James E. Ricks and Professor David Busath, Zoology
The existence of multicellular organisms is dependent on the concerted activities of scores of individual cells. These vital communication links are supplied by hosts of receptors, hormones, and trans-membrane channels. One of these key links is the gramicidin ion channel, a helical peptide which when dimerized forms an uncharged channel spanning its host membrane.1
Under normal conditions, the gramicidin pore exhibits size dependent cation-selective properties; it allows the passage of certain ions, while restricting the entry of others.2 Occasionally, one of the larger species will block the channel, resulting in a reduction or halt to the flow of ions through it. Initially, it was thought that these blockages occurred at the advent of the pore’s opening, however, further studies have shown that these large classes of ions become fixed within the channel.3 These findings indicate that the pore’s geometry is not that of a simple cylinder, rather, a more complicated topography within the channel exists.
To illuminate structural characteristics of the channel within the region of dimerization, a simulated cellular membrane was formed using lipid bilayers produced on the aperture of a pipette and immersed in a potassium chloride bath.3 Malonyl gramicidin added to the bath was then allowed to diffuse to the bilayer at a frequency low enough to allow the observation of single channels.2 To restrict the number of possible channel formations to manageable quantities, the aperture diameter utilized was no greater than 100 μm.
Channel activity was measured using an applied electric potential of 100 mV across the membrane. Utilizing a commercial amplifier and a filter to reduce background noise and allow accurate measurements of the block durations, the current through the channel was plotted and recorded. This information was then digitized for analysis with 12 bit resolution at 1,000 samples/second using the IGOR® conversion program to produce the binary waves. Such a high sample frequency was necessary since block durations could be as short as 1 millisecond. Block periods were designated as those deviating from the standard channel conductances (see Figures 1 & 2). The block intervals were measured from the last data point at which the channel opened to the last data point preceding the re-establishment of pre-block current flow. Statistical analysis of block durations was conducted using TAC® and TACFit® analysis programs.
The first four months were spent developing the aforementioned lipid bilayer creation, introduction of gramicidin A channels, control of electrical trans-channel flow, and statistical analysis methods. Once these techniques were perfected, work on the malonyl gramicidin experiments commenced. The initial controls focused on analyzing the block durations of potassium ions in malonyl gramicidin channels under conditions similar to those in previous experiments utilizing the more common gramicidin A. These experiments were characterized by blocks of short duration, not unlike those present in gramicidin A channels under similar conditions. Gramicidin A controls (as exemplified in figure 1) were prepared by analyzing block durations in the presence of guani linium to use as a comparision for the malonyl gramicidin experiments.
When guanidinium ions were introduced to the malonyl gramicidin channel bath, the block durations varied greatly. However, a new phenomenon appeared, as illustrated in figure 2: clusters of prolonged blocks nearing ten times the duration of those found in the control runs.
Statistical comparisons of the block durations proved difficult, even with duplicated runs due to the variance in durations in the guanidinium blocks. This variability may be due to multiple versions of the covalent bonding in malonyl gramicidin preparation, and merits further inquiry. These results, despite their randomness, indicate that blocks do lengthen, illustrating that the absence of elastic Hbonds at the dimer junction increases malonyl gramicidin’s resistance to guanidinium ion passage.
References
- Anderson, O.S. 1984. Gramicidin Channels. Annu. Rev. Physiol. 46:531-548.
- Hemsley, G., and D. Busath. 1991. Small iminium ions block gramicidin channels in lipid bilayers. Biophy. J. 59:901-908.
- Busath, D., and G. Szasbo. 198 1. Gramicidin forms multi-state conducting channels. Nature (Lond.). 294:371-373.