animation of the series of minimum-energy orientations. The low permeability of the channel to guanidinium is tentatively ascribed to an entropic barrier resulting from the restrictions on the ion motion in the channel.