Functional modifications of alamethicin ion channels by substitution of glutamine 7, glycine 11 and proline 14.

Alamethicin is a 20 amino acid, potentially helical peptaibol which forms voltage-dependent ion channels in bilayer systems. Two aspects of alamethicin structure have been suggested to be of particular functional significance for stabilization of alamethicin channels. (i) Proline 14 inducing a helix kink is together with glycine at position 11 responsible for an appropriate orientation of the molecules in the conducting associates. (ii) Glutamine 7 lining the channel interior is assumed to stabilize the channel structure by forming inter-helix hydrogen bonds. The functional importance of these residues was probed in macroscopic and single-channel experiments with alamethicin analogs containing polar, side chain bearing residues at position 11 (glutamine, asparagine) or at position 14 (glutamine). In order to investigate the crucial role of glutamine 7 for the stabilization of channel aggregates, this residue was substituted by alanine. The conformation of the lipid bound peptides was determined by circular dichroism spectroscopy. The results show that glutamine 7, glycine 11 and proline 14 are not essential for channel formation but substitution of any residue reduced the number of conductance levels and significantly reduced their lifetimes. Channel stabilization by the introduction of residues with potential hydrogen bonding capacity at positions 11 and 14 was not observed. Differences in the conformation of the lipid bound peptides, their orientation in the bilayer and their affinity for the lipid membrane appear thus to contribute to the modulation of functional properties.

Proline at position 14 of alamethicin is essential for hemolytic activity, catecholamine secretion from chromaffin cells and enhanced metabolic activity in endothelial cells.

Alamethicin is known to lyse different biological cells and to induce voltage dependent ion channels in lipid bilayers. A set of analogs with proline shifted from position 14 in the native peptide towards the N- and C-terminus was used to investigate the role of proline in: (i) alamethicin induced hemolysis of human red blood cells, (ii) stimulation of catecholamine secretion from bovine adrenal chromaffin cells and (iii) induction of metabolic activity in bovine aortic endothelial cells. Half maximal hemolytic activity was found at 30 microM alamethicin concentration, complete lysis occurred at 100 microM. The stimulation of catecholamine secretion in the presence of extracellular Ca2+ was concentration dependent up to 50 microM alamethicin. At this high concentration mild secretion was also found in the absence of Ca2+ indicating cell membrane damage. Alamethicin transiently stimulated the metabolic rate of endothelial cells in a concentration dependent mode up to 20 microM while the inhibition of metabolism at higher concentrations pointed to a toxic effect. The alamethicin analogs were completely inactive in all the biological assays. The effects correlated with a loss of dye release inducing activities on phosphatidylcholine vesicles and reduction of channel forming properties in lipid bilayers and were associated with modifications of membrane affinity rather than conformational changes of the peptides. The results indicate that proline at position 14 of the native peptide is essential for the interaction with different membrane systems.

Influence of proline position upon the ion channel activity of alamethicin.

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. To study relationships between the proline-14-induced kink region and the channel-forming behavior of the peptide, a set of alamethicin analogs with proline incorporated at positions 11, 12, 13, 14, 15, 16, and 17, respectively, as well as an analog with alanine instead of proline at position 14 were synthesized. Macroscopic conductance experiments show that the voltage dependence of the peptides is conserved although slightly influenced, but the apparent mean number of monomers forming the channels is significantly reduced when proline is not located at position 14. This is confirmed in single-channel experiments. The analogs with proline next to position 14 (i.e., 13, 15, 16) show stable conductance levels, but of reduced number, which follows the order Alam-P14 > Alam-P15 > Alam-P16 > Alam-P13. This reduction in the number of levels is connected with changes in the lifetime of the channels. Analogs with proline at position 11, 12, or 17 produce erratic, extremely short-lived current events that could not be resolved. The changes in functional properties are related to structural properties as probed by circular dichroism. The results indicate that proline at position 14 results in optimal channel activity, whereas channels formed by the analogs bearing proline at different positions are considerably less stable.

The transmembrane domains of the nicotinic acetylcholine receptor contain alpha-helical and beta structures.

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue contains both alpha-helical and beta structures. The secondary structure was investigated by Fourier transform infrared (FTIR) spectroscopy after the extramembrane moieties of the protein from the extracellular and intracellular sides of the membrane were removed by proteolysis using proteinase K. The secondary structure composition of this membrane structure was: alpha-helical 50%, beta structure and turns 40%, random 10%. The alpha-helices are shown to be oriented with respect to the membrane plane in a way allowing them to span the membrane, while no unidirectional structure for the beta structures was observed. These findings contradict previous secondary structure models based on hydropathy plots alone.