STRUCTURE-FUNCTION-RELATIONSHIPS IN HELIX-BUNDLE CHANNELS PROBED VIA TOTAL CHEMICAL SYNTHESIS OF ALAMETHICIN DIMERS - EFFECTS OF A GLN(7) TO ASN(7) MUTATION

Alamethicin channels are prototypical helix bundles that may serve as tractable models for more complex protein ion channels. Solid-phase pe ptide synthesis of alamethicin analogues using FMOC-amino acid fluorid es followed by chemical dimerization of these peptides facilitates str ucture-function studies of particular channel states in bilayer membra nes. State 3 in particular, tentatively assigned to a hexameric helix bundle, is sufficiently long-lived that current-voltage measurements c an be made during the lifetime of an individual channel opening. Molec ular models of hexameric helix bundles, generated using restrained mol ecular dynamics with simulated annealing, indicate that a Gln(7) --> A sn(7) (Q7 --> N7) mutation will increase channel diameter locally. Exp erimentally, the conductance of state 3 of the N7-alm channel is found to be larger than that of the Q7-alm channel when ion flow is in the usual direction (cations entering the C-terminal end of the channel). When ion flow is in the opposite direction, no difference in the condu ctances of state 3 of Q7 and state 3 of N7 channels is observed. These results indicate that the effect of a change in pore diameter at posi tion 7 is dependent on the magnitude of other barriers to permeation a nd that these barriers are voltage-dependent.